ENHANCED ERK1/2 ACTIVITY A CENTRAL FEATURE OF CYSTOGENESIS IN ARPKD. IMPLICATIONS FOR ION TRANSPORT PHENOTYPE. ILIR ELIAS VEIZIS

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1 ENHANCED ERK1/2 ACTIVITY A CENTRAL FEATURE OF CYSTOGENESIS IN ARPKD. IMPLICATIONS FOR ION TRANSPORT PHENOTYPE. by ILIR ELIAS VEIZIS Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis adviser: Dr CALVIN U. COTTON Department of Physiology & Biophysics CASE WESTERN RESERVE UNIVERSITY January, 2005

2 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Ilir Elias Veizis candidate for the Ph.D. degree *. Richard Eckert (signed) (chair of the committee) Calvin Cotton Faramarz Ismail-Beigi Ulrich Hopfer Cathleen Carlin Stephanie Orellana Michael Simonson (date) 11/19/2004 *We also certify that written approval has been obtained for any proprietary material contained therein.

3 Dedication To my wife Mitsi and my daughters Elsa and Athena, for being the inspiration in my life. To my parents for giving me the foundations that last forever.

4 TABLE OF CONTENTS Table of contents.1 List of tables...3 List of Figures. 4 Acknowledgments...6 List of Abbreviations...7 Abstract...8 CHAPTER I. Introduction..10 A. Significance 11 B. Literature survey 12 i. Genetics of ADPKD and polycystins...12 ii. Genetics of ARPKD and fibrocystin..14 iii. Cystogenesis...15 iv. In vivo and in vitro models to study the disease...18 v. Cyst growth and attempts to slow disease progression vi. Abnormal ion transport in cystic cells Mechanism of fluid secretion in ADPKD. 22 vii. ARPKD a developmental disease..24 C. Hypothesis and Rationale 30 1

5 D. Figures...31 CHAPTER II. Ion transport phenotype in ARPKD. 33 A. Introduction B. Methods C. Results D. Discussion E. Figures CHAPTER III. Abnormal EGF regulation of Na absorption in ARPKD..75 A. Introduction B. Methods C. Results D. Discussion E. Figures CHAPTER IV. Summary and Futuredirections.112 A. Summary B. Future directions BIBLIOGRAPHY 130 2

6 List of Tables Chapter II Table 2.1 Table 2.2 Primers used for RT-PCR reactions. 52 Phenotype of Hoxb7/EGFP +/? cystic and non-cystic mice and Balbc cystic mice 53 Table 2.3 Summary of transepithelial bioelectric properties in normal and cystic principal cell monolayers

7 Chapter 1 List of Figures Fig. 1.1 Model of ion transport phenotype of ADPKD...31 Chapter 2 Fig. 2.1 Illustration of the strategy 56 Fig GFP expression in kidneys from normal and cystic mice...58 Fig. 2.3 GFP is expressed in principal and intercalated collecting duct cells Fig. 2.4 Fluorescence activated cell sorting (FACS) of isolated renal cells..62 Fig. 2.5 Quantitative analysis of primary CD cell isolation.. 64 Fig. 2.6 Transmitted and fluorescent microscopy of GFP-positive cell monolayers.66 Fig. 2.7 RT-PCR analysis of expression of cell specific markers...68 Fig. 2.8 Ultrastructure of primary cultures of GFP-positive CD cells..70 Fig. 2.9 Ion transport properties of normal and cystic primary cell monolayers...72 Fig camp- and Ca 2+ -induced Cl - secretory currents in normal and cystic primary cell monolayers. 74 Chapter 3 Fig. 3.1 Analysis of EGFR expression in non-cystic and cystic mouse kidney.95 Fig. 3.2 Expression and phosphorylation of extracellular-signal regulated kinase in non-cystic and cystic mouse kidneys...97 Fig. 3.3 In vivo activation of MAP kinase signaling cascade in kidneys from non-cystic and cystic mice..99 Fig. 3.4 Effects of exposure to EGF on amiloride-sensitive short-circuit 4

8 current in primary cultures of CD cells isolated from non-cystic mice.101 Fig. 3.5 Effects of exposure to EGF on amiloride-sensitive short-circuit current in primary cultures of CD cells isolated from cystic mice Fig. 3.6 Summary of the acute effects of unilateral addition of EGF on amiloride-sensitive short-circuit current in CD cells isolated from non-cystic and cystic BPK mice Fig. 3.7 Polarized EGFR localization and EGF effects in primary cultures of non-cystic and cystic collecting duct cells 107 Fig. 3.8 Effects of long-term treatment of non-cystic and cystic collecting duct cells with EGF on amiloride-sensitive sodium absorption 109 Fig. 3.9 Effects of long-term treatment of non-cystic and cystic collecting duct cells with EGF on steady-state mrna levels for ENaC subunits.111 Chapter 4 Fig. 4.1 ERK1/2 a central converging point in PKD.124 Fig. 4.2 ERK phosphorylation post treatment with MEK1/2 inhibitor in cystic kidney slices Fig. 4.3 ERK phosphorylation is a self-sustained event in cystic kidneys.128 Fig. 4.3 ERK1/2 activity modulates MR regulated gene transcription

9 Acknowledgements This work would not have been possible without the excellent mentorship and enthusiastic support and friendship of Dr. Calvin U Cotton. I would like to express my sincere gratitude to all the people that were helping me along this path: members of my thesis committee, Drs. Cathy Carlin, Ulrich Hopfer, Farmarz Ismail-Beigi, Stephanie Orellana, Michael Simonson and Richard Eckert for their objective criticism and guidance; I would especially like to express my gratitude to Drs. Carlin and Hopfer for sharing their knowledge with me and the support they offered throughout my education; I would lie to thank members of Dr. Cotton s lab Mike Haley, Elizabeth Carrol, Mike Wilson and Rebeka Falin, for their assistance, friendship and support. I would like to thank the Department of Physiology and Biophysics for being a very good educational environment and its chairman Dr Antonio Scarpa for giving me the opportunity of being part of Physiology & Biophysics Department. 6

10 List of Abreviations PKD ARPKD ADPKD EGFR EGF RTK ENaC polycystic kidney disease autosomal recessive polycystic kidney disease autosomal dominant polycystic kidney disease epidermal growth factor receptor epidermal growth factor receptor tyrosine kinase epithelial sodium channel NaKATPase sodium potassium ATPase (the pump) Na + K + Cl - Ca ++ I sc Ι sc MR GR CD PC AP BL sodium potassium chloride calcium short-circuit current amiloride-sensitive short circuit current mineralocorticoid receptor glucocorticoid receptor collecting duct principal cells (cell type of collecting ducts) apical membrane basolateral membrane 7

11 Enhanced ERK1/2 Activity a Central Feature of Cystogenesis in ARPKD. Implications for Ion Transport Phenotype. Abstract By Ilir Elias Veizis The purpose of this work was to identify the abnormalities of ion transport in ARPKD cystic cells and to determine the mechanisms that might be responsible for functional abnormalities that lead to fluid accumulation inside cysts. We examined and compared the ion transport phenotype of primary collecting duct cystic and non-cystic monolayers. To that end, a transgenic mouse (Hoxb7/GFP) in which enhanced green fluorescent protein (GFP) is specifically expressed in collecting ducts was bred with an ARPKD mouse (BPK), and GFP-positive cells from non-cystic and cystic mice were selected by fluorescence activated cell sorting (FACS). We evaluated the bioelectrical properties of cystic and non-cystic monolayers and found that Cl - -secretory responses elicited by elevation of camp or calcium were not significantly different between normal and cystic monolayers. In contrast, the amiloride-sensitive short-circuit current was significantly reduced in cystic cell monolayers. These studies suggest that cystic cells have an abnormal phenotype reflected by the decrease in ENaC-mediated sodium absorption. 8

12 We examined the EGF/EGFR signaling pathway (implicated in pathogenesis of the disease) in vivo. Analysis of cystic kidneys revealed mis-localization of EGF receptor, hyperphosphorylation of EGFR and excessive activation of the p42/44 extracellular signal-regulated protein kinase pathway (ERK1/2) particularly in cyst mural cells. Primary monolayer cultures of non-cystic and cystic collecting duct principal cells were used to determine whether there is a potential role for abnormal EGF/EGFRdependent regulation of Na + transport in ARPKD. Addition of EGF acutely and chronically to the basolateral bathing solution of non-cystic or cystic monolayers led to p42/44 phosphorylation and inhibition of Na + transport; whereas apical EGF was effective only in monolayers derived from cystic mice. p42/44 phosphorylation and inhibition of Na + transport were prevented by prior treatment of the cells with a MEK inhibitor. Chronic EGF addition to the apical bathing solution of cystic monolayers led to inhibition of Na + transport and decreased ENaC steady state mrna levels. The results of these studies reveal that the mis-localized apical EGF receptors are functionally coupled to the ERK pathway and that abnormal EGF-dependent ERK1/2-mediated regulation of ENaC function and expression may contribute to PKD pathophysiology. 9

13 CHAPTER I. Introduction 10

14 I. Significance Polycystic kidney disease (PKD) is a common disorder that causes renal failure in children and adults as a result of accumulation of fluid-filled cysts in the kidney. There are two main genetic forms of PKD: A) Autosomal dominant polycystic kidney disease (ADPKD) with an incidence of 1 in (30; 34). The disease appears and becomes clinical during the fourth decade of life and affects all ethnic groups. B) Autosomal recessive polycystic kidney disease (ARPKD) with an incidence 1 to live births. ARPKD usually presents as perinatal and infantile disease (8), but rarely has juvenile or even adult onset. Renal cysts can develop in patients subjected to chronic dialysis (43). They comprise an increasing group of patients with the acquired form of PKD. Kidney cystic disease is associated with other genetic diseases such as juvenile nephrophtisis (124) or tuberous sclerosis (87). All forms of PKD result in end stage renal disease (ESRD) requiring chronic dialysis or kidney transplant (39; 42). This is a result of a continuous decline in the number of functioning nephrons. This decline is due to cyst enlargement which destroys kidney architecture (40). As cysts grow, mechanical pressure on functional nephrons together with remodeling of the extracellular matrix associated with interstitial fibrosis results in the functional kidney demise. Cystogenesis and cyst enlargement occur primarily due to increase in proliferation of the epithelial cells lining cyst and fluid accumulation inside them. Active fluid secretion from cystic cells has been implicated in ADPKD but we still do not know what are the mechanisms involved in fluid accumulation inside the cysts in the infantile form of the disease, ARPKD. 11

15 Understanding the cellular biology of the cystic cells hopefully will shed light on its pathogenesis and ultimately to improvements in diagnosis and treatment. II. Literature Survey i. Genetics of ADPKD and Polycystins ADPKD can arise from mutations in two genes, PKD1 and PKD2 (60; 107). Mutations in PKD1 located on chromosome 16p13.3 are responsible for 85% of the cases, whereas mutations of PKD2 on chromosome 4q21-23 are responsible for 15% of the cases. Mutations of PKD1 and PKD2 produce identical renal and extra renal manifestations. The PKD1 gene distributes over 52 kb of genomic DNA and contains 46 exons (54). The gene encodes a 14.1 kb mrna that is translated into a protein composed of 4302 aminoacids. Different types of mutations have been identified including splice site, inframe and out-of-frame deletions or insertions, nonsense mutations, missense mutations, and most of those likely represent inactivating mutations (110; 111). The protein encoded by PKD1, polycystin-1 is very large (~500 kd) and complex containing distinct protein motifs (54; 114). Many of these motifs are involved in protein-protein interactions (74) and suggest polycystin-1 may function as a receptor for an unidentified ligand. Polycystin-1 is expressed in many tissues including kidney, brain, heart and bone (35). It has been found to be localized in plasma membrane sometimes associated with other proteins in large cell junctional complexes of tubular epithelial cells especially collecting duct cells (36; 52; 55). Several hypothetical functions have been ascribed to polycystin-1 such as: 1) cell surface receptor (74); 2) regulator of exocytosis in kidney- 12

16 derived cells (18); 3) regulator of cell cycle by inducing cell cycle arrest at G0/G1 transition (7); and 4) regulator of G-protein signaling (58). The PKD2 gene encodes a 968 amino acid protein called polycystin-2 (72). Most of the mutations identified in this gene are truncating mutations that would inactivate the gene product. Polycystin-2 shares structural features with transient receptor potential (TRP) channels as well as voltage gated Ca 2+ channels. It is widely expressed in many tissues particularly kidney, ovary, testis and heart vascular smooth muscle (69). It is found to be localized in plasma membrane and primarily in ER membrane (88). The carboxy-terminal domain of polycystin-1 contains a coil-coil motif that binds to the carboxy-terminal domain of polycystin-2 (105) and this interaction is important since it seems to trigger G-protein signaling (98). Several functional studies have shown that polycystin-2 conducts divalent cations including calcium (37) and more recently a study showed that polycystin-2 can amplify calcium release from intracellular stores in response to hormone stimulation that transiently increases cytosolic calcium (61). The studies of genes responsible for ADPKD and the proteins they encode reveal several possible aberrations of signaling pathways that might be responsible for cystic phenotype in epithelial cells. It also raises the hypothesis that polycystin-1, a plasma membrane receptor that receives information from the environment (yet unidentified stimuli) transmits it to the cell interior through its interaction with polycystin-2 (82). This signal causes an increase in cytosolic calcium concentration, triggering exocytosis (of ion channels or membrane receptors) and changes in gene expression. 13

17 ii. Genetics of ARPKD and fibrocystin Autosomal recessive polycystic kidney disease typically affects infants and its major manifestations include ectasia of renal collecting duct and hepatic billiary ducts and fibrosis of both liver and kidney (8; 81; 157). The gene responsible for the disease PKHD1 (polycystic kidney and hepatic disease 1) is identified (97). It is localized on human chromosome 6p within a 472 kb genomic span and encodes a predicted protein of 4074 amino acids named fibrocystin (or polyductin by another group) (79; 94; 147). Fibrocystin appears to be a membrane associated protein with only one transmembrane domain and a short stretch of cytoplasmic tail composed of 192 amino acids (92; 93). A splice variant of the protein lacking the transmembrane domain has been identified and might be a secreted version of the protein (79). The structure of fibrocystin suggests that it might be a cell surface receptor. Analysis of PKHD1 protein during development reveals that it is expressed during embryogenesis in epithelial cell derivatives including kidney, neural tube, gut, pulmonary bronchi, and hepatic cells (148). It is also highly expressed in mature kidney collecting duct cells (71). So far there is not enough data to examine any relationship between the nature of the mutations and the clinical course of the disease. Fibrocystin acts as a membrane associated-protein affiliated with the basal bodies/primary cilia in renal epithelial cells (148; 158). Cilia and PKD Epithelial cells in the kidney and elsewhere contain central cilia. Cilia are long thin tubular structures that originate from the basal body (103). Central cilia likely serve a mechanosensory function in kidney epithelial cells (102). The involvement of cilia in 14

18 PKD was first suggested by studies in the orpk mouse model of ARPKD that was created by insertional mutagenesis (75). Recently PC1 and PC2 (101) have been found to be localized in cilia of kidney epithelial cells where they co-localize with ciliary tubulin. Fibrocystin also is localized in cilia and in the vicinity of the basal body which raises the possibility that it may act as an intraflagelar motor-binding protein (99; 100). In two murine models of PKD, cilia abnormalities (shorter and probably immotile) were detected (350,361). These observations have led some researchers to believe that abnormalities of ciliary function may play a role in cystic disease. To examine whether there is a direct link between cilia and cystogenesis Lin et al. specifically inactivated Kif3A in the kidney (64). Kif3A is a subunit of kinesin II, a protein necessary for cilia formation. This resulted in kidney cystic disease where epithelial cells lacked primary cilia, had increased proliferation, apoptosis and apical mis-localization of EGFR. Clinical phenotype and cell biology were similar to PKD. This finding directly implicates cilia function in lumen-forming epithelial cell differentiation. Further studies are needed to elucidate the mechanism of how alterations in ciliary function lead to cyst formation. iii. Cystogenesis Micro dissection studies from early stages of ADPKD revealed that cysts begin as focal aneurismal enlargement of tubules and with time they lose tubular connection (almost 85% of cysts in one study) (45). This focal tubular dilatation in theory could arise from an obstruction downstream producing an increase in intraluminal pressure, 15

19 aberration in basal membrane compliance, or primary abnormality in cell proliferation. Several models were considered over the years to explain the cyst formation and growth: a) Obstruction theory. One of the earliest models proposed to explain cyst formation and growth was the obstruction model. Tubular obstruction caused by polyp-like growth inside the lumen or circumferential hyperplasia was supposed to underlie cystogenesis (6; 27; 28). However experiments in animal models demonstrated that many cystic nephrons remain patent and connected with the tubular system and in most of the cysts hydrostatic pressure was not different compared to tubules of wild type animals (134; 135). b) Compliance theory. According to this theory, defects in the elasticity of the basement membrane could explain cyst creation. This was supported by the presence of connective tissue type defects in ADPKD such as cerebral aneurisms, intestinal diverticulae, and cardiac valve abnormalities (17; 44). Evidence from animals and humans with PKD showed biochemical alterations in basement membrane (16; 56; 65) such as a reduction in sulfated proteoglycan content, increase in fibronectin and the presence of matrix-degrading proteases luminaly. However direct measurements of basement membrane compliance found similar values in cystic and normal nephrons. It is likely that biochemical and morphological matrix changes are secondary to the abnormal cystic epithelial phenotype. Recently an animal model knock-out of laminin 5, an integral protein of basal membrane, produced a cystic phenotype (Shanon B.M. et al ASN abstract). This finding suggests that even basal membrane changes alone can create the microenvironment favorable for cystogenesis. 16

20 c) Tumor like cellular proliferation. Cellular proliferatin was hypothesized as the only alternative to explain cyst formation and cyst growth. It has been proven that an increase in cellular proliferation occurs in all the animal models and human disease. Sometimes cysts show polyp like hyperplasia but this is rare or not enough 3D studies have been performed to confirm polyp-like tumorigenesis. A series of in vivo and in vitro studies demonstrated that cystic cells have an intrinsic propensity to proliferate as do the immature undifferentiated kidney epithelial cells. d) Net fluid secretion in cystic tubule segments. Renal cysts in ADPKD or aneurismal tubular enlargements in ARPKD can grow up to several times over the normal tubule diameter (in centimeters in ADPKD). Mathematical models predict that even with a complete outflow obstruction, a proliferating cyst (tubule) with a normal absorptive state would not increase beyond 2-mm in diameter. Thus an imbalance in fluid transport across cystic epithelium that results in net fluid secretion must occur for the cysts to enlarge. Theoretically, impaired absorption and/or enhanced secretion can be the mechanism that would result in net fluid secretion. There is at least an agreement recently derived from data showing that two factors play the central role in cystogenesis and cyst growth: 1) increased cell proliferation (41; 68; 151; 153); and 2) abnormalities in ion transport that lead to an increase in fluid inside the cysts or enlarged tubule segments (125; 127; 128; 146). The questions that remain to be solved are: a) what determines the proliferative phenotype of epithelial cells; b) how is this process regulated and what triggers it (involvement of mitogens such as EGF, TGF- 17

21 α, HGF and their receptors); c) what are the abnormalities in ion transport phenotype; d) how the hormones, receptors and ligands affect the regulation (or dysregulation) of ion transporter activity and/or expression that would lead to net fluid secretion or retention in the lumenal space. iv. In vivo and in vitro models to study the disease. To elucidate the mechanisms that underlie PKD pathophysiology several in vivo and in vitro model systems have been utilized. Below is a general description of some of them. Animal models There are multiple murine models in which the phenotype closely resembles human PKD with respect to cyst location, morphology and disease progression. They were generated by: A. Spontaneous mutations: 1) congenital polycystic kidney in C57BL/6J strain called cpk, transmitted as a recessive trait (33); 2) BALB/c polycystic kidney called bpk, transmitted as a recessive trait (83); and 3) polycystic kidney disease (pcy) mutation occurred on the diabetic prone KK mouse strain. Initially presented as ADPKD phenotype but when mutant locus was transferred to DBA/2J strain the disease was fully penetrant as a recessive trait (134). B. Chemical induction: 1) juvenile congenital polycystic kidney disease mutation (jcpk) was discovered in a chlorambucil mutagesis program. 30 % of heterozygous +/jcpk mice also develop a late onset cystic disease but involve only glomeruli (32). 18

22 C. Insertional mutagenesis: 1) oak ridge polycystic kidney (orpk) where the gene interrupted encodes for polaris and mimics ARPKD, but the genetic background modulated significantly the disease severity (75). D. Targeted mutagenesis: 1) target mutagenesis of human ADPKD orthologues PKD1 and PKD2 in mice (9; 150). Disease progression is rapid with embryonic mortality occurring in almost all homozygotes; 2) renal specific inactivation of Hepatocite Nuclear Factor 1β (HNF1β) was associated with cystic disease of the kidney (48); 3) kidney specific inactivation of Kif3A a subunit of kinesin II a protein associated with cilia, resulted in cystic kidney disease (64). Compared with mice, only a few rat models have been described: 1) The Han:SPRD-cy rat is the best characterized (20). The mutation arose spontaneously in Sprague-Dawley strain and initial analysis revealed a dominant pattern of transmission. The disease appears less severe and progresses very slowly, but it is more pronounced in male heterozygotes than in age matched females. It is considered (based on features) as the best model for ADPKD; 2) the Winstar polycystic kidney (wpk) mutation arouse spontaneously in an outbred Winstar strain. Wpk rats exhibit histopathology that is similar to ARPKD but is lacking the hepato-biliary symptoms; 3) the polycystic kidney (pck) mutation developed spontaneously in the Crj: CD/SD strain and is transmitted as an autosomal recessive trait (63). Ward et.al (147) identified that the same gene PKHD1 mutated in pck rats, was also the gene that caused the disease in human ARPKD. There is a plethora of models to study the disease and although the genetics are different they are remarkably similar in the basic abnormalities of cell biology in cystic 19

23 cells. This prompts the argument that multiple genes define similar pathways to cystogenesis. BPK mouse a model of ARPKD The model used in studies described below is the bpk mouse. The Bpk mutation in a BALB/c background is transmitted as a fully penetrant recessive trait. Heterozygotes are normal while homozygotes develop cystic dilatation of renal collecting ducts as well as biliary dysgenesis (84; 132; 141). Death occurs within the fourth week after birth presumably due to renal insufficiency. The model has been extensively characterized from the pathology point of view but recently the responsible gene was identified as the bicaudal C gene (Bicc1) (19). It is postulated that the protein encoded by this gene plays a role in protein-rna interactions. In vitro: Several in vitro models have been used to study cystic cells in culture. Those include: 1) collecting duct cell lines generated from cystic Immortomouse (132); 2) conditionally immortalized cells derived either from human fetal tissues (cystic) or human adult cystic kidneys (109); and 3) primary cells generated from dissected human kidney cysts (128). Each of these models has its own advantages and limitations. But two of the limitations with respect to the disease are: we do not know how the immortalization process effects the phenotype of the disease and in the case of dissected cysts the segmental origin and homogeneity of cells is unknown. 20

24 v. Cyst growth and efforts to inhibit disease progression. Research in different model systems has identified several abnormalities in tubular cystic epithelial cells. These abnormalities include: 1) abnormally high cell proliferation(68; 77; ); 2) aberrant EGF, TGF-α (and other EGF-like growth factors) expression and processing (23; 155); 3) apical mislocalization of EGFR (25; 104); 4) overexpression of extracellular matrix remodeling enzymes, matrix metalloproteinases (MMP s) (106) ; 5) increased production of proinflamatory cytokines; 6) alterations in steroid and bioactive lipid metabolism; and 6) enhanced expression of the vasopressin receptor. Although these findings are not necessarily present in all disease models, they provide direction to target specific processes and pathways involved in PKD pathogenesis. I will briefly discuss two interventions that are relevant to the work described in later chapters. A. Tyrosine kinase inhibitors. A number of studies have hinted at a potential role for EGFR and its ligands, EGF and TGF-α, in renal hyperplasia and cyst enlargement. EGFR is the only receptor protein so far with abnormal localization and expression (95; 108; 130). There is evidence that EGF concentration is high in luminal fluid of cystic tubules (23; 24) since the urine of cystic animals is mitogenic. Taking into account these facts, specific (EKI- 785) and non specific (tyrphostin and genistein) EGFR inhibitors were used to determine whether they can effect cyst growth (24; 130; 131) Indeed the disease was remarkably slowed in bpk mice and kidney function improved. Besides pharmacologic manipulation, in a proof-of-principle experiment, +/orpk mice were crossed with mice heterozygous for 21

25 waved-2 (wa-2) a hypomorphic allele that attenuates EGF tyrosine kinase activity. Mutant homozygous for both orpk and wa-2 had significant reduction in collecting duct cysts and improved kidney function compared with their orpk/orpk littermates. B. Reducing ligand availability. Transforming growth factor-alpha (TGF-alpha) expression is abnormal in polycystic kidney disease(23; 84). The therapeutic potential of inhibiting TGF-alpha in ARPKD was examined using a novel inhibitor of tumor necrosis factor-alpha converting enzyme (TACE), the metalloproteinase that cleaves membrane-bound TGF-alpha to release the secreted ligand (24). Use of the inhibitor in the bpk mouse model of ARPKD resulted in improvement of the disease as indicated by reduction in cystic index in treated animals when compared with non treated littermates (130; 131). These findings support a pathophysiological role for the TGF-alpha/EGF/EGFR axis in murine ARPKD. vi. Ion transport in ADPKD Mechanism of fluid secretion in ADPKD The focus of research for many years has been on the function of cystic epithelium. Most of the studies have been performed in tissues derived from ADPKD human kidneys. As such, the accepted model of ion transport in cystic cells, is based on the dominant form of the disease. The hypothesis is that active transcellular chloride secretion drives fluid accumulation in the lumen of renal cysts (38; 46; 47; 125). In the model depicted in Fig1.1, NaKATPase located on the basolateral (BL) membrane acting 22

26 in concert with K channels establishes the chemical and electrical gradient utilized by chloride transporters. The NaK2Cl cotransporter, also located on the basolateral membrane, brings Cl - into the cell. A Cl - channel in the apical (AP) membrane provides the efflux pathway. Secretagogues, often acting through the camp signal transduction system activate the chloride channel (CFTR has been implicated). The activation of Cl channels in the apical membrane and the increased efflux of K across basolateral membrane establish a transepithelial electrical gradient (lumen negative) that drives net Na + flux through the paracellular pathway. Addition of NaCl to the luminal fluid provides the osmotic force to drive fluid secretion. Studies supporting this model were performed on dissected segments of cysts walls mounted on Ussing chambers and ADPKD cells derived from collagenase digested human cystic kidneys (47; 126; 128; 129). In another study, intact cysts dissected from human kidneys were filled with culture medium and incubated in culture medium (47). Fluid secretion was measured upon addition of forskolin to the BL side and oubain on the apical side. Forskolin (which increases camp) stimulated fluid secretion, while oubain on the apical side did not have any effect on fluid secretion. In contrast oubain added to the BL side inhibited fluid secretion. This is an indication that NaKATPase is accessible only from the basolateral surface. Kidney epithelia are predominantly absorptive epithelia. Sodium reabsorption electrogenic or not occurs in all segments from proximal tubule to collecting ducts. In most studies of ion transport by ADPKD cystic cells, net fluid Na absorption is not observed and even high doses of amiloride do not inhibit any significant portion of the 23

27 short-circuit current in Ussing chambers. This has suggested that maybe ADPKD cystic cells in vitro: 1) do have Na absorptive capacity due to the lack of Na transporters and/or abnormal regulation of Na transport; 2) are able to produce enough secretagogue to activate Cl channels that in turn inhibit Na transport. vii. ARPKD a developmental disease. ARPKD begins with rapid embryonic cyst expansion, first in proximal tubules and next in all collecting tubules. Failure of the differentiation program during nephrogenesis may lead to diverse pathological conditions that can perturb renal structure and function. I believe that the process of nephrogenesis holds very important clues relevant to cystogenesis. Kidney development requires inductive interaction between two different tissues, namely the nephrogenic (mesenchymal) and the ductogenic (ureteric) tissues. The latter includes the collecting duct system which is the site of the lesion (cysts) in ARPKD. That is not to say that mesenchymal derived tissue is normal (the initial lesions in murine ARPKD are in proximal tubule) because the appropriate maturation depends on interactions between signaling molecules secreted by mesenchymal primordial tissue and ureteric bud (CD precursors) receptors. I would like to discuss briefly an important signaling pathway in normal kidney development and also the switch in ion transport believed to occur during kidney maturation. Those are two important factors I believe to be involved in cyst formation in ARPKD. 24

28 Kidney development and the importance of ERK1/2 signaling Normal kidney development is dependent on inductive interactions between the metanephric blastema a mesenchymal tissue and the ureteric bud an epithelial structure. Signals from the metanephric blastema stimulate ureteric bud growth as a lateral extension of the Wolffian duct and invasion of the metanephric blastema at 5 weeks of gestation in humans and E10.5 in the mouse. Cells of the metanephric blastema adjacent to the tip of ureteric bud are induced by the ureteric bud to undergo a mesenchymal-toepithelial transformation. In reciprocal fashion, the metanephric blastema signal the ureteric bud to grow and branch in a process termed branching morphogenesis. Ongoing reciprocal inductive tissue modeling results in formation of the proximal epithelial nephron segments (glomerulus, proximal and distal tubules, loop of Henle) from blastemal progenitors and cortical and medullary collecting ducts from the ureteric bud (51). Development of tubular epithelium is regulated by a large number of molecules produced by the mesenchyme including: growth factors, proteases, and components of the extracellular matrix. Epithelial cells integrate these signals to produce the appropriate response. One of the major signaling networks that plays an important role in branching and morphogenesis of the collecting ducts is the mitogen-activated protein (MAP) kinase pathway (31). The growth factor (GDNF) which is secreted by the nearby metanephrogenic mesenchyme is so powerful that GDNF-soaked beads can elicit a large number of ureteric branch tips in culture but also necessary since GDNF -/- transgenic mice fail to form ureteric buds (73). The receptor tyrosine kinase (RTK) that binds GDNF also binds related ligands (growth factors), and most of these signals are 25

29 postulated to integrate at the ERK1/2 levels signaling cascade. As described in a series of elegant experiments by Davies at al, ERK1/2 is normally active in ureteric bud and inhibition of ERK activity with a MEK inhibitor (PD98059) reversibly blocks branching and morphogenesis (31). Furthermore, when ERK is inhibited ureteric bud tips show less cell proliferation than controls and also produce fewer laminin rich processes penetrating the mesenchyme. MAPK (ERK1/2) is considered therefore essential for normal branching and morphogenesis of the precursors of collecting ducts and lies downstream of significant extracellular regulators of ureteric bud development. The ERK1/2 pathway is generally, but not exclusively, responsive to activators of both receptor tyrosine kinases and G-protein coupled receptors. Principal mammalian ERKs, p44 and p42, are activated by MEK1/2 kinases that phosphorylate a threoninetyrosine motif specific to ERKs. Effectors of activated ERK include transcription factors (e.g. elk-1, m-myc), adaptor proteins and nuclear receptors (estrogen receptor) (137). Characterization of physiological consequences of ERK signaling has been facilitated by pharmacological inhibitors PD (2) and U1206 (29). Both appear to block ERK signaling indirectly through inhibition of its upstream activator, MEK. The ERK pathway in the kidney CD plays an important role in many physiological processes, such as growth factor induced proliferation, CD branching and development, urea signaling in IMCDs and the osmotic stress response. The requirement for ERK activity during nephrogenesis is demonstrated by the failure of CDs to develop in organ culture when treated with PD98059 (Davies et.al..1999). ERK1/2 in CD cells can be activated by EGF- EGFR (EGF is produced upstream of CD), bradykinin (G-protein coupled receptor), 26

30 purinergic agonists (NTPs) and endothelin. Continuous environmental and hormonal challenges make ERK and important signaling pathway in kidney CD. Ontogenesis of CD Na + transport The capacity for net Na + absorption by the CD epithelium is acquired as the kidney matures. Embryonic ureteric bud cells (E17) express constitutively active outwardly rectifying Cl conductance whereas perinatal and early postnatal ureteric bud/cortical collecting duct cells acquire the expression of mature type swelling activated chloride conductance around postnatal day 1 before the onset of vectorial ion transport. During this period the embryonic type chloride conductance is downregulated (51). Ontogenic acquisition of vectorial transport should be functional at the onset of glomerular filtration to prevent the loss of Na + in urine. Electrogenic Na + transport in CDs mediated by the amiloride-sensitive epithelial sodium channel (ENaC), begins at the end of nephrogenesis (embryonic day 17 in mice) (51; 53). During early nephrogenesis neither ENaC activity nor mrna is present. ENaC mrna, first detected in kidneys of E17 mice, is increased by a factor of 2 in postnatal CD (P1-P6) and by 5 fold 7-28 days post birth. This is an indication that the onset of Na + absorption occurs toward the end of nephrogenesis and increases with the process of CD maturation. Na + absorption in the kidney CD is controlled by the mineralocorticoid receptor ligand, aldosterone (70; 96; 145). Differentiated (mature) kidney CDs are aldo-sensitive; whereas, immature kidney CDs (e.g... premature infants) are aldo-insensitive (139; 140). 27

31 Apico-basal polarization in CD cells. Epithelial cell ontogeny from the ureteric bud to differentiated CD cells is characterized by a polar distribution of membrane proteins such as transporters and receptors. Ureteric bud cells maintain a nonpolar distribution of receptors such as EGFR but also of transporters such as those carrying Cl - ions (50; 53). In differentiated CD principal cells, EGFRs are localized predominantely on the basolateral membrane. The mature polarization pattern includes AP distribution of ENaC, AQP2 and basolateral distribution of NaKATPase and AQP3. In ARPKD there is a partial loss of polarity (associated with the so-called dedifferentiated PC phenotype) since the EGFR is localized to the AP as well as BL membrane. This is not a generalized defect since in many animals models of the disease immunolocalization studies have found that other membrane receptors and transporters are localized appropriately. It has been proposed that fluid secretion in ARPKD results from active Na + secretion on the apical membrane of the cystic epithelium caused by misslocalization of a functional sodium pump. Expression of NaK ATPase on the AP membrane of cystic cells is still a point of contention between researchers in the field. In 1991 Wilson et al (149) proposed that Na + is the ion actively secreted by cystic tissue and that it occurs in the context of a complete reversal in polarity of cystic cells. The author investigated activity and localization of NaKATPase in slices of very early-stage cystic kidneys and found higher activity of the pump in cystic kidneys and detected NaKATPase only on the apical membranes. Several studies have disputed that theory on the following basis: a) although the Na + pump can be found apically in tissues that secrete Na + such as choroids plexus and retinal epithelium and also transiently in nephrogenic zones of developing 28

32 kidneys it is not consistently found in PKD tissues with several studies disputing its misslocalization in cystic epithelia (64; 136). b) even in studies where misslocalization was demonstrated it occurred only in about 25 % of the cysts indicating that it is not necessary for progressive cyst enlargement (15). 29

33 C. Hypothesis and Rationale We hypothesize that aberrant signaling pathways in cystic epithelial cells cause abnormalities in ion transport that have as a consequence an increase in lumenal fluid accumulation and tubule enlargement. Rationale. Mutations in cystic kidney disease genes despite their clinical and histopathological heterogeneity result in a similar outcome: dilatation of tubules and cystogenesis. This indicates that the gene defects underlying these disorders might influence a common pathway. These mechanisms involve loss of control over cell cycle (cellular proliferation), partial loss of cell polarity, abnormalities in fluid and electrolyte transport which result in net secretion and fluid accumulation in enlarged tubules. Net fluid accumulation could occur as a result of either active secretion or inhibition of absorption. Mature CD cells have the capacity and the appropriate transporters to both secrete and absorb. Of course they have to choose either process otherwise it would be energetically extremely unfavorable. Cystic collecting duct cells resolve this dilemma by enhancing signaling pathways that inhibit Na absorption. Turning off functional Na + transport channels (ENaC) might play an important role in cyst fluid acumulation. ERK1/2 activity is an important focal point of intracellular cross-talk through which cell surface receptors (such as EGFR) and cellular stress pathways can control a basic function of those cells namely Na + reabsorption. 30

34 Figure 1.1 Model of ion transport phenotype in ADPKD. Secretagogoues such as camp stimulate Cl secretion from apically located Cl channels with the driving force provided by NaKATPase and chloride entry into the cell by NaK2Cl cotransporter. As noted, Na flux occurs through the paracellular pathway and water follows NaCl movement toward the lumen. Note the lack of apical Na + conductance (ENaC?) in the model supported by the data that short circuit current is insensitive to high amiloride doses. 31

35 Na + H 2 O Cl CCl? CFTR Lumen + + C ca K + Na + K + Cl Na + K + NaK2Cl cotransporter NaKATPase K Blood 32

36 Chapter II Decreased amiloride-sensitive Na + absorption in collecting duct principal cells isolated from bpk ARPKD mice. 33

37 Introduction The renal cystic disease typically begins in utero and manifests as fusiform dilatation of the collecting ducts that radiate from the medulla to the cortex (8). Detailed studies of salt and water transport in renal cysts detached from the nephron of origin and primary cultures of renal epithelial cells isolated from ADPKD patients suggest that fluid accumulation in the cysts is the result of NaCl secretion. Grantham and coworkers demonstrated that camp-stimulated, CFTR-dependent Cl - secretion contributes to fluid accumulation in renal cysts (21; 128). Since the cysts are detached from the segment of origin, it is difficult to identify the precise alterations in tubule transport that accompany development of a renal cyst in ADPKD. In contrast, the predominant site of renal disease in ARPKD is the collecting duct (CD) and late in the disease most of the kidney is composed of dilated, fluid-filled CDs rather than isolated, detached cysts. The collecting duct of the mammalian kidney is a cytologically diverse segment comprised of principal cells (PC) and intercalated cells (IC). Intercalated cells account for 10-30% of the cells in the collecting duct and are responsible for H + /HCO - 3 excretion in the distal nephron (11; 59). Principal cells are more numerous (70-90%) and are characterized by hormonally-regulated (e.g., aldosterone and vasopressin) sodium, potassium, and water transport (86; 96). Principal cells play a vital role in salt and water homeostasis via regulated alterations in sodium absorption and water permeability. The expression and activity of epithelial sodium channel (ENaC), located in the apical plasma membrane of CD principal cells (13), and is the rate-limiting step for CD sodium absorption. ENaC expression, although established early in nephrogenesis (51; 53), is developmentally regulated and is important for postnatal sodium homeostasis (115). 34

38 ARPKD is generally considered to be a disorder with developmental arrest or cellular dedifferentiation to a less mature phenotype. Therefore, ENaC-mediated sodium absorption capacity, which is considered an indication of CD maturation, represents an important ion transport pathway that may not fully develop or might be lost from less mature cystic collecting duct cells. ARPKD cystic CDs are comprised almost exclusively of principal cells; however, almost nothing is known about the ion transport properties of cystic CD principal cells. A limiting factor in the study of ion transport pathophysiology in ARPKD is the lack of relevant biological preparations, since the cystic CDs are extremely dilated and are not suitable for conventional tubule perfusion. Cell lines generated from human (109) and murine (132) ARPKD kidneys as well as freshly isolated or primary cultures of epithelial cells represent important reagents for the study of disease-related alterations in cell function. In this study, we developed an efficient method to isolate renal CD principal cells from normal and ARPKD mice that can be grown in primary culture and are suitable for analysis of transepithelial ion transport. The results of these studies suggest that amiloride-sensitive sodium absorption is significantly reduced in cystic CD principal cells, whereas agonist-induced Cl - secretion is similar in normal and cystic cells. These observations highlight a fundamental difference between ion transport dysregulation in autosomal dominant and autosomal recessive PKD. 35

39 Methods Generation of the animal model. The primary disease model is the BPK mouse, which arose as a spontaneous mutation on a BALB/c background and mimics the phenotype of ARPKD (84). Confirmed BPK heterozygotes (bpk +/- determined by breeding) were crossed with a transgenic mouse (Hoxb7/GFP; B6xCBA) (123) in which green fluorescent protein (GFP) expressed under the control of Hoxb7 promoter is specifically expressed in the ureteric bud and its derivatives collecting ducts. GFP +/? offspring (F 1 ) were bred with confirmed BPK heterozygotes (bpk +/- ) in order to determine BPK status of the F 1 pups (Fig 2.1). GFP +/? /bpk +/- animals were identified and bred with one another. All of the resulting F 2 pups were genotyped for the GFP transgene and examined at postnatal day to identify cystic pups which have a characteristic abdominal distention. Age-matched, cystic (GFP +/?, bpk +/+ ) and normal (GFP +/?, bpk +/- or bpk -/- ) littermates between days were used for these studies. The offspring were genotyped by polymerase chain reaction (PCR) analysis of DNA extracts from tail sections to identify animals that carried the GFP transgene (GFP +/? ). The primers used to screen for the Hoxb7/EGFP transgene are primers E and primer K (see Table 1), which amplify a band of 321 bases as described by Srinivas at al. Primary cell isolation and cell culture. Kidneys were dissected under sterile conditions from CO 2 -anesthetized normal and cystic animals and the renal capsule was removed. The kidneys were minced thoroughly with a razor blade, rinsed with sterile phosphate buffered saline (PBS), and resuspended in 10 ml of collecting tubule media (CT media) supplemented with collagenase (1.5 mg/ml Type IV; Worthington). The minced tissue was digested for minutes at 37ºC in a shaking water bath to obtain a 36

40 homogenous suspension of individual cells and tubule fragments. The cellular material was collected by centrifugation, re-suspended in CT media, plated on plastic tissue culture dishes and placed in a humidified tissue culture incubator (37ºC and 5% CO 2 ) for hours. The unattached cells were removed, centrifuged, resuspended in fresh CT media and plated onto additional tissue culture dishes. Media was changed every 48 hours thereafter. Primary cell cultures were expanded for 4-6 days before sorting. CT media was composed of: 1:1 mix of Dulbecco s modified Eagle s medium (DMEM) and Ham s F12 medium (Life technologies) supplemented with 1.3 µg/l sodium selenite, 1.3 µg/l triiodo-l-thyronine, 5 mg/l insulin, 5 mg/l transferrin, 25 µg/l prostaglandin E1, 2.5 mm glutamine, 50 nm dexamethasone, U/l nystatin, 50 mg/l streptomycin, and 30 mg/l penicillin G. Fluorescence Activated Cell Sorting (FACS). After 4-6 days in culture the cells were detached from the tissue culture dish (0.25% trypsin and 0.5 mm EDTA), resuspended in CT medium that contained fetal calf serum (10%) and passed through a 40 µm mesh to remove debris. The cells were recovered by centrifugation (400 x g, 5 min) and the pellet was re-suspended in an appropriate volume of ice-cold Hepes-buffered salt solution (~15 million cells/ml). The preparation was subjected to FACS using an Elite ESP (Beckman Coulter, Miami) FACS sorter equipped with an argon ion laser tuned to 488 nm. Data were processed with Expo32 software (version 1.2b, Beckman Coulter, Miami) analysis program. GFP-positive cells were identified by their high fluorescence intensity compared to GFP-negative cells. The dot plot analysis revealed that the population of GFP-positive cells was well separated from the GFP-negative population. The sorting gate was positioned well into the GFP-positive population to minimize 37

41 contamination of the preparation by GFP-negative cells. The GFP-positive CD cells were collected under sterile conditions, re-suspended in CT media supplemented with EGF (2ng/ml) and FBS (2.5%), and plated on collagen-coated permeable supports (see below). In addition to the initial FACS, CD cells were subjected to a second analysis after electrophysiology experiments to validate the purity of the experimental preparation. Electrophysiological studies. CD primary cells were seeded (1.5-2 X 10 5 cells/filter) on collagen coated, permeable supports (12-mm Millicel-CM filter). The filter surface was coated with calfskin collagen as described (133). The GFP-positive cells were grown in CT media supplemented with 2.5% FBS and 2 ng/ml EGF for 4-5 days at 37ºC in a humidified 5% CO 2 atmosphere. FBS and EGF were omitted from CT media at least 24 h prior to electrophysiological analysis. Confluent monolayers were mounted in thermostatically controlled Ussing chamber equipped with gas inlets and separate reservoirs for the perfusion of the apical and basolateral compartments. Both sides were bathed with an equal volume of Krebs-Ringer bicarbonate solution containing (in mm): 115 NaCl, 25 NaHCO 2, 5 KCl, 2.5 Na 2 HPO 4, 1.8 CaCl 2, 1 MgSO 4 and 10 glucose. The solutions were circulated through the water-jacketed glass reservoir by gas lifts (95% O 2-5%CO 2 ) to maintain solution temperature at 37ºC and ph at 7.4. Transepithelial voltage difference (V T ) was measured between two Ringer-agar bridges; each positioned 3 mm from the monolayer surface. Calomel half-cells connected the bridges to a high impedance voltmeter. Current through an external direct current source was passed by silver-silver chloride electrodes and Ringer-agar bridges to clamp the spontaneous V T to 0 mv. The current required [short-circuit current (I sc )] was corrected 38

42 for solution and filter series resistance. Monolayers were maintained under short circuit conditions except for brief 3-5 s intervals when the current necessary to clamp the voltage to a nonzero value was measured to calculate transepithelial resistance (R T ). RT-PCR analysis of gene expression from kidneys and primary cultures. RNA was obtained from cystic and normal kidneys (100 mg tissue) and primary cell cultures (1-2x10 6 cells) with the RNeasy Mini Kit which includes an on-column DNase digestion with RNase free DNase set (Qiagen, Valencia, CA). The concentration and quality of mrna was determined photometrically (260/280nm). Reverse transcriptasepolymerase chain reaction was performed by using MMLV (Moloney Murine Leukemia Virus), a reverse transcriptase system (Life Technologies, Rockville, MD) according to manufacturer s instructions. Appropriate primers (Table 2.1) were used to amplify cdnas from whole kidney and primary cell cultures for: GFP (123), mineralo-corticoid receptor (MC-R) (5); α-subunit of epithelial sodium channel (α ENaC) ; anion exchanger 1 (AE1, band 3) (67), and C- and N-termini of β1-subunit of the hydrogen pump (H-ATPase, β1) (85). PCR reactions were performed on a thermalcycler (94ºC denaturing 1 min/ 55ºC annealing 1 min / 72ºC elongation 1 min per cycle). PCR products were resolved by electrophoresis in 1% agarose gels and stained with ethidium bromide. The size of PCR products was compared with DNA low-mass ladder (Gibco BRL). Appropriate control PCR reactions were carried out in the absence of RT. Microscopy. GFP expression was examined in sections of kidney from cystic and normal littermates cut with a manual microtome and observed with standard fluorescein filters on a Zeiss LSM confocal microscope (Zeiss, Gottingen, Germany). Acquired images were processed with Adobe Photoshop 6.0. GFP expression in cystic CD s was 39

43 also examined in tubule fragments from a preparation of partially digested kidneys. To identify different cell types in CDs, GFP-positive cells were evaluated for expression of aquaporin 2 (AQP2) and H-ATPase 70 kda subunit, markers of principal cells and intercalated cells, respectively. Mice were anesthetized and kidneys were removed, washed, and fixed by immersion in ice cold 3.7% paraformaldehyde in PBS for 30 min at 4 C. The kidneys were washed with running water for 1 hour, dehydrated through serial ethanol and xylene solutions, and embedded in paraffin. Staining for AQP2 and H- ATPase was carried out on 4 µm sections. Deparaffinized, rehydrated sections were treated for 5 min with 1%SDS, an antigen retrieval procedure used to improve antigen exposure. The sections were blocked with 5% BSA, 0.1% Triton X-100 in PBS and incubated with the AQP2 (1:400 dilution) primary antibody (Santa Cruz, CA) and anti H + -ATPase (1:500 dilution) 70 kda subunit (kindly provided by Dr. Xia-Song Xie, U. T. Southwestern) for 2 hours at room temperature. The immune serum against the 70 kd catalytic subunit of the vacuolar H + -ATPase labels all intercalated cell subtypes in mouse. Sections were washed three times for 10 min with PBS, blocked with 5% normal serum from secondary antibody species in PBS and incubated for 1 hour with the secondary antibody coupled to fluorophore Texas-Red or rhodamine (Jackson Immunoresearch). The sections were washed three times for 10 min with PBS and mounted with SlowFade reagent (Molecular Probes, Eugene, OR). Sections were examined with a confocal Zeiss LSM 410 microscope (Zeiss, Gottingen, Germany) by using 488 to 568 nm wavelength lines of an argon-krypton laser. Confluent monolayers of CD primary cells grown on collagen-coated permeable supports were fixed with 3% paraformaldehide-pbs for 10 min on ice, and permeabilized with 40

44 0.5% SDS for 5 min. Cells were incubated with anti ZO-1 antibody (Zymed, CA) diluted 1:1000 in PBS for 45 min at room temperature. The fluorophore conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Fort Washington, PA) was applied for 45 min at room temperature. After three washes with PBS for 15 min, membranes were cut from their plastic support and mounted on slides. Cells were examined with a Nikon microscope using phase contrast and 488/568-nm wavelength lines. Images were collected using a 40x Plan-Neofluor objective and processed in Adobe Photoshop. Scanning electron microscopy. After electrophysiology experiments were completed, monolayers (n=8) were washed with Ringer solution and fixed in 2.5% glutaraldehyde containing 0.1M cacodylate buffer, ph 7.4. The filter membranes were cut from their supports and postfixed for 1 hr in 2% osmium tetroxide, stained en block with uranyl acetate, dehydrated in graded ethanol, and embedded in Epon (Electron Microscopy Sciences, Ft. Washington, PA). Membranes were stained with uranyl acetate and lead citrate prior to examination with a scanning electron microscope at 3000X magnification. 41

45 Results Characterization of GFP +/? normal and cystic mice. The offspring of confirmed GFP +/? / bpk +/- parents (Fig2.1) were evaluated by PCR for GFP transgene and by visual observation for abdominal distention on postnatal day to identify cystic and normal GFP +/? pups. Cyst development and disease progression in outbred GFP +/? cystic mice are indistinguishable from inbred BALBc cystic BPK mice (Table 2.2). Mean survival of cystic GFP +/? mice is 23±3 days and kidney weight/total body weight is 22±2.5%. Furthermore, the GFP +/? cystic mice exhibit the urine concentrating defect characteristic of this animal model of ARPKD (Table 2). All experiments described in this study were performed on age-matched cystic and normal littermates between days old, an age that represents a late-stage in the disease (End Stage Renal Disease, ERDS) in this murine model of ARPKD. Histologic examination of sections of GFP +/? normal mouse kidneys revealed a GFP expression pattern consistent with CD s radiating from the cortex to medulla (Fig. 2.2A). On the other hand, cystic kidneys and isolated cystic nephron fragments revealed that the majority of the dilated renal tubules (cysts) were lined by a single layer of GFPpositive epithelial cells (Fig. 2.2B,C). Immunolocalization of principal cell aquaporin 2 (Fig. 2.3C) and intercalated cell H-ATPase (Fig. 2.3D) demonstrated that nearly all of the cells (>95%) lining the GFP-positive cysts are principal cells, consistent with previous reports. In contrast to cystic mice, the normal littermates have the expected distribution of intercalated and principal cells in the GFP-positive collecting ducts (Fig. 2.3 A,B). Isolation of GFP-positive collecting duct cells. Individual cells and tubule fragments obtained from a collagenase digest of minced kidneys from cystic and normal 42

46 mice were plated on plastic tissue culture dishes and maintained in defined, serum-free CT medium. After 4-6 days in culture the cells were collected (15-24x10 6 cells/mouse; Fig.2.5A) and subjected to fluorescence-activated cell sorting. Representative sorting profiles of cystic GFP +/? cystic GFP -/- and re-analysis of GFP- positive cell population from age-matched littermates are shown in Figure 2.4. The cells derived from normal and cystic kidneys are similar in terms of forward scatter (FSC) and side scatter (SSC), which indicate size and viability, respectively. However, based on GFP fluorescence intensity, the histogram revealed a bimodal distribution of cells derived from either GFP +/? normal or cystic mice, but not from GFP -/- animals. Mean fluorescence intensity of GFP-positive cells was approximately 100 fold greater than that of GFP-negative cells. The GFP-positive cells comprise 18 % (n=29) of the sorted cells in cultures from normal mice and 22% (n=32) of the cells derived from cystic mice (Fig. 2.5B). From the total population of kidney cells we were able to isolate x 10 6 (n=28-32) GFP-positive cells/animal from cystic or normal littermates (Fig. 2.5C). Reanalysis GFP-positive collecting duct cells indicated a very low contamination levels by GFP-negative cells (<3%). Characterization of cells obtained by FACS. Primary cultures of CD cells selected by FACS were grown on collagen-coated permeable supports for 5-6 days. The confluent monolayers developed a typical cobblestone appearance (Fig.2.6A) and normal and cystic monolayers were morphologically indistinguishable. Flouresence microscopy revealed that 97% (n=60 cells/monolayer in 4 different preparations) of the cells in the monolayer are GFP-positive, consistent with a nearly pure population of collecting duct cells (Fig. 2.6B). The cells differentiate and form junctional complexes as 43

47 demonstrated by ZO-1 staining (Fig. 2.6C) and develop high electrical resistance (1-1.4 kωcm 2 ; Table 2.3). Since both PC and IC are derived from the ureteric bud and are GFPpositive,we sought to determine by RT-PCR the gene expression of marker proteins that are characteristic of PC or IC. RT-PCR analysis of RNA isolated from cystic or normal monolayers, revealed the presence of mrna for PC-specific proteins (mineralocorticoid receptor and epithelial sodium channel α-subunit of ENaC) but not for IC-specific proteins (H-ATPase β1 subunit and anion exchanger 1 band 3) (Fig. 2.7). RNA isolated from normal and cystic kidneys was used as a positive control for expression The eponymous morphologic characteristic of the PC is the presence of a single central cilium (30) that is absent from IC. Scanning electronmicroscopy revealed that nearly all of the cells (57 out of 60 cells, in 10 fields) in monolayers derived from cystic or normal mice have a central cilium and no morphologic differences were noted between the cystic and the normal principal cells (Fig. 2.8). The average length of the cilium (1-2.5 µm, n=30 cells) was the same in cystic and normal principal cells. These results indicated that the primary cultures of CD cells isolated by FACS of GFP +/? mice and cultured for 5-6 days on permeable supports prior to Ussing chamber experiments, consisted almost exclusively of cells with features characteristic of mammalian principal cells. Bioelectric properties of normal and cystic PC monolayers. The transepithelial bioelectric properties of primary cultures of CD principal cells are listed in Table 2.3. Cells derived from either normal or cystic mice formed polarized, highresistance epithelial monolayers. Under basal conditions, the transepithelial electrical resistance (R T ) was not different between normal and cystic monolayers (Table 2.3). However, the short circuit current (I sc ) was significantly lower in cystic cells compared to 44

48 normal principal cell monolayers. In addition, the open-circuit transepithelial voltage difference (V T ) of monolayers of principal cells isolated from cystic mice (-20.0±3.4mV, n=10) was significantly reduced compared to monolayers comprised of normal principal cells (-32.6±7.6mV, n=12, p<0.005 unpaired t-test ). As illustrated in figures 2.9A and B, addition of the sodium channel inhibitor amiloride (100 µm; a maximally effective inhibitory concentration) to the apical bathing solution caused a rapid increase in R T and a decrease in I sc (Table 2.3). Nearly all (95%) of the basal I sc in normal monolayers was inhibited by amiloride, in contrast to cystic monolayers where a significantly smaller fraction (83%) of I sc in cystic monolayers was sensitive to amiloride. Thus, the absolute and fractional inhibition of I sc (Fig. 2.9C) were significantly greater in monolayers derived from normal compared to cystic mice, a response suggestive of reduced amiloride-sensitive sodium absorption in cystic collecting duct principal cells. Furthermore, the amiloride-induced conductance decrease ( G T ) in normal monolayers was nearly twice as large as the decrease observed in cystic monolayers (Table 2.3 and Figure 2.9D). Net Cl - secretion, mediated by either camp- and/or calcium-dependent apical Cl - channel activation has been implicated in transepithelial fluid secretion in ADPKD. Since collecting duct cells are known to express a number of Cl - channels including camp-and calcium-activated channels, we examined the potential role of enhanced Cl - secretion in ARPKD. We determined the effect of elevated camp (forskolin/isobutylmethylxanthine; 10 µm/100 µm) on I sc in cystic and normal monolayers which had been pretreated with amiloride. Addition of the camp-agonists to the basolateral bathing solution caused a small, sustained increase in I sc. The steady- 45

49 state (reached after 10 min exposure to the agonist) I sc during elevation of camp in normal and cystic monolayers was 4.7±0.2 and 5.5±0.3 µamp/cm 2, respectively (Table 2.3). As expected, R T was also significantly decreased in response to camp (Table 2.3). Neither the increase in I sc nor the decrease in R T were significantly different in normal compared to cystic monolayers. Calcium-activated Cl - secretion was elicited by addition of ATP (100 µm) to the apical bathing solution of epithelial monolayers pretreated with amiloride and forskolin/ibmx. Both normal and cystic monolayers responded with a large, transient increase in I sc. The peak currents were observed at ~ 30 seconds after exposure to ATP and were not significantly different between normal and cystic monolayers (Table 2.3). The I sc of both cystic and normal monolayers returned to pre- ATP values within 3-5 minutes. Thus, neither the magnitude (Fig. 2.10A, B) nor the duration of the secretory response to extracellular ATP was abnormal in cystic cells. 46

50 Discussion The dilated CDs (referred as CD cysts due to the analogy with ADPKD) are lined with a single layer of highly proliferative CD principal cells. Anatomically the dilated nephron segments retain up- and down-stream connections but they appear to result in functional cysts. Hypertension frequently accompanies ARPKD but the precise mechanisms responsible for high blood pressure in this disease remain unknown. Since ARPKD is a complex disease hypertension might develop as a result of renin-angiotensin- aldosterone axis overactivity, local and systemic effects of substances released in response to kidney hypoxia or aberrant renal tubule ion transport. CDs are the site of the kidney lesion in ARPKD and ion transport phenotype changes in the disease might provide clues to fluid retention in those dilated tubules and/or to etiology of hypertension. Renal CD principal cells isolated from normal and ARPKD mice form high resistance, polarized monolayers in primary culture. The Cl - secretory responses due to elevation of camp or Ca ++ are the same in normal and cystic cells, whereas amiloridesensitive sodium absorption is significantly reduced in cystic cells. These results suggest that dysregulation of PC sodium absorption may contribute to the CD dilatation and fluid retention in the kidney characteristic of ARPKD. Aberrant ion transport in the kidney is not linked directly to the genetic defects that cause PKD, but may play an important role in the rate of disease progression. In the dominant form of the disease (ADPKD) fluid accumulation driven by NaCl secretion, increases cyst size leading to kidney parenchyma destruction and ultimately renal failure. Little is known about ion transport in ARPKD mostly due to the lack of relevant 47

51 experimental systems. The goal of these studies was to develop a method for isolating CD principal cells and to examine the ion transport phenotype of primary collecting duct cells derived from the BPK mouse model of ARPKD. To this end, we crossed the Hoxb7/GFP mouse line with the BPK murine model of ARPKD to yield cystic and noncystic mice that specifically expressed EGFP in collecting duct cells. The Hoxb7/EGFP +/? x bpk +/+ mice had the same timecourse of disease progression, site of cystic lesions, and degree of renal failure due to cyst enlargement as the inbred BALBc bpk +/+ mice, indicating that disease phenotype is independent of the mouse strain. Similar results were obtained when BALBc bpk +/+ mouse was bred with other mouse lines (e.g. CFTR knockout and ImmortoMouse) and there was no change in disease phenotype (80; 132). It is clear from our immunolabeling experiments that normal and cystic animals express GFP in both PCs and ICs. However, nearly all of the GFP-positive cells in the collecting duct cysts are PCs, in agreement with previous studies (130). Thus, GFP expression provides an easily visualized marker for the CDs in whole cystic kidney sections in organ culture, in kidney section immunohistochemistry, and more importantly a nearly pure population of CD cells can be isolated by FACS. Even though we can not ascertain the sub-segment origin of the isolated cells, the phenotypic characterization reveals that they are CD principal cells. GFP-positive cells were easily detected and isolated by FACS because of uniform, high-level of expression of the transgene (no evidence of intracellular GFP aggresome formation) with approximately 100 fold increase in mean fluorescence intensity compared to GFP-negative cells. A similar approach was described recently by Nelson and coworkers in which PC-specific GFP 48

52 transgene expression was driven by the aquaporin 2 promoter (159). Their model provides a unique opportunity to study cell-specific AQP2 promoter activity; however, sustained high-level expression of GFP appears to require maneuvers such as dehydration of the animals or exposure of the cells to camp agonists. The relatively large number of cells obtained from each Hoxb7/EGFP +/? normal or cystic mouse precludes the need for multiple passages to expand the cell number and thereby reduces the problems associated with long-term cell culture. Importantly, GFP expression status is faithfully retained in culture for at least two weeks. As discussed above, both PC and IC are GFP-positive in vivo but the epithelial monolayers after days in culture are comprised exclusively of principal cells (e.g. posses a central cilium and express GFP, α-enac, MC-R, and AQP2, but not HATPase β1 subunit and AE1 mrna). It is not known if the culture conditions utilized in our studies do not support IC survival or if there is conversion from ICs to PCs ex vivo. There is evidence of phenotypic plasticity in CD cells, in particular interconversion of α- and β- ICs and PCs in vitro (1); however, the molecular mechanisms responsible for this behavior have not been elucidated. The initial hypothesis for reversal of Na,K-ATPase polarity in PKD (149) is clearly not supported by our findings in primary cultures of ARPKD principal cells, since cells form high resistance epithelial monolayers with the appropriate polarization of Na + absorption and Cl - secretion. The importance of CFTR-dependent Cl - secretion in ADPKD is widely accepted (21) and recent observations suggest that extracellular ATP may be a paracrine mediator of calcium-dependent Cl - secretion in ADPKD cells (120). Similar studies have not been carried out in ARPKD cells. Our results indicate that the Cl - secretory responses, elicited by camp or Ca ++ were small and similar in monolayers 49

53 derived from normal and cystic mice. This is in agreement with a previous study in which the BPK (ARPKD) mouse was crossed with the CFTR knockout mouse and demonstrated that cystic disease progression was not affected by loss of CFTR-dependent anion secretion (80); however the contribution of alternative Cl - channels was not excluded. Based in these results it appears that Cl - secretion may not play a critical role in lumenal fluid accumulation in ARPKD cystic collecting ducts. The most striking finding in this study is the lower (approximately 50%) basal I sc recorded from cystic monolayers compared to normal monolayers. Furthermore, the absolute magnitude of amiloride-sensitive current I sc as well as the fractional inhibition of the current by amiloride (82±2% and 95±1% inhibition for the cystic and normal respectively) are lower in cystic than normal monolayers, so we conclude that electrogenic sodium absorption is significantly reduced in cystic cell monolayers. There are several possible explanations for the decrease in amiloride-sensistive I sc in cystic cell monolayers including: 1) contamination of the primary cultures with nonprincipal cells, 2) perturbed activity or partial mis-localization of the Na,K-ATPase from the basolateral to the apical plasma membrane, 3) differences in the electrochemical driving force for sodium entry across the apical membrane, and 4) reduced activity of apical ENaC channels. Significant contamination by non-pcs is unlikely since in the high resistance monolayers more than 98% of the cells are GFP-positive (CD cells), have a central cilium (PC) and expression of IC markers are undetectable by RT-PCR. It was initially proposed that reversal of polarity was an important component of altered salt and water transport in PKD including mislocalization of the Na,KATPase from the basolateral to the apical plasma membrane (3; 4; 149); however this remains 50

54 controversial (129). A more recent report (109) confirms the presence but not the activity of Na,KATPase in the apical side of a cell line derived from human fetal ARPKD kidney. Furthermore, they found that the immortalized cystic cells had a higher rate of apical to basolateral 22 Na flux compared to a cell line derived from and age matched normal human kidney. It is unclear at present the reason for these disparate observations. Electrodifussive entry of Na + across the apical plasma membrane via the ENaC is the rate-limiting step for Na + absorption by the mammalian PC and changes in apical membrane potential and/or intracellular Na + would alter the rate of Na + entry, independent of changes in Na + permeability. Since, the amiloride-sensitive conductance was reduced by approximately 50% ( G T =0.34 vs G T =0.16 ms/cm 2 for normal and cystic monolayers, respectively) in cystic monolayers compared to normals, it is likely that reduced apical Na + permeability (ENaC activity) in cystic cells is responsible for the decrease in Na + transport. Postnatal maturation of CDs is associated with an increase in Na + absorptive capacity (53; 115) which parallels ENaC expression. It is possible that in cystic disease the highly-proliferative PCs do not fully differentiate (12), and as such they do not develop a mature Na + absorptive capacity. The reduced ENaC activity might be due to lower expression or aberrant signaling processes that regulate ENaC activity such as EGFR axis overactivity (108; 130) or some combination of alterations that lead to a steady-state decrease of ENaC activity in cystic PC cells. Additional studies will be required to elucidate the mechanisms responsible for reduced PC sodium absorption in ARPKD. A thorough understanding of the ion transport abnormalities associated with all forms of PKD may provide important markers of disease progression and suggest therapeutic interventions to reduce or delay the loss of renal function. 51

55 Table 2.1 Primers used for RT-PCR reactions. Primer name Sequence Reference or gene acc number EGFP Primer E Primer K 5 -AGC GCG ATC ACA TGG TCC TG-3 5 -ACG ATC CTG AGA CTT CCC ACA CT-3 (23) Mineralo-corticoid receptor MC-R 5 -GTG GAC AGT CCT TTC ACT ACC G-3 5 -TGA CAC CCA GAA GCC TCA TCT C-3 (25) Epithelial sodium channel (ENaC) α-subunit 5 -CTA ATG ATG CTG GAC CAC ACC-3 5 -AAA GCG TCT GTT CCG TGA TGC-3 (26) H-ATPase (kidney specific) β1 isoform (C-terminal) 5 -ACG GAG ATG TTT CCA ACC AG-3 5 -AAT GCG CTT CAG CAT CTC TT-3 Acc#BC H-ATPase (kidney specific) β1 isoform (N-terminal) 5 -GGC CAC AAC AGT AGA CAG CA-3 5 -TGG CCA CTC CTC TGA GTA CC-3 Acc#BC Anion exchanger 1 band 3 AE1 5 -CTG TTC AAA GCC ACC CAA GTA-3 5 -TCA CAC AGG CAT GGG CAC TT-3 (27) 52

56 Table 2.2. Phenotype of Hoxb7/EGFP +/? cystic and normal mice and Balbc cystic mice. Phenotype, Genotype & animal background Total body weight (gr) Kidney weight (gr) (KW/TBW)x100 (%) Urine osmolarity (mosm/kg) Animal survival (days) Normal GFP+/?,BPK±/- Balbc X B6CBA (n=28) Cystic GFP+/?, BPK+/+ Balbc X B6CBA (n=28) Normal, BPK±/- Balbc (n=21) Cystic, BPK+/+ Balbc (n=21) 11.3± ± ± ±95 NM 10.9± ±0.3 22± ±35 23±3 10.6± ± ± ±54 NM 10.9± ± ± ±67 23±3 indicates significantly different compared to normal animals (p<0.001, unpaired t- test) 53

57 Table 2.3. Summary of transepithelial bioelectric properties in normal and cystic principal cell monolayers. Group Basal Amiloride Forskolin/Ibmx ATP I sc R T I sc R T G T I sc R T G T I sc R T G T µa/cm 2 Ωcm 2 µa/cm 2 Ωcm 2 ms/cm 2 µa/cm 2 Ωcm 2 ms/cm 2 µa/cm 2 Ωcm 2 ms/cm 2 Normal ±3.1 ±132 ±0.1 ±242 ±0.03 ±0.2 ±152 ±0.04 ±1.6 ±50 ±0.1 (n=12) (n=12) (n=8) (n=8) Cystic 15.6 ±1.7* 1234 ±78 (n=10) 2.3 ±0.2 * 1554 ±101 (n=10) 0.16 ±0.02* 5.5 ± ±109 (n=8) 0.08 ± ± ±154 (n=7) 0.56 ±0.1 A Amiloride (100µM) was added to the mucosal bathing solution, five minutes later, forskolin (10µM) and IBMX (100µM) were added to the basolateral, followed fifteen minutes later byatp (100µM) added to the luminal bathing solution. The steady-state I sc and R T following the addition of amiloride and forskolin/ibmx and the peak increase in I sc in response to ATP are reported. The drug-induced change in conductance ( G T ) was calculated from the measured transepithelial resistances before and after addition of the drug. B Values are the means ± SE from the number of independent experiments indicated. Drug induced changes are statistically significant in the normal group (paired t-test, p<0.05). Drug induced changes are statistically significant in the cystic group (paired t-test, p<0.05).* Basal value or drug-induced changes are statistically different between normal and cystic (unpaired t-test, p<0.05). 54

58 Figure 2.1 Experimental strategy. 55

59 I. Breeding: Hoxb7/EGFP (+/+) BPK (+/-) BPK (+/-) II. Genotyping for EGFP Observation for detection of cystic disease Hoxb7/EGFP (+/+) Hoxb7/EGFP (+/?) BPK (+/+) Wild type Cystic (ARPKD model) III. Cell culture and FACS (Isolation of CD primary cells) Culture for 4-5 days FACS IV. Investigating the phenotype of the disease Sodium and chloride transport AQP2 localization in Cystic cells, 56

60 Figure 2.2 Expression of GFP in kidneys of normal and cystic mice. (A). Confocal fluorescence microscopy indicated that EGFP (green) is expressed throughout the collecting ducts in normal Hoxb7/EGFP animals from the cortex to the medulla and all the cells comprising the collecting ducts express GFP. The image was reconstructed with LSM Image Browser software. (B). GFP expression in cystic kidneys. Dilated (cystic) collecting ducts of bpk +/+ mice are lined by GFP positive cells. (C) Cystic tubule fragments in collagenase preparation. Scale bars are: for A 20 µm and B&C 50µm. 57

61 58

62 Figure 2.3 GFP is expressed in principal and intercalated collecting duct cells. Sections of normal (A,B) and cystic (C,D) kidneys were stained for immunodetection of AQP2 and H + ATPase 70 kda subunit. Both AQP2 (A and C, red; PC marker) and H + ATPase (B and D, red: IC marker) positive cells express GFP (A-D, green) in either normal or cystic kidneys. Note the paucity of intercalated cells in cystic tubules (D). Scale bars are: for A,B and C is 50µm and for D is 100 µm 59

63 60

64 Figure 2.4 Fluorescence activated cell sorting (FACS) of isolated renal cells. The GFP intensity profiles for cells isolated from normal and cystic mouse kidneys are indistinguishable. The dot plots show side scatter vs forward scatter (A1,B1,C1), forward scatter vs GFP intensity (A2,B2,C2), and the histograms indicate the analyzed events vs GFP intensity (A3,B3,C3). (A) FACS distribution of cells derived from GFP +/? cystic mouse. (B) FACS distribution of cells derived from GFP -/- cystic mouse. (C) FACS reanalysis of cells grown for 5-6 days in collagen-coated permeable supports and used for electrical measurements. The monolayers are composed of > 97% GFP-positive cells. 61

65 62

66 Figure 2.5 Quantitative analysis of primary CD cell isolation. GFP-positive cells were sorted from mouse renal cell population grown for 4-6 days in culture. (A) Total number of kidney cells obtained per mouse after expanding the renal cell population for 4-6 days in culture (n=28-32 ). (B) FACS analysis reveals that 17.7± 3.5 % of the cells isolated from the normal mouse are GFP +/? (n=29) and 21.6 ± 4.3% of cells isolated from a cystic mouse are GFP-positive (n=32). (C) The yield of GFP-positive cells from normal or cystic mice is x10 6 (n=28-32). The bars represent means ± SE. 63

67 A. Total cell number ( x10 6 /animal) normal cystic B. Percentange of GFP(+) cells C normal cystic normal cystic GFP-positive cell number (x10 6 /animal) 64

68 Figure 2.6 Transmitted and fluorescent microscopy of GFP-positive cell monolayers grown on permeable supports. Collecting duct cells form confluent, polarized, well differentiated epithelial monolayers. (A) Phase contrast microscopy reveals the cobblestone appearance characteristic of epithelial cells and almost all the cells in the monolayer are GFP-positive as indicated by fluorescence microscopy (B). CD cells differentiate in culture and form confluent, polarized monolayers as indicated by tight junction formation (C, ZO1 red). Scale bars are 50µm. 65

69 66

70 Figure 2.7 RT-PCR analysis of expression of cell specific markers. GFP-positive cells grown for 4-6 days on permeable supports are principal cells. RT-PCR analysis of FACS isolated cells derived from normal or cystic mice express αenac and mineralo-corticoid receptor mrnas (PC marker proteins) but do not express H-ATPase kidney specific β1 subunit and anion exchanger 1 band 3 mrnas (IC marker proteins). Whole-kidney mrna isolated from normal or cystic mice was used as a positive control for each of the 6 PCR primer sets. Control reactions without the addition of RT were negative (data not shown). 67

71 68

72 Figure 2.8 Ultrastructure of primary cultures of GFP-positive CD cells. Cells isolated by FACS, seeded on collagen-coated permeable supports, and grown to confluence for 4-6 days. Scanning electron microscopy (SEM) of CD cells from normal and cystic mice showed that all of the cells are characterized by the presence of a central cilium, a feature of principal cells but not intercalated cells of the collecting duct. Cilia length varies from µm in both preparations. Calibration bars are 10 µm in both Panels A and B. Magnification: 3000X. 69

73 Non-cystic monolayer Cystic monolayer 70

74 Figure 2.9 Ion transport properties of normal and cystic primary cell monolayers. Normal (A) and cystic (B) monolayers were placed on Ussing chambers and bathed on both sides with Krebs-ringer bicarbonate solution. Transepithelial voltage (V T ) was clamped to 0 to measure I sc and clamped to +4mV at 1min intervals to calculate transepithelial resistance. The representative traces illustrate the effects of amiloride, forskolin/ibmx and ATP on I sc. (C and D) Amiloride-sensitive Na + absorption in primary principal cell monolayers derived from normal and cystic animals. Short-circuit current (I sc ) and transepithelial conductance (G T ) were measured before and after the addition of amiloride (100µM) to the mucosal bath solution and the amiloride-induced changes in I sc and G T were calculated. (C) I sc values (µa/cm 2 ) for normal and cystic monolayers were 27.3±3.1 (n=12) and 12.9±1.6 (n=11), respectively. (D) G T values (ms/cm 2 ) for normal and cystic monolayers were 0.34±0.04 (n=12) and 0.16±0.03 (n=11), respectively. The bars represent means ± SE. *significantly different from normal values (p<0.005; unpaired t-test). 71

75 A. 40 B. 40 Amiloride 100µM AP Isc (µa/cm 2 ) ATP 100 µm AP I sc (µa/cm 2 ) Amiloride 100µM AP ATP 100 µm AP Forskolin/IBMX 10/100 µm BL Forskolin/IBMX 10/100 µm BL C. D I sc (µa/cm 2 ) * G T (ms/cm 2 ) * normal PC cystic PC normal PC cystic PC 72

76 Figure 2.10 camp- and Ca 2+ -induced Cl - secretory currents in normal and cystic primary cell monolayers. (A) After treatment with amiloride, forskolin/isobutylmethylxanthine (10/100µM) were added to the serosal bath solution and the steady-state change in short-circuit current (Isc) was recorded after min. camp-stimulated I sc was 2.9±0.3 and 3.2±0.2 µa/cm 2 for normal (n=8) and cystic (n=8) monolayers, respectively. (B) ATP (100µM) was added to the mucosal bathing solution and I sc was recorded as peak current. ATP-induced I sc was 13.4±0.8 and 15.3±1.6 µa/cm 2 for the normal (n=8) and cystic (n=7) monolayers, respectively. The bars represent means ± SE. Drug induced changes were significant within a group (p<0.05; paired t-test) but the responses of normal and cystic monolayers were not significantly different from each other (unpaired t-test). 73

77 A. B. Forskolin stimulated I sc (µa/cm 2 ) ATP stimulated I sc (µa/cm 2 ) normal PC cystic PC 0 normal PC cystic PC 74

78 Chapter III. Abnormal EGF-dependent regulation of sodium absorption in ARPKD collecting duct cells 75

79 INTRODUCTION Cyst epithelial cells in human PKD exhibit abnormalities in epithelial cell polarity. The EGFR localized to basolateral membrane of mature renal tubules is found in both apical and basolateral membranes of cyst epithelial cells (25; 95; 130). Similar mislocalization was observed in several murine models of PKD such as: cpk, orpk, bpk, Pkd1 mutant mice (49) and a recently developed Kif3A knock-out model (64). The aberrant localization of EGFR does not reflect a generalized defect in cell polarity because aquaporins 2 and 3 (AQP2 and AQP3) and NaKATPase are localized appropriately (10; 64) in postnatal kidney. Furthermore, overactivity of EGF/EGFR axis contributes to PKD pathophysiology, since EGF receptor inhibition slows disease progression in ARPKD models (108; 131; 138). Kidney collecting duct is predominantly an absorptive epithelium and electrogenic Na + entry into the principal cells (PC) is mediated by epithelial sodium channel (ENaC) (26; 112). ENaC is composed of three homologous subunits α, β, and γ (13; 14) and channel expression, trafficking, and gating are highly regulated. While aldosterone is recognized as the major positive regulator of sodium transport in the colon and distal nephron (66; 70; 96) several signaling pathways appear to modulate ENaC mediated Na + transport in CD including those activated by growth factors (EGF). In contrast to steroid hormones that increase Na + transport, EGF inhibits CD Na + transport by a poorly defined mechanism(s) (121; 141; 142). Interaction of EGF with its receptor elicits receptor dimerization and phosphorylation, recruitment of accessory proteins, and initiation of several downstream signaling pathways including sequential activation of Ras, Raf-1, Mek-1, and ERK1/2 kinases (117; 137). In parotid salivary epithelial cells, 76

80 protein kinase C-dependent activation of ERK1/2 leads to transcription downregulation of the α-enac expression (156). Furthermore, it was shown that ERK1/2 phosphorylation antagonized glucocorticoid-dependent activation of α-enac gene transcription {73} which is evidence for a crosstalk between nuclear receptor and ERK1/2 pathways. Rapid, non-genomic inhibitory effects of EGF on Na + absorption have been reported (141); however the precise mechanism of ENaC inhibition remains to be defined. The present study was undertaken to examine the effect of EGF on regulation of Na + transport in CD principal cells isolated from non-cystic and cystic kidneys. Shortterm and long-term exposure to EGF from either apical (AP) or basolateral (BL) surface was evaluated. The results of these studies demonstrate that acute addition of EGF to the BL bathing solution of non-cystic or cystic CD principal cells stimulates phosphorylation of ERK1/2 and inhibition of amiloride-sensitive Na + transport. In contrast, addition of EGF to the AP bathing solution had no effect on non-cystic CD monolayers, yet elicited robust activation of ERK1/2 and inhibition of Na + transport in cystic cell monolayers. Long-term exposure to BL EGF was associated with a decrease in Na + transport and steady-state ENaC mrna expression in non-cystic and cystic cells. A similar response was observed in cystic cells but not in non-cystic cells when EGF was added to the AP bathing solution. These findings support the concept that mislocalization of EGF receptors to the AP membrane of cystic cells allows access to activating ligand present in the luminal fluid and thereby contributes to elevated ERK1/2 phosphorylation in cystic tubules and may be responsible for enhanced cellular proliferation and ion transport abnormalities associated with PKD. 77

81 METHODS Collecting duct cell isolation and primary culture. The mice used in this study were obtained by breeding the Hoxb7/EGFP transgenic line (123) with the BPK mouse model of ARPKD and primary cultures of collecting duct principal cells were prepared from kidneys taken from GFP-positive non-cystic (Hoxb7/GFP-bpk -/? ) transgenic mice and cystic (Hoxb7/GFP-bpk +/+ ) mice as described previously (143). Briefly, kidneys were dissected from day old cystic and non-cystic GFP-positive littermates. Kidneys were washed in ice-cold sterile PBS, minced to small fragments, and incubated in 10 ml of collagenase solution (Type IV, 1 mg/ml in CT media, Sigma) for minutes. The digest was collected by centrifugation and small tubular fragments and cells were seeded in 10 cm tissue culture dishes and maintained in CT medium consisting of a 1:1 mixture of Dulbecco s-modified Eagle s medium and Ham s F-12 medium (Life Technologies), supplemented with: 5 mg/l insulin, 2.5 mm glutamine, 25 µg/l prostaglandin E1, 1.3 µg/l sodium selenite, 5 mg/l transferrin, 1.3 µg/l triiodothyronine, 50 nm dexamethasone, 30 mg/l penicillin G, 50 mg/l streptomycin and 5% fetal bovine serum (FBS) (GIBCO- BRL, Gaithersburg, MD) at 37 o C. Five to seven days later cells were subjected to Fluorescence Activated Cell Sorting (FACS) to collect GFP-positive CD cells. Cells were seeded on collagen-coated permeable supports (Millicell CM filter; 12 mm diameter) at a density of 2.5 x 10 5 cells/filter and grown in CT media containing 5% FBS, 2ng/ml EGF and 10 nm aldosterone for 4-6 days. At this point serum and EGF were removed from CT media for at least 24 hrs before electrophysiological experiments or chronic treatment protocol. 78

82 Electrophysiological studies and experimental protocol. Transepithelial bioelectric properties of CD cell monolayers were evaluated as described previously (143). Briefly, confluent monolayers were mounted in thermostatically-controlled Ussing chambers equipped with gas inlets and separate apical and basolateral bath solution reservoirs. Both sides were bathed with an equal volume (10 ml) of Krebs- Ringer bicarbonate solution circulated through the water-jacketed glass reservoir by gas lifts (95% O 2-5%CO 2 ) to maintain solution temperature at 37ºC and ph at 7.4. Transepithelial voltage (V T ) was measured and the current required to clamp V T to 0 mv was determined. The I sc was corrected for solution and filter series resistance. Monolayers were maintained under short circuit conditions except for brief 3-5 s intervals when the current necessary to clamp the voltage to a non-zero value was measured to calculate transepithelial resistance (R T ). The experimental protocol for determining the effects of acute exposure to EGF on electrogenic sodium absorption consisted of the following sequence: transfer the monolayers to Ussing chamber, 5-10 min period of equilibration, exposure to vehicle or PD98059 (30 µm AP and BL) for min, addition of EGF (20 ng/ml, AP or BL) for min, and finally amiloride was added to the AP bathing solution. The long-term effects of EGF on sodium transport and ENaC subunit mrna levels were determined in cultures treated for 24 hrs with EGF. Confluent monolayers of collecting duct cells isolated from non-cystic and cystic mice were treated by addition of EGF (20 ng/ml) to the AP or BL media either with or without an ERK kinase inhibitor (30 µm PD98059 was added to the apical and basolateral media 30 min prior to the addition of EGF). On the day of analysis, the cultures used for ion transport 79

83 measurements were transferred to Ussing chambers, allowed to stabilize for min and then amiloride was added to the AP side. The cultures used for RT-PCR analysis were washed and the cells were lysed for isolation of RNA. Immunohistochemistry. Kidneys were dissected from 18 day old animals (cystic and non-cystic GFP-negative littermates) and one kidney was fixed in ice-cold 3.7% paraformaldehyde in PBS and the contralateral kidney was quickly frozen for western blot analysis (see below). Kidneys were washed for 1 hr under running water, dehydrated through serial ethanol and xylene solutions and embedded in paraffin. Tissue sections (3µm) were cut from paraffin embedded kidneys. Immunohistochemistry was performed on deparaffinized, rehydrated sections as previously described (143). Sections were probed with antibodies to epidermal growth factor receptor (EGFR; RDI), E- cadherin (E-cadherin; Zymed), and anti phospho-erk1/2 (p-erk1/2; Cell Signalling). Secondary antibodies (from Molecular Probes) were conjugated to either Alexa Fluor 488 or Texas Red. Photomicrographs were obtained with a confocal Zeiss LSM microscope. To examine EGFR localization in vitro, immunohistochemistry was performed as described (105) on confluent monolayers of cystic and non-cystic CD cells grown on collagen-coated permeable supports. Image stacks were acquired with a Zeiss 200 M inverted microscope equipped with a DG4 light source (Sutter Instrument Co) and a 12- bit CoolSnapHQ camera (Roper Scientific) under control of Metamorph v4.5 (Universal Imaging Corp). Images were deconvolved by Autoquant s Autodeblur software (AutoQuant Imaging, Inc). Immunoprecipitation and western blot analysis. Kidneys were dissected from non-cystic and cystic littermates, capsula was removed, and tissues were washed with 80

84 PBS and quickly frozen on dry ice. Afterwards 1 ml of ice-cold RIPA buffer (150 mm Tris, ph 8.0, 150 mm NaCl, 1% IGEPAL, 0.1% Triton-X-100, 1 mm Na orthovanadate, 2.4 mm EDTA, 0.5 mm PMSF) plus protease and phosphatase inhibitor cocktail (according to manufacturer s instructions, Sigma;) was added to the non-cystic kidney and 2 ml to cystic kidney. Tissues were homogenized on ice and the lysates were cleared by centrifugation (10 min to 10,000xg). The supernatant was collected and protein concentration was determined using bicinchoninic acid protein assay (BCA; Peirce, Rockford, IL). EGFR was precipitated from 500 µg of cell lysate protein by 2 hr incubation at 4ºC with 10 µg of anti-egfr antibody. Complexes were bound to 25 µl of protein A-agarose (Santa Cruz) by co-incubation overnight at 4ºC. Immune complexes were recovered by centrifugation (14,000xg for 20sec), washed three times with 1 ml of ice-cold lysis buffer, and prepared for SDS/PAGE and probing with anti-egfr antibody or anti-phosphotyrosine antibody. The remaining portion of the kidney lysate (not used for IPs) was retained for SDS/PAGE and western blot analysis of MAP kinase signaling components. Cell lysates were also prepared from primary monolayer cultures of CD cells, treated the same as described under electrophysiological studies (acute EGF exposure), for SDS/PAGE analysis of total and phosphorylated ERK1/2. Samples of kidney lysates (20 µg), primary cell culture lysates (10 µg) and protein recovered by immunoprecipitation were boiled in SDS sample buffer containing 50 mm Tris-HCl (ph=6.8), 2% SDS, 5% β-mercaptoethanol, 10% glycerol, and 0.1% bromophenol blue for 10 min. The denatured proteins were separated by either 7.5% or 10% SDS/PAGE. The protein was electrophoretically blotted onto a pure nitrocellulose transfer and immobilization membrane (Schleicher & Schuell, Keene, NH). Membranes were 81

85 blocked 1 h at room temperature in Tris-buffered saline (TBS) that contained 5% dried milk (wt/vol), 0.1% polyoxyethylenesorbitan monolaurate (Tween 20), and 0.01% sodium azide or TBS-3%BSA. After a brief wash to remove the Tween 20, the membranes were incubated overnight at 4ºC with specific primary antibodies [anti-egfr (Research Diagnostics Inc.); RC20 anti-p-tyr (BD Biosciences); anti-praf, anti-p-mek, anti-p-erk1/2, anti-erk1/2, anti-p-elk1 (Cell Signalling)] in TBS-5% died milk (or TBS-3%BSA for anti-p-tyr). The membranes were then incubated with secondary antibody (horseradish peroxidase-conjugated) at room temperature for 1 hr. Membranes were rinsed three times and peroxidase-labeled membranes were developed by enhanced chemiluminescence (Amersham, Arlington Heights, IL) and protein bands were visualized on X-ray film (X-O-Mat, Kodak, Rochester, NY). Molecular mass estimation of detected bands was determined by using Precision Plus Protein Standards (Bio-Rad). Quantification of the intensity of the bands on the luminograms was determined with a Versa Doc Imaging Systems (model 3000, Bio-Rad). The Quantity One (version 4.4.0) density scan program (Bio-Rad) was used to analyze the relevant densitities of protein bands. Quantitative RT- PCR analysis of ENaC mrna. Cystic and non-cystic CD cells were grown on collagen-coated filter inserts and divided in two groups: a) non-treated (controls); b) EGF treatment (20 ng/ml) for 24 hours (AP or BL). Total RNA was extracted using the RNeasy Mini Kit which includes an on-column DNase digestion with RNase free DNase (Qiagen, Valencia, CA). The concentration and quality of mrna was determined photometrically (260/280nm). RT-PCR was performed by using 0.5-1µg of RNA, random hexamer primers and MMLV (Moloney Murine Leukemia Virus) an RT 82

86 system (Life Technologies, Rockville, MD) in 25 µl reaction volume. 2-5 µl out of 25µl of cdna reaction was used for real-time PCR amplification (RT-PCR) using DNA amplification kit (SYBER Green I, Roche Diagnostics Systems). Transcript levels of the housekeeping gene, glyceraldehyde-phosphate dehydrogenase (GAPDH), and ENaC α, β and γ subunit were quantified by real-time PCR on a LightCycler (Roche Diagnostics, Indianapolis, IN). The following primer pairs were used: mouse GAPDH forward 5 - CGT CTT CAC CAC CAT GGA GA -3, reverse 5 - CGG CCA TCA CGC CAC AGT TT -3 ; mouse α-enac forward 5 - GCC AGT GCT CCT GTC A -3, reverse 5 - GGG GTA CAG GGT ACC AA -3 ; mouse β-enac forward 5 - CTC CGA TGT TGC CAT AAA G-3, reverse 5 - TCT CTC TCT GGG TCA CAC TC -3 ; mouse γ-enac forward 5 - CTC GTC TTC TCT TTC TAC ACC G -3, reverse 5 - TTC CCA CTG ATT TTC CGC-3. Hot start PCR was performed and reactions continued for cycles with 94ºC denaturing 5s/ 68~60º C annealing 5s / 72ºC elongation 16s per cycle. Water instead of cdna was used as a control for PCR contamination and primer-dimer formation. To further eliminate possible variation due to genomic DNA contamination, primers were designed to span intron/exon boundaries. Several ENaC primer pairs were investigated and the selected ones had good efficiency and no primer- dimer formation for up to 40 cycles. The amplified products were of the predicted size as demonstrated by electrophoresis (data not shown). The various transcript levels were determined by using a standard curve method for the gene of interest. Briefly, duplicates of five ten-fold dilutions ( copies) of full length cdna for mouse GAPDH, or α,β,γ ENaC were included in each RT-PCR run. GAPDH and α,β, and γ ENaC mrna (copy number) were measured for each EGF 83

87 treated sample and the respective control sample. Real-time PCR analysis of all samples (non-treated and EGF-treated cystic; non-treated and EGF-treated non-cystic) was performed in a single run for each transcript. Variation in cdna concentration in different samples was corrected by using the housekeeping gene concentration-gapdh in each sample. The relative amount of ENaC mrna subunit (calculated as a ratio of α or β or γ ENaC/ GAPDH) present in EGF treated samples was normalized to the amount of ENaC mrna subunit (calculated as a ratio of α or β or γ ENaC/ GAPDH) in the control samples. The studies with mice were performed in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee of Case Western Reserve University School of Medicine. Statistical analysis. All results are expressed as mean ± SE and statistical significance was evaluated by either unpaired or paired Student s t-test. P< 0.05 was considered significant. 84

88 RESULTS EGFR and ERK1/2 activation in non-cystic and cystic kidneys. EGF/EGFR axis over activity is a feature of cystic tubules in dominant and recessive PKD and disruption of EGFR slows disease progression in animal models of ARPKD (131; 138), but the activity of potential downstream signaling pathways such as Ras/Raf/MEK/ERK has not been evaluated. As previously described, EGFR distribution in non-cystic renal tubules is primarily at the basolateral membrane and overlaps that of E-cadherin (Figure 3.1A-C). There is basolateral colocalization of EGFR and E-cadherin in cystic tubules, but there is also strong apical staining in these cells (Figure 3.1D-F). The localization patterns of the EGFR in collecting ducts from non-cystic and cystic kidneys were retained in polarized primary cultures of collecting duct principal cells as illustrated in Figures 3.7A&C. The expression levels of EGFR are similar in cystic and non-cystic kidney, but the amount of the tyrosine-phosphorylated form of EGFR is significantly elevated in cystic kidneys compared to non-cystic (Figure 3.1G). Immunolocalization of p-erk1/2 in sections of non-cystic and cystic kidneys revealed a high level of p-erk1/2 in epithelial cells lining the cystic tubules with almost no detectable p-erk1/2 in sections from non-cystic kidney (Figure 3.2A-B). Furthermore, western blot analysis of lysates from non-cystic and cystic kidney show a dramatic increase (6-9 fold, n=16) in the amount of p-erk1/2 with no difference in tot ERK1/2 (Figure 3.2C). In vivo phosphorylation status of additional components of the EGFR-MAP kinase signaling pathway was evaluated by western blot analysis of kidneys from 3 noncystic mice and 3 cystic littermates. As illustrated in Figure 3, the phosphorylated forms of MAP kinase kinase kinase (craf), MAP kinase kinase (MEK) and MAP kinase 85

89 (ERK1/2) were significantly increased in cystic compared to non-cystic kidneys. Phosphorylation of a downstream transcription factor (Elk1), known to be a substrate of active ERK1/2, was also found to be increased in cystic kidneys. A sustained high level of MAP kinase signaling might be expected to impact cellular proliferation and function in cystic epithelial cells. Acute effects of EGF on non-cystic and cystic collecting duct monolayers. Primary cultures of cells isolated by FACS from Hoxb7/GFP-bpk -/? transgenic mice (noncystic) and Hoxb7/GFP-bpk +/+ (cystic) form polarized epithelial monolayers and express marker proteins and ion transport functions attributable to distal nephron principal cells. Cultures derived from non-cystic mice had mean short-circuit current of 29±4.5 µa/cm 2 (n=17); due primarily (90%) to amiloride-sensitive sodium absorption. Monolayer cultures derived from cystic mice exhibited ~50% lower amiloride-sensitive sodium absorption (I sc = 14.2±4.8 µa/cm 2 ; n=18). The bioelectric properties of normal and cystic cell cultures used in the studies reported herein are similar to those reported previously (143). Addition of EGF (20 ng/ml) to the apical (AP) bathing solution of non-cystic CD cell monolayers mounted on Ussing chambers had no effect on I sc (Figure 3.4A). In contrast, addition of EGF to the basolateral (BL) bathing solution of non-cystic CD cell monolayers elicited a monotonic decrease in I sc as illustrated in Figure 3.4B. Pretreatment of the monolayer with an ERK kinase inhibitor (PD98059; 30 µm; 15 min) completely prevented the EGF-induced inhibition of I sc (Figure 3.4C). The data from multiple experiments are summarized in Figure 3.6A. 86

90 Apical mislocalization of the EGF receptor is a common feature in PKD, but the coupling to specific signal transduction pathways and the effects of apical EGF receptor signaling on cellular physiology in cystic cells have not been reported. In order to determine whether apical EGF receptors are functional and can modulate ion transport, monolayers of cystic CD principal cells were exposed to EGF added to either the apical or basolateral bathing solution. Similar to what was observed with non-cystic CD cell monolayers, addition of EGF to the BL bathing solution reduced amiloride-sensitive I sc by ~35 % (Figure 3.5C). In contrast to the lack of response of non-cystic cells to apical EGF, treatment of cystic monolayers caused inhibition of I sc (Figure 3.5A) that was indistinguishable from the response to basolateral EGF (Fig 3.6C). Furthermore, the inhibitory effect of apical or basolateral EGF on I sc, is completely prevented by pretreatment with an ERK kinase inhibitor (PD 98059; 30 µm) (Figures 3.5B&D). Treatment with PD has no significant effect on baseline amiloride-sensitive I sc in non-cystic or cystic monolayers (Figures 3.6A&B) likely because ERK1/2 phosphorylation is very low under baseline conditions. The ion transport data from multiple experiments are summarized in Figure 3.6B. In another set of experiments, EGF was added simultaneously to both apical and basolateral chambers and the inhibitory effects of EGF on I sc were not additive (data not shown). Most likely this is due to full stimulation of ERK1/2 signaling from either apical or basolateral EGF and/or to saturation of a downstream response element. The results presented in the preceding sections showed that the inhibitory effect of EGF on amiloride-sensitive Na + current was completely abolished by pretreatment with PD98059, presumably due to a reduction in EGF-induced ERK1/2 phosphorylation and activation. In order to test this, the effect 87

91 EGF added to either the apical or basolateral bathing solution on p42/44 phosphorylation in non-cystic and cystic CD cell monolayers was examined. Total and phosphorylated ERK1/2 in lysates from cystic and non-cystic cell monolayers treated with vehicle, EGF (20 ng/ml; AP or BL), and EGF plus PD98059 (30 µm; AP and BL) was measured. Whereas total ERK1/2 did not vary between the conditions, basolateral exposure to EGF dramatically increased the phosphorylation of ERK1/2 in both non-cystic and cystic cells (Figure 3.7B&D). In contrast, addition of EGF to the AP side of non-cystic CD cell monolayers did not increase ERK1/2 phosphorylation, but treatment of cystic CD cell monolayers elicited a robust phosphorylation of ERK1/2. Pretreatment with PD98059 completely blocked EGF-induced ERK1/2 phosphorylation (Figure 3.7B&D). Modulation of electrogenic sodium transport by long-term exposure to EGF. Previous work from our laboratory demonstrated that prolonged exposure of mct1 cells (mouse collecting duct cell line) to EGF inhibits amiloride-sensitive Na + absorption (121) due to a reduction in ENaC-mediated apical Na + entry. The effects of chronic exposure to EGF in non-cystic and cystic primary CD cell monolayers were determined. Confluent epithelial monolayers of non-cystic or cystic cells were exposed to either apical or basolateral EGF (20 ng/ml) for 24 hrs and amiloride-sensitive I sc was measured. As summarized in Figure 3.8A, addition of EGF to the apical side of non-cystic monolayers had no effect on I sc, while basolateral EGF reduced I sc by ~50% (28.7±3.2 to 15±2.6 µacm 2 ; n=4). Chronic exposure of cystic cell monolayers to EGF from either the apical or basolateral surface significantly inhibited I sc (Figure 8B) by 60-65% (control 16.7±2.4 n=4; apical EGF 8.2±1.6 n=5; basolateral EGF 6.7±2.1 µa/cm 2 n=4). The inhibitory 88

92 effects of EGF on amiloride-sensitive I sc were not observed in monolayers pretreated with the ERK1/2 kinase inhibitor PD Effects of long-term EGF treatment on α,β, γ-enac mrnas. The acute effects of EGF on I sc probably reflect a change in channel gating or trafficking; whereas, the sustained reduction of amiloride-sensitive I sc with prolonged exposure to EGF may involve genomic regulation. To investigate the mechanism of long term (24hr) EGFdependent, ERK1/2-mediated ENaC regulation of sodium transport, steady state mrna levels of all three ENaC subunits were measured in primary monolayer cultures of noncystic and cystic collecting duct principal cells. Real-time RT-PCR was used to quantify the mrnas for the three ENaC subunits and for GAPDH. For each treatment (EGF) and control sample, GAPDH mrna and α,β,γ ENaC mrna were measured, the data are normalized to GAPDH and expressed as a percentage of control (no treatment). The steady state mrna levels for GAPDH were not changed by EGF treatment of the monolayers [(the mean values for GAPDH mrna of treated non-cystic cultures were 103±5 % (n=4) and cystic 104±4 % (n=6)]. The results are summarized in Figure 3.9. Chronic exposure of primary CD cells to EGF from the basolateral side (20ng/ml; 24 hr) decreased the abundance of all three ENaC subunits in non-cystic (α,β and γ ; Figure 3.9A) and cystic cells (α,β and γ; Figure 3.9B). In contrast, exposure to EGF from the apical side caused downregulation of ENaC mrna in cystic (α,β and γ; Figure 3.9B) but not in non-cystic collecting duct cell cultures (Figure 9A). 89

93 DISCUSSION. The primary objective of this study was to determine the impact of mislocalized EGF receptors on MAP kinase signaling and regulation of sodium absorption in primary cultures of normal and ARPKD collecting duct cells. Previous studies demonstrated that genetic or pharmacologic modulation of EGFR activity results in attenuation or slowing of the development of renal disease. Mislocalized apical receptors can bind EGF, autophosphorylate, and are mitogenic but the signaling pathway(s) downstream from the mislocalized apical receptors have not been delineated. While our studies do not demonstrate directly that EGFR signaling is responsible for in vivo activation of MAP kinase cascade in cystic kidneys, it is a likely mechanism since ERK1/2 phosphorylation was prominent in cystic tubules known to express apical EGF receptors (Figures 1&2). Enhanced in vivo phosphorylation ERK1/2 in cystic kidneys (Figures 3.2&3) suggests a role for MAP kinase signaling in cell growth and function. Activation of basolateral EGF receptors in perfused rabbit collecting ducts (76) elicited rapid inhibition of sodium absorption and chronic treatment of a mouse collecting duct cell line with EGF caused ERK1/2-dependent inhibition of sodium transport (121). It is clear that both non-cystic and cystic cells respond to exogenous EGF added to the basolateral surface with robust phosphorylation of ERK1/2 (Figure 7) and acute inhibition of amiloride-sensitive sodium absorption (Figure 6). Perhaps more importantly, mislocalization of EGF receptors to the apical membrane in cystic cells results in acquisition of sensitivity to EGF added to the apical bathing solution (Figures 5&6). Phosphorylation of ERK1/2 and inhibition of I sc by EGF are prevented by pretreatment with an ERK kinase inhibitor; therefore, MAP 90

94 kinase signaling is at least necessary for acute inhibition of sodium transport by EGF. Since primary cultures of cystic collecting duct cells do not exhibit substantially enhanced ERK1/2 phosphorylation in the absence of exogenous ligand, the dramatically higher level of phosphorylation observed in vivo (Figures 3.2&3) is probably due to enhanced ligand availability and/or aberrant expression of apical EGF receptors that can be activated by ligand constituitively present in the luminal fluid. Alteration in ligand processing, increase in EGF receptor independent signaling, or decrease in phosphatase activity could contribute to enhanced MAP kinase signaling in cystic kidneys. The finding that prolonged (24 hrs) exposure of cultured collecting duct cells to EGF causes a significant reduction in amiloride-sensitive sodium transport and expression of ENaC subunits is consistent with ERK1/2-dependent changes in gene expression in collecting duct principal cells. Similar effects would be predicted in vivo since: 1) EGFR receptors are mislocalized to that apical membrane in cystic collecting ducts, 2) EGF and EGF-like molecules are present in urine, 3) there is overactivity of EGF/EGFR axis, 4) phospho-erk1/2 is elevated in cystic collecting duct cells, and 5) MAP kinase cascade signaling is substantially increased in cystic kidneys. Thus abnormal regulation of amiloride-sensitive sodium transport by apically localized EGF receptors seems to be a feature of ARPKD. The molecular mechanisms responsible for acute and chronic inhibition of sodium transport by EGF are unknown. Since both the acute and the chronic inhibitory effects of EGF are prevented by pretreatment with an ERK kinase inhibitor, ERK1/2 phosphorylation is required for EGF-dependent regulation of ENaC activity. The characteristic rapid stimulatory effect of EGF on ERK1/2 phosphorylation and resultant 91

95 inhibition of amiloride-sensitive I sc suggests a change in ENaC gating (i.e., decrease in channel open probability, perhaps due to phosphorylation of the channel or a regulatory protein), or an increase in endocytosis and decrease of the number of active channels present in apical membrane. Indeed, ERK1/2 dependent phosphorylation of ENaC near the PY motif present in the C terminus of the β and γ subunits has been suggested to increase its affinity for a ubiquitin ligase implicated in channel retrieval from the plasma membrane (22; 116). The inhibitory effect of long-term exposure to EGF is probably mediated by transcriptional downregulation or a decrease in mrna stability since EGF leads to a decrease in steady-state mrna levels for all 3 ENaC subunits. Sustained ERK1/2 activity may interfere with nuclear receptor function (glucocorticoid or mineralocorticoid receptor) by either direct phosphorylation of nuclear receptors (62)} or by activation of a repressor of the ENaC transcription complex (156). Additional studies will be required to define the precise mechanisms of acute and chronic regulation of ENaC activity by EGF. We reported previously that primary cultures of collecting duct principal cells isolated from normal and BPK mice differ primarily in the magnitude of amiloridesensitive sodium absorption, with approximately 50% reduction in cystic monolayers. The molecular basis for the lower rate of amiloride-sensitive sodium transport in the cystic cells is not known. However, synthesis of EGF or EGF-like ligands may be increased in primary cultures of cystic cells or cystic cells may be more sensitive to autocrine/paracrine regulation by released ligands; but there is not an obvious increase in ERK1/2 phosphorylation in cystic cell monolayers under basal in vitro conditions (Figure 3.7). 92

96 Many of the epithelial cysts present in advanced ADPKD lack upstream connections and fluid accumulation must be driven by net secretion of salt and water (125; 128). Since ADPKD is a slowly progressing disease and cysts are thought to form at multiple sites along the nephron, it is difficult to ascertain with certainty the segment of origin of a particular cyst and document changes in ion transport that accompany cyst development. In contrast, ARPKD progresses rapidly, the cysts are derived primarily from collecting tubules, and actually represent dilated nephrons with upstream and downstream connections, rather than anatomically isolated cysts. Net fluid secretion is not required for cyst enlargement, since an increase in secretion or a decrease in absorption would favor retention of luminal fluid and enlargement of cystic tubules in face of destruction of kidney architecture and pseudo-obstruction. We recently reported that primary cultures of non-cystic and cystic collecting duct principal cells maintained under basal conditions (i.e. without exogenous EGF) had similar Cl - secretory responses to elevation of camp or calcium (143). Elimination of a camp-regulated Cl - channel, implicated in ADPKD fluid secretion, did not affect renal cystic disease in a murine model of ARPKD (80). Non-CFTR Cl - channels could make a significant contribution to in vivo fluid secretion that is not apparent when cells are placed in primary culture. (e.g., hyperactivity of the EGF/EGFR axis and enhanced ERK1/2 activation may promote fluid secretion). These observations suggest that abnormal regulation of sodium reabsorption, perhaps in combination with enhanced Cl - secretion, would be expected to contribute to luminal fluid retention and tubule dilatation in ARPKD. 93

97 Figure 3.1. Confocal laser scanning microscopy (CLSM) analysis of E-cadherin and EGFR expression in non-cystic and cystic mouse kidney. Sections (3 µm) of paraffin-embedded kidney were prepared and incubated with primary antibodies against E-cadherin (A and D) and EGFR (B and E) followed by incubation with secondary antibodies conjugated to either Alexa Fluor 488 (EGFR-green) or Texas Red (Ecadherin-red). Overlays of EGFR and E-cadherin immunofluorescence are shown in C and F. Images of non-cystic and cystic kidney sections were collected and processed in an identical fashion. Note the extensive colocalization of E-cadherin and EGFR in the basal and lateral membrane of non-cystic tubules (A-C). E-cadherin and EGFR are also colocalized in the lateral and basal membrane of cystic tubules, but there is also prominent apical expression of EGFR but not E-cadherin (D-F).. G. Western blot analysis of total and phosphorylated EGFR from non-cystic and cystic kidney (each lane represents a different animal). EGFR was immunoprecipitated from kidney lysates and proteins were separated by SDS/PAGE. In the upper panel, samples were probed with an anti-egfr antibody (IB: EGFR) and in the lower panel the same samples were probed with an anti-phosphotyrosine antibody (IB: p-tyr). Bar=25 µm. 94

98 95

99 Figure 3.2. Expression and phosphorylation of extracellular-signal regulated kinase in non-cystic and cystic mouse kidneys. Sections (3 µm) of paraffin-embedded kidney were prepared and incubated with a primary antibody against p-erk1/2 (A and B), followed by a secondary antibody conjugated to Texas Red and nuclei were labeled by exposing the sections to DAPI. Images of non-cystic and cystic kidney sections were collected and processed in an identical fashion. Note the extensive labeling of p-erk1/2 in epithelial cell lining the cysts compared to renal tubules from the non-cystic mouse kidney. Bar=40 µm. C. Western blot analysis of total and phosphorylated ERK1/2 from non-cystic and cystic kidney (each lane represents a different animal). Kidney lysates were prepared and proteins were separated by SDS/PAGE. In the upper panel samples were probed with an anti-p-erk1/2 antibody and in the lower panel the same samples were probed with an anti-total-erk1/2 antibody. 96

100 97

101 Figure 3.3. In vivo activation of MAP kinase signaling cascade in kidneys from non-cystic and cystic BPK mice. Equal amounts of protein (20µg/lane) isolated from three pairs of cystic and non-cystic 21 day old littermates were loaded, electrophoresed, and probed. Immunoblots of p-craf, p-mek, p-erk, tot-erk, and p-elk are shown. Each of the active (phosphorylated) components of the MAP kinase signaling cascade is dramatically upregulated in kidneys from cystic mice compared to non-cystic littermates. 98

102 99

103 Figure 3.4. Effects of exposure to EGF on amiloride-sensitive short-circuit current in primary cultures of CD cells isolated from non-cystic mice. A. EGF (20 ng/ml) was added to the apical bathing solution at the time indicated by the first arrow, and 15 min later amiloride was added to the apical bathing solution to block remaining sodium absorption. B. EGF was added to the basolateral bathing solution followed by addition of amiloride to the apical bathing solution at the times indicated. C. The CD cell monolayer was treated with an ERK kinase inhibitor (PD98059, 30µM, apical and basolateral addition) for min, followed by addition of EGF to the basolateral bathing solution, and then finally amiloride to the apical bathing solution. At 1 min intervals, V T was clamped to a non-zero value (2-5 mv) to calculate R T 100

104 Acute polarized EGF inhibition of Na absorption in non-cystic cells Isc (µa/cm 2 ) A. B. C EGF AP 10 Amil I sc (µa/cm 2 ) Amil I sc (µa/cm 2 ) EGF BL 20 PD98059 EGF BL 10 Amil

105 Figure 3.5. Effects of exposure to EGF on amiloride-sensitive short-circuit current in primary cultures of CD cells isolated from cystic mice. A. EGF (20 ng/ml) was added to the apical bathing solution at the time indicated by the first arrow, and 20 min later amiloride was added to the apical bathing solution to block remaining sodium absorption. B. The CD cell monolayer was treated with an ERK kinase inhibitor (PD98059, 30µM, apical and basolateral addition) for 25 min, followed by addition of EGF to the apical bathing solution, and then finally amiloride to the apical bathing solution. C. EGF was added to the basolateral bathing solution at the time indicated by the first arrow, and 20 min later amiloride was added to the apical bathing solution to block remaining sodium absorption. D. The CD cell monolayer was treated with an ERK kinase inhibitor (PD98059, 30µM, apical and basolateral addition) for min, followed by addition of EGF to the basolateral bathing solution, and then finally amiloride to the apical bathing solution. 102

106 Acute non-polarized EGF inhibition of Na absorption in cystic cells Isc (µa/cm 2 ) Isc (µa/cm 2 ) A. B Isc (µa/cm 2 ) EGF AP PD EGF AP C. D. 25 Amil Amil EGF BL 5 I sc (µa/cm 2 ) PD98059 EGF BL 5 Amil Amil

107 Figure 3.6. Summary of the acute effects of unilateral addition of EGF on amiloride-sensitive short-circuit current in CD cells isolated from non-cystic and cystic BPK mice. Confluent monolayers of collecting duct cells isolated from non-cystic and cystic mice were treated with vehicle PD alone, EGF (AP or BL), or PD98059 plus EGF (AP or BL) as described in Figures 4 and 5. The EGF-induced change in shortcircuit current ( I sc ) is taken as a measure of EGF-dependent inhibition of electrogenic sodium absorption. A. Collecting duct cell cultures isolated from non-cystic mice (n= 3-5 for each condition). The mean I sc prior to addition of drugs was 29±4.5 µa/cm 2 n=17. B. Collecting duct cell cultures isolated from cystic mice (n=3-5 for each condition). The mean I sc prior to addition of drugs was 14.2±4.8 µa/cm 2 (n=18). *, EGF-induced change is significantly different from zero (P<0.05; paired t-test). C. Time course of EGF-induced inhibition of amiloride-sensitive I sc in non-cystic and cystic monolayers. I sc at the time of EGF addition (considered time 0) was given the value of 1. As shown Ι sc /Ι sc time 0 presented in the graph are not significantly different (paired t test). 104

108 Summary of acute EGF induced ERK1/2 mediated effects on Na absorption in non-cystic and cystic CD cells I sc (µa/cm 2 ) A. B EGF PD98059 non-cystic cystic 2 AP BL BL 1 AP AP BL BL * -5-6 * * I sc (µa/cm 2 ) C. Isc / Isc time 0 (time 0 is EGF addition) Time course of polarized EGF effects on amiloride-sensitive Isc Time (min) EGF AP on non-cy EGF BL on non-cy EGF AP on cy EGF BL on cy Non-cystic CD Principal cell Cystic PC cells Na + Na + EGF PD ENaC - Ras Raf Mek X perk1/2 Na + EGFR PD Ras Raf Mek X perk1/2 - ENaC Na + EGF EGFR Na + EGF EGFR Na + BL 105

109 Figure 3.7. EGFR localization and effects of EGF on ERK1/2 phosphorylation in primary cultures of non-cystic and cystic collecting duct cells. A. Localization of EGFR on the basolateral membrane as detected on non-cystic primary cell monolayers (bar=10 µm, x-y plane is at approximately middle of the cell height). C. EGFR abnormal localization in cystic monolayers. (bar=20 µm, x-y plane is at subapical supranuclear slice). Confluent monolayers of collecting duct cells isolated from non-cystic and cystic mice were treated for 15min with EGF (20 ng/ml) added to either the AP or the BL bathing solution. Some samples that received EGF were pretreated with an ERK kinase inhibitor (30 µm PD98059; apical and basolateral) for 15-25min prior to addition of EGF. The cells were lysed and prepared for western blot analysis of total and phosphorylated ERK1/2. Each lane was loaded with 20 µg of protein and blots were probed for phospho-erk1/2, stripped and reprobed for total ERK1/2. These blots are representative of 3 independent experiments. 106

110 A. Normal monolayers x-y x-z C. Cystic monoalyers x-y x-z 107