Activation of Mitogenic Pathways by Albumin in Kidney Proximal Tubule Epithelial Cells: Implications for the Pathophysiology of Proteinuric States

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1 J Am Soc Nephrol 10: , 1999 Activation of Mitogenic Pathways by Albumin in Kidney Proximal Tubule Epithelial Cells: Implications for the Pathophysiology of Proteinuric States RICK DIXON* and NIGEL JOHN BRUNSKILL* *Department of Cell Physiology and Pharmacology, and Department of Nephrology, Leicester University School of Medicine, Leicester, United Kingdom. Abstract. Albumin is filtered into the proximal tubule in large quantities in nephrotic states. It has been proposed that this protein may have a toxic effect on tubular epithelial cells and may be responsible for the initiation of interstitial inflammation and scarring. The mitogenic effect of recombinant human albumin in wild-type opossum kidney cells and in similar cells transfected with a dominant negative p85 subunit ( p85) of phopshatidylinositide 3-kinase (PI 3-kinase) has been studied. This study demonstrates that recombinant human albumin stimulates proliferation of opossum kidney cells in culture. This effect is mediated via PI 3-kinase, and is inhibited by wortmannin and p85 expression. Albumin stimulates PI 3-kinase activity in opossum kidney cells as determined by three different experimental procedures. Recombinant albumin also stimulates pp70 s6 kinase activity in a kinase cascade downstream of PI 3-kinase. Activity of pp70 s6 kinase is essential for albumin-induced proliferation of opossum kidney cells. It is proposed that this mitogenic pathway may have a critical role in proximal tubular homeostasis and pathophysiology of proteinuric states. Once initiated, renal impairment tends to be relentlessly progressive, terminating in end-stage renal failure and the need for dialysis. In the majority of renal diseases, protein most notably albumin is able to enter the proximal tubular fluid as a consequence of altered glomerular permselectivity. Clinicians have long recognized that proteinuria represents an adverse prognostic factor in patients with many different renal diseases, and those individuals with significant proteinuria are much more likely to develop progressive impairment of renal function (1,2). Furthermore, even in the primary glomerular diseases, developing renal insufficiency correlates best with pathologic changes observed in the tubulointerstitium of the kidney rather than in the glomeruli themselves (3,4). In light of these clinical observations, it has been proposed that proteinuria per se may have a toxic effect on the proximal tubular epithelium, thus stimulating the induction of interstitial inflammation. Consequently, it has been suggested that the abnormal proteinuric environment prevailing in the proximal tubule as a consequence of altered glomerular permeability may be directly responsible for progressive tubulointerstitial pathology in renal disease (5,6). Nevertheless, this suggestion of a causal link between proteinuria and renal disease progression still remains controversial. The traditional view holds that proteinuria is simply a Received December 8, Accepted February 1, Correspondence to Dr. Nigel Brunskill, Department of Cell Physiology and Pharmacology, University of Leicester, P.O. Box 138, Maurice Shock Medical Sciences Building, University Road, Leicester LE1 9HN, United Kingdom. Phone: ; Fax: ; njb18@le.ac.uk / Journal of the American Society of Nephrology Copyright 1999 by the American Society of Nephrology marker of a more severe renal lesion, which by virtue of this greater severity is more likely to progress and worsen, and thus many investigators refute a direct link between proteinuria and renal inflammation and scarring. More recently, a body of evidence has begun to accumulate suggesting that albumin is not simply a biologically inert molecule serving primarily to exert oncotic pressure in the circulation. The latest indications are that albumin may be able to exert effects on the function of the cells that it comes into contact with in the manner of a signaling molecule. The potentially important effects of albumin on cell function have been investigated by numerous groups. Circulating albumin is able to modulate vascular permeability possibly via changes in intracellular [Ca 2 ] (7,8), and is able to act as a survival factor for serum-starved endothelial cells in vitro (9). Albumin interaction with the binding protein gp60 in bovine pulmonary microvascular endothelial cells is not only a prelude to its transcytosis, but also results in activation of an intracellular kinase system catalyzing tyrosine phosphorylation of a number of proteins (10). Other authors have proposed a pathophysiologic role for albumin in cerebral scarring after breakdown of the blood brain barrier such as occurs in cerebral hemorrhage. This contention is also based on the observation of changes in intracellular [Ca 2 ] seen in astrocytes when treated with albumin (11,12). In proximal tubule cells, treatment with albumin has been demonstrated to induce the synthesis and release of endothelin (13), monocyte chemoattractant protein-1 (MCP-1) (14), and the chemoattractant chemokine RANTES (15). Phenotypic changes occur in vitro when proximal tubular cells are cultured with nephrotic urine and are represented by changes in the expression of cell surface integrins (16).

2 1488 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 Taken together, these results indicate that albumin may possess the hitherto unexpected ability to signal in multiple cell types. Of particular interest to us are the effects observed in proximal tubular cells. We previously examined the binding of albumin to proximal tubule cells and have described the characteristics of albumin-binding proteins in opossum kidney (OK) cells (17). Our studies have shown that albumin is absorbed by receptor-mediated endocytosis after binding to OK cells, and that this process is subject to complex regulation (18,19). Of particular note is the observation that albumin endocytosis in OK cells is controlled by the activity of the p85/p110 phosphatidylinositide (PI) 3-kinase (19), an enzyme with a well-documented role in mitogenesis (20 23). We therefore hypothesized that the endocytic machinery involved in the uptake of albumin by these cells may impinge on pathways of cell growth and mitogenesis, and as such we presented preliminary evidence that albumin itself is able to stimulate PI 3-kinase activity in OK cells (19). Because of the capacity of circulating albumin to bind various serum components (drugs, lipids, etc.) that may have bioactivity in cell culture, in these experiments we have predominantly used yeast recombinant human albumin (rhsa), which is uncontaminated by serum constituents or other circulating molecules. In the current study, we extend our earlier observations by showing definitively that albumin stimulates proliferation of OK cells through the activation of PI 3-kinase, and subsequently the p70 ribosomal protein S6 kinase (pp70 s6 kinase). We propose that this signaling pathway may underlie many of the observed effects of albumin in proximal tubular cells (and possibly other cell types), and that manipulation of this pathway may have clinically important implications for nephrology. Materials and Methods Reagents and Materials Wild-type OK cells were obtained from J. Caverzasio (Cantonal Hospital, Geneva, Switzerland). p85 OK cells express a dominant negative form of the p85 regulatory subunit of p85/p110 PI 3-kinase in an isopropylthiogalactopyranoside (IPTG)-inducible vector. The production, characterization, and maintenance of this cell line have been described in detail previously (19). Wortmannin was obtained from Calbiochem (Nottingham, United Kingdom) and stored at 20 C as concentrated stock in dimethylsulfoxide. Anti-p85 and anti-phosphotyrosine (PY20) antisera were obtained from Transduction Laboratories (Lexington, KY). Anti-pp70 S6 kinase antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant human albumin (Recombumin ) was kindly donated by Delta Biotechnology (Nottingham, United Kingdom) and was supplied as a 25% solution dissolved in NaCl buffer with 1.5 mg/100 ml Tween 80. In all experiments, appropriate diluent vehicle controls were performed, and in no instances were they able to reproduce the effect of rhsa. [ 3 H]-myoinositol (70.0 Ci/mmol) was from Amersham Life Science (Amersham, Buckinghamshire, United Kingdom), [ 32 P]-orthophosphate and -[ 32 P]-ATP were from New England Nuclear Life Science Products (Hounslow, United Kingdom). All other reagents were obtained from Sigma (Poole, United Kingdom). Cell Culture Wild-type OK cells were maintained in Dulbecco s modified Eagle s media Ham s F12 mix (DMEM F-12) supplemented with 10% fetal calf serum (FCS), 2 mmol/l L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. p85 cells were maintained in the above media with the addition of 200 g/ml hygromycin and 300 g/ml G418. All cells were incubated at 37 C in a humidified atmosphere of 5% CO 2 /95% air, and split at confluence approximately once per week for wild-type cells and once every 10 d for p85-transfected cells, which grow slightly more slowly. For induction of p85 expression in transfected cells, monolayers were treated with 1 mm IPTG for 18 h (19). [ 3 H]-Thymidine Incorporation Assay These studies were performed in both wild-type and p85-transfected OK cells essentially as described previously (24). Briefly, OK cells were plated in 24-well plates and grown to 70 to 90% confluence. They were then incubated in serum-free and thymidine-free DMEM F-12 for 24 h. The medium was then replaced with fresh serum-free DMEM F-12 alone (control), or serum-free DMEM F-12 supplemented with 10% FCS or various concentrations of albumin. After an additional 24 h, 2 Ci of [ 3 H] thymidine was added to all wells. After 2 h, the cells were washed 3 times with serum-free DMEM and processed as described (24). For those experiments using wortmannin, this agent was added for 15 min at a concentration of 100 nm to the cell monolayers before the stimulation of proliferation by the addition of FCS or albumin. The media was then changed and wortmannin was not present during the subsequent incubation with the stimulators of proliferation, or any other parts of the experiment. In those experiments using rapamycin, a selective inhibitor of pp70 s6 kinase (25), cells were incubated with 30 nm rapamycin for 20 min before the addition of 10% FCS or rhsa. Rapamycin was then present throughout the subsequent period of the experiment. Anti-Phosphotyrosine Immunoprecipitations Wild-type OK cells were grown to confluence in 6-well plates and serum-starved overnight before experiments. Cells were then exposed to serum-free DMEM F-12 containing albumin for 10 min at 37 C. For the preparation of immunoprecipitates for immunoblotting, monolayers were washed twice with ice-cold phosphate-buffered saline (PBS), ph 7.4, and then lysed in an ice-cold lysis buffer composed of 1% Triton X-100, 0.5% Nonidet P-40, 150 mm NaCl, 10 mm Tris, ph 7.4, 1 mm ethylenediaminetetra-acetic acid, 1 mm ethyleneglycolbis( -aminoethyl ether)-n,n -tetrra-acetic acid, 200 M sodium vanadate, and 200 M phenylmethylsulfonylfluoride for 30 min on ice. To this lysate was added 5 g of PY20 anti-phosphotyrosine antiserum followed by incubation at 4 C for 1 h. An aliquot of rabbit anti-mouse Ig was then added, and the immune complexes were immunoprecipitated using protein A-Sepharose. [ 32 P]-Orthophosphate Metabolic Labeling of OK Cells and Immunoprecipitation with Anti-pp70 S6 Kinase Wild-type and p85 OK cells were grown to confluence in 6-well plates and serum-starved overnight. On the morning of the experiment, cells were washed 3 times with serum-free, phosphate-free DMEM F-12 and then incubated in this medium containing 200 Ci/ml [ 32 P]-orthophosphate for 4hat37 C. Cells were then stimulated for varying times with various concentrations of albumin. After stimulation, cell monolayers were washed twice with ice-cold PBS, ph 7.4, and lysed in an ice-cold lysis buffer composed of 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate

3 J Am Soc Nephrol 10: , 1999 Albumin Mitogenesis in Proximal Tubule Cells 1489 (SDS), 200 M phenylmethylsulfonylfluoride, and 200 M sodium vanadate, all in PBS, ph 7.4. After incubation on ice for 20 min, large aggregates were disrupted by repeated aspiration through a 21-gauge needle and then insoluble material was removed by centrifugation. The supernatant was precleared with protein A-Sepharose for 30 min at 4 C and then incubated with an aliquot of 10 l of anti-pp70 S6 kinase antibody for 1 h. Immune complexes were immunoprecipitated with protein A-Sepharose. The beads were then washed 3 times with lysis buffer and prepared for polyacrylamide gel electrophoresis (PAGE) by the addition 30 l of Laemmli buffer containing 60 mm Tris, ph 6.8, 10% glycerol, 2% SDS, 100 mm dithiothreitol, and 0.01% bromphenol blue. Immunoprecipitated proteins were then separated on 12% polyacrylamide gels, and radiolabeled proteins were detected by autoradiography. Immunoblotting For the detection of p85 proteins in anti-phosphotyrosine immunoprecipitates, the Sepharose beads were resuspended in Laemmli buffer and the proteins were separated by PAGE, then transferred to nitrocellulose membranes. The membranes were probed with anti-p85 antibodies and anti-rabbit peroxidase-linked second antibodies, and bound antibodies were detected using enhanced chemiluminescence (ECL) development. For the detection of gel shift of pp70 S6 kinase, OK cells were grown to confluence and stimulated with several concentrations of albumin for various times. Monolayers were washed with ice-cold PBS and lysed with Laemmli buffer. Equivalent quantities of cell lysate from each experimental condition were loaded onto cm, 8% polyacrylamide gels and separated for8hat75ma. After transfer to nitrocellulose membranes, pp70 S6 kinase proteins were detected using anti-pp70 S6 kinase antibodies and ECL as described above. Assay of PI 3-Kinase Activity in Immunoprecipitates and Intact Cells PI 3-kinase activity was measured in anti-phosphotyrosine immunoprecipitates of both wild-type and p85-transfected cells by in vitro kinase assay, using phosphatidylinositol (PtdIns) and PtdIns(4,5) bisphosphate (PtdIns(4,5)P 2 ) as substrates, as described previously (19). In addition to the immune complex PI 3-kinase assay, we measured PI 3-kinase activation in response to albumin in intact cells labeled with [ 3 H]-myoinositol. Wild-type and p85-transfected OK cells were passaged into inositol-free DMEM F-12 containing 10% FCS and allowed to grow to confluence. Two days before experiments, confluent cells in 6-well plates were washed twice with serumfree, inositol-free DMEM F-12 and then incubated in this same serum-free media containing 400 Ci/ml [ 3 H]-myoinositol. For stimulations, this media was then removed and replaced with prewarmed serum-free, inositol-free DMEM F-12 containing various concentrations of albumin. After 10 min, the media was quickly aspirated and the incubation was terminated by placing on ice with the addition of 1 ml of ice-cold 2.4 M HCl. The cell monolayers were scraped and left on ice for 10 min. This mixture was then transferred to a polypropylene tube and the wells were rinsed with 0.25 ml of PBS. This was combined with the acid extract, and then 1.5 ml of chloroform/ methanol (1:2 vol/vol) was added followed by 1.0 ml of chloroform. The mixture was vortexed, the phases were separated by centrifugation, and the upper aqueous phase was removed and reextracted twice with 1.0 ml of chloroform. The chloroform layers were then combined and dried under nitrogen. The dried lipid films were redissolved in 500 l of a mixture containing methylamine (25 to 30% aqueous solution), methanol, and water in the ratio 4:4:1 (by volume) and deacylated for 30 min at 53 C, to produce water-soluble glycerophosphoinositol phosphates from their corresponding phosphoinositides. Samples were then cooled and dried under a vacuum. The deacylated samples were redissolved in 1.0 ml of water and washed twice, first with 1.2 ml and then with 0.75 ml of a mixture composed of butan- 1-ol/petroleum ether (boiling point 40 to 60 C)/ethyl formate (20:4:1 by volume). The upper organic phase was discarded after each wash. Glycerophosphoinostiol phosphates remaining in the aqueous phase were then separated by HPLC, using a Partisphere SAX HPLC column (BAS Technicol, Congleton, United Kingdom) eluted with a continuous gradient made using water and 1.25 M (NH 4 ) 2 HPO 4,pH 3.8, with H 3 PO 4, as described previously (19,26). Radiolabeled reaction products were detected using a [ 3 H] flow-through detector (Canberra Packard, Pangbourne, United Kingdom). Statistical Analyses Curve fitting was performed by nonlinear regression using curve fitting software (GraphPad Prism, San Diego, CA). Data are generally represented as mean values together with the SEM. Where multiple comparisons were required, statistical analysis was performed by one-way ANOVA, and the significance of differences between individual group means was determined with the Duncan multiple range test at P 0.05 and P Results Characterization of p85-transfected OK Cells p85-transfected OK cells can be induced to express the p85 protein by IPTG. This induction is maximal when cells are incubated with 1 mm IPTG for 18 h. After induction under these conditions, p85 is expressed at levels 10-fold greater than the endogenous protein, and results in complete abolition of PI 3-kinase activity in anti-phosphotyrosine immunoprecipitates in response to 10 g/ml insulin. The results of the extensive characterization experiments relating to this transfected cell line have been published previously (19). Albumin Stimulates Proliferation in OK Cells We first investigated whether albumin was able to stimulate proliferation of serum-starved OK cells. Addition of rhsa to culture media resulted in a significant increase in proliferation of wild-type OK cells as measured by [ 3 H]-thymidine incorporation (Figure 1A). This effect was most marked at an incubated rhsa concentration of 1 mg/ml when an approximately threefold increase in [ 3 H]-thymidine incorporation was observed compared with controls. The effect of addition of 10% FCS is shown for comparison. A modest proliferative effect was apparent with concentrations of rhsa as low as 10 g/ml, but this failed to reach statistical significance. The proliferative effect observed in response to rhsa was completely abolished by pretreatment with wortmannin, whereas the proliferative effect of 10% FCS, which contains multiple proliferative stimuli, was only partially prevented. rhsa was also capable of eliciting a proliferative response in p85- transfected OK cells, and this response was completely prevented by the induction of p85 expression by IPTG (Figure 1B). In a pattern similar to the results seen with wortmannin, induction of p85 expression was only partially successful in preventing 10% FCS-induced proliferation. These results

4 1490 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 therefore suggested that rhsa was able to stimulate OK cell proliferation dependent on p85/p110 PI 3-kinase activity. Activation of PI 3-Kinase Activity by Albumin To determine whether treatment of OK cells with albumin was able to activate PI 3-kinase activity in OK cells, we first looked for evidence of a p85 translocation event. Wild-type OK cells stimulated with either fatty acid-containing albumin, fatty acid-free albumin, or rhsa were immunoprecipitated with anti-phosphotyrosine antibodies, and the immunoprecipitate was probed for the presence of p85 subunits of PI 3-kinase. Under control conditions, p85 protein was barely detectable in these immunoprecipitates, but after 10 min of stimulation, by all forms of albumin tested, p85 protein becomes associated with tyrosine phosphorylated proteins (Figure 2). To confirm that PI 3-kinase activity became associated with anti-phosphotyrosine immunoprecipitates in response to rhsa, the immunoprecipitate was used to phosphorylate PtdIns and PtdIns(4,5)P 2 in the presence of -[ 32 P]-ATP. The HPLC elution profiles of [ 32 P]-labeled deacylated reaction products from representative experiments are depicted in Figure 3. Under control conditions, no phosphorylated reaction products are observed, but when wild-type OK cells are stimulated by rhsa, a number of peaks are resolved by HPLC. The first peak represents deacylated PtdIns(3)P, and the last peak represents deacylated PtdIns(3,4,5)P 3. These products of PI 3-kinase activity are also observed in response to rhsa in noninduced p85-transfected cells, but are abolished when p85 is expressed following IPTG induction. Similar peaks are also observed when cells are treated with non-defatted human serum albumin (results not shown). There were no statistically significant differences between the activity of PI 3-kinase in anti-phosphotyrosine immunoprecipitates derived from wildtype or noninduced p85 cells for any given concentration of rhsa. Figure 1. Proliferation of opossum kidney (OK) cells measured as [( 3 H)]-thymidine incorporation in response to serum or various concentrations of yeast recombinant human albumin (rhsa). (A) [ 3 H]- thymidine incorporation in wild-type OK cells. Seventy to 90% confluent cells were serum-starved and incubated with [ 3 H]-thymidine under control conditions, after treatment with various concentrations of rhsa, or after readdition of 10% fetal calf serum (FCS). In some experiments, cells were pretreated with 100 nm wortmannin just before agonist addition (p). (B) [ 3 H]-thymidine incorporation in p85-transfected OK cells. Seventy to 90% confluent noninduced p85 cells were serum-starved and incubated with [ 3 H]-thymidine under control conditions, after treatment with various concentrations of rhsa, or after readdition of 10% FCS (f). In some experiments, cells were induced to express p85 with isopropylthiogalactoside (IPTG) before agonist addition (p). Data represent means SEM for n 4 experiments. **P 0.05; *P 0.01, compared with control values. Figure 2. Translocation of p85 to tyrosine phosphorylated proteins in wild-type OK cells after treatment with various albumins. Wild-type OK cells were grown to confluence, serum-starved, and then treated with either 1 mg/ml rhsa (Rec Alb), fatty acid-free human serum albumin (FAF Alb), or fatty acid-replete human serum albumin (FAR Alb) for 10 min at 37 C. Cells were washed and lysed, and the lysate was immunoprecipitated with anti-phosphotyrosine antibodies. Immunoprecipitates were subjected to polyacrylamide gel electrophoresis (PAGE) and Western-blotted using anti-p85 antibodies. Bound antibodies were detected using an enhanced chemiluminescence (ECL) development system followed by exposure to photographic film. The blot shown is representative of three separate experiments.

5 J Am Soc Nephrol 10: , 1999 Albumin Mitogenesis in Proximal Tubule Cells 1491 Figure 3. In vitro kinase assay for phopshatidylinositide 3-kinase (PI 3-kinase) activity in anti-phosphotyrosine immunoprecipitates derived from wild-type and p85 OK cells. The activity of PI 3-kinase was measured in anti-phosphotyrosine immunoprecipitates derived from either wild-type cells or p85 OK cells with or without IPTG induction, and with or without prestimulation with 1 mg/ml rhsa. (A) Wild-type cells with no agonist stimulation. (B) Wild-type cells pretreated with rhsa. (C) p85 induced with IPTG and pretreated with rhsa. (D) Noninduced p85 cells pretreated with rhsa. The substrates provided were phosphatidylinositol (PtdIns) and PtdIns(4,5) bisphosphate (PtdIns(4,5)P 2 ). Lipid reaction products were deacylated and separated by HPLC. Representative elution profiles are depicted above. Stimulation of wild-type and noninduced p85 cells results in the elution of two main peaks. The peak at 29 min represents PtdIns(3)P, and that at 73 min PtdIns(3,4,5)P 3. Treatment of wild-type OK cells with IPTG before rhsa stimulation had no effect on PI 3-kinase activity in these immunoprecipitates (not shown). These results indicated that rhsa was able to stimulate the association of PI 3-kinase activity with tyrosine phosphorylated proteins in OK cells. To confirm that rhsa stimulation resulted in a rise in the intracellular levels of the products of in vivo activity of PI 3-kinase, we examined the levels of PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 extractable from [ 3 H]-myoinositol-labeled cells under control conditions and after stimulation by rhsa. Figure 4 depicts representative elution profiles of deacylated [ 3 H]-labeled lipids extracted from both wild-type and p85 OK cells under control conditions, or after agonist stimulation. A number of peaks are observed, and have been labeled according to the elution time of known deacylated standard [ 3 H]-phosphoinositides. Under control conditions, PtdIns(3,4,5)P 3 is present at barely detectable levels in OK cells. After treatment of OK cells with rhsa, the levels of PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 both rise appreciably, and become readily detectable in [ 3 H]-phosphoinositide extracts from the cells. Activation of PI 3-kinase by rhsa is blocked after p85 induction by IPTG. Treatment of wild-type OK cells with IPTG has no effect on activation of PI 3-kinase activity (data not shown). Figure 5A depicts the dose response profile for rhsa activation of PtdIns(3,4,5)P 3 production. The results are expressed as % PtdIns(3,4,5)P 3 /PtdIns(4,5)P 2 and demonstrate that PtdIns(3,4,5)P 3 levels rise by approximately 10-fold after maximal stimulation by 1 mg/ml rhsa. It is noteworthy, however, that measurable elevations in cellular PtdIns(3,4,5)P 3 levels are observed after stimulation with rhsa concentrations as low as 10 g/ml. The activation of PI 3-kinase activity measured in this way in response to rhsa is also inhibited by p85 expression (Figure 5B). Fold increases in PtdIns(3,4,5)P 3 /PtdIns(4,5)P 2 ratio in response to albumin were very similar in wild-type and p85 cells, and significant differences in PtdIns(3,4,5)P 3 accumulation were not observed between these cell types in response to albumin. Albumin Stimulates pp70 s6 Kinase Activity in OK Cells Confluent wild-type and p85-transfected OK cells were radiolabeled with [ 32 P]-orthophosphate, stimulated with rhsa,

6 1492 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 Figure 4. Accumulation of PtdIns(3,4,5)P 3 in response to rhsa in wild-type and p85-transfected OK cells. Total phosphoinositides were extracted from [ 3 H]-myoinositol-labeled OK cells under control conditions and after stimulation with various concentrations of rhsa. Extracted lipids were deacylated, and the resulting glycerophosphoinositol phosphates were separated by HPLC. Relevant areas of the elution profiles are displayed. (A) Wild-type OK cells under control conditions demonstrate negligible levels of PtdIns(3,4,5)P 3. (B) Wild-type OK cells after stimulation with 100 g/ml rhsa demonstrate an appreciable rise in intracellular levels of both PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3, as indicated. (C) Wild-type OK cells after stimulation with 1 mg/ml rhsa show an even greater rise in PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3. (D) Noninduced p85-transfected cells under control conditions show tiny basal levels of PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3. (E) Noninduced p85 cells demonstrate a rise in PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 after stimulation with 1 mg/ml rhsa. (F) In p85-transfected OK cells, the rise in PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 after stimulation by rhsa is completely blocked by induction of p85 expression with IPTG. These elution profiles are representative of at least three experiments for each condition. lysed, and immunoprecipitated with anti-pp70 s6 kinase antibodies. Equivalent amounts of immunoprecipitate were subjected to PAGE, and phosphorylated pp70 s6 kinase was detected by autoradiography. A representative autoradiograph from an experiment with p85 cells is depicted in Figure 6. It is apparent that treatment of the cells with increasing concentrations of rhsa results in a concomitant increase in pp70 s6 kinase phosphorylation, indicating its activation by this treat-

7 J Am Soc Nephrol 10: , 1999 Albumin Mitogenesis in Proximal Tubule Cells 1493 Figure 6. Phosphorylation and activation of pp70 s6 kinase in response to rhsa. p85-transfected OK cells were labeled with [ 32 P]-orthophosphate, treated with various concentrations of rhsa, and immunoprecipitated with anti-pp70 s6 kinase. Immunoprecipitates were subjected to PAGE followed by autoradiography. The labeled band represents phosphorylated pp70 s6 kinase protein. In some experiments, cells were pretreated with IPTG before rhsa stimulation. The bottom panel depicts densitometric evaluation of this gel, with pp70 s6 phosphorylation under control conditions being arbitrarily assigned a value of 1.0. The gel is representative of three separate experiments. Figure 5. Stimulation by rhsa of PI 3-kinase activity in [ 3 H]- myoinositol-labeled wild-type and p85-transfected cells expressed as percentage of PtdIns(3,4,5)P 3 cpm/ptdins(4,5)p 2 cpm. Wild-type and p85-transfected OK cells were labeled with [ 3 H]-myoinositiol, and total inositol phospholipids were extracted under control conditions, or after stimulation with various concentrations of rhsa. Lipids were deacylated and then separated by HPLC. Levels of PtdIns(3,4,5)P 3 produced were normalized to the level of PtdIns(4,5)P 2, which does not change on agonist stimulation. The results are expressed as percentage of PtdIns(3,4,5)P 3 cpm/ptdins(4,5)p 2 cpm. (A) Dose response of PtdIns(3,4,5)P 3 production in response to rhsa. Increasing concentrations of rhsa result in greater production of PtdIns(3,4,5)P 3, indicating a dose-dependent stimulation of PI 3-kinase activity. (B) Effect of induction of p85 protein expression by IPTG in [ 3 H]-myoinositol-labeled p85-transfected cells. Labeled inositol phospholipids were extracted from p85 cells under control conditions, and after stimulation by 1 mg/ml rhsa with and without IPTG pretreatment. p85 expression completely blocks rhsa-stimulated PtdIns(3,4,5)P 3 production. Results represent means SEM of n 3 separate experiments. ment. Maximal activation of pp70 s6 kinase is seen when cells are incubated with 1 mg/ml rhsa. In p85-expressing cells, the pp70 s6 kinase activation seen in response to rhsa is abolished, suggesting that PI 3-kinase activity is required upstream for rhsa-induced stimulation of pp70 s6 kinase activity. An identical stimulation of pp70 s6 kinase is seen in wild-type cells in response to rhsa, and a similar inhibitory effect is exhibited after wortmannin pretreatment of the cells (data not shown). The increase in phosphorylation of pp70 s6 kinase seen on activation results in a gel shift of its molecular weight. This gel shift is clearly seen in response to rhsa and is depicted in Figure 7. p85-transfected OK cells were treated for various times with 1 mg/ml rhsa. A gel shift of pp70 s6 kinase to higher molecular weight species is apparent after 2 min of treatment of serum-starved cells with rhsa, and is maximal after 15 min. Induction of p85 expression by IPTG abolishes this gel shift, and restricts the phenotype of pp70 s6 kinase proteins to the lowest molecular weight forms. Again, results of these experiments are the same if performed in wild-type cells, with wortmannin pretreatment of cells abolishing this gel shift of pp70 s6 kinase in response to albumin (not shown). Proliferation of OK Cells in Response to Albumin Is Dependent on pp70 s6 Kinase Activity OK cells were prepared for [ 3 H]-thymidine incorporation assays and pretreated with rapamycin as described above. Under these conditions, rhsa-stimulated OK cell proliferation is completely abolished, indicating that pp70 s6 kinase activity

8 1494 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 Figure 7. Gel shift of pp70 s6 kinase after stimulation of p85 cells by rhsa. Serum-starved p85-transfected OK cells were stimulated with 1 mg/ml rhsa for various lengths of time. In some experiments, cells were induced to express p85 by IPTG pretreatment before rhsa stimulation. Cells were lysed, and lysates were subjected to Western blotting with anti-pp70 s6 kinase antisera with subsequent ECL development. Increasing time of stimulation by rhsa results in a progressive shift of pp70 s6 kinase to higher molecular weight forms as indicated. This shift is prevented by p85 expression. This blot is representative of three separate experiments. 3-kinase acting via pp70 s6 kinase, and that inhibition of either of these enzymes is able to block this proliferative effect. Figure 8. Prevention of OK cell proliferation by pp70 s6 kinase inhibitor rapamycin. [ 3 H]-thymidine incorporation assays were used to assess proliferation of serum-starved OK cells stimulated with 1 mg/ml rhsa. Cells were either subjected to no pretreatment (f) or were pretreated with 30 nm rapamycin before the addition of rhsa (p). Data represent means SEM of n 4 experiments. *P 0.01 compared with control. is necessary for the induction of OK cell proliferation by rhsa (Figure 8). Proliferation of cells provoked by readdition of serum is only partially blocked by rapamycin, indicating that this treatment is not simply toxic to the cells. These results therefore indicate that rhsa is able to stimulate proliferation of OK cells via stimulation of the p85/p110 PI Discussion The nephrology community has for several years debated the potential pathophysiologic significance of albuminuria in patients with renal disease. A consensus view still has not emerged, and one of the major stumbling blocks has been a reluctance on the part of some nephrologists to abandon the traditional concept of albumin as a benign or inert molecule. The data presented in the current study clearly indicate that albumin may have a crucial role in the maintenance of proximal tubular integrity and function. Indeed, the demonstration that rhsa is able to stimulate PTC proliferation via PI 3-kinase and pp70 s6 kinase provides a plausible explanation for a number of previously unexplained effects of albumin on cell biology in kidney tubular, and other cell types. Earlier work from our laboratory indicated that albumin is reabsorbed from the proximal tubule by receptor-mediated endocytosis after binding to both high- and low-affinity sites (17,18), and that this process is regulated by PI 3-kinase (19). In the course of this work, we made a preliminary observation that albumin had the unexpected ability to activate p85/p110 PI 3-kinase in OK cells (19). Other workers in our laboratory have also made preliminary observations of increases in proximal tubular cell growth in response to albumin and proteinuric urine (27). We have now extended this work by demonstrating unequivocally, using a recombinant preparation, that albumin is able to activate PI 3-kinase, causing translocation of activity to tyrosine phosphorylated proteins, and a rise in the cellular levels of the important lipid signaling intermediate PtdIns(3,4,5)P 3. The PI 3-kinase enzyme activated by albumin appears to be the p85/p110 form. We base this contention on the observed inhibition of PI 3-kinase enzyme activity by expression of p85, which would not be expected to inhibit other members of the PI 3-kinase enzyme family (28). Also, the intracellular products of PI 3-kinase stimulation observed in response to albumin, PtdIns(3,4)P 2, and PtdIns(3,4,5)P 3, are characteristic of p85/p110 PI 3-kinase activity (29). The importance of this observation in the context of the current study relates to the prototypic action of the p85/p110 PI

9 J Am Soc Nephrol 10: , 1999 Albumin Mitogenesis in Proximal Tubule Cells kinase enzyme, i.e., mitogenic and growth factor-related signaling (20 23). Hence, we investigated the possibility that albumin may possess growth factor-like effects when applied to cultured OK cells. Earlier studies using serum-derived albumin in miscellaneous experiments are complicated because the albumin molecule is sticky and binds a number of substances in the circulation, especially lipids, and thus it is not possible to completely exclude the possibility that material other than albumin may be responsible for any observed biologic effects. Using rhsa, uncontaminated by bioactive bloodderived molecules, we have shown that albumin itself has a mitogenic effect on OK cells in culture, stimulating DNA synthesis and cell proliferation. The recombinant albumin product is described as % pure and devoid of attached ligands by the manufacturers and, to our knowledge, is the most pure preparation of human albumin available. All of the relevant rhsa diluent vehicle control experiments failed to reproduce the rhsa effects. Hence, we are confident that the responses observed are evoked by the rhsa and not another molecule in the preparation. This response is mediated via the activation of p85/p110 PI 3-kinase since DNA proliferation is inhibited by wortmannin and p85 expression. The reason for the slightly smaller proliferative response seen in p85 cells is not clear. It may relate to the fact that the p85 cells are a stably transfected clone derived from a single cell of the wild-type population that may possess slightly different growth characteristics compared to the wild-type population as a whole. Alternatively, the presence of G418 and hygromycin in the culture medium may slow the growth of these cells. We next wanted to examine possible downstream targets of albumin-stimulated PI 3-kinase activity. The signaling pathways initiated by PI 3-kinase are thought to involve the protein kinase B (PKB), and PKB activity in turn is regulated by the phosphoinositide-dependent kinase-1 (PDK-1). The lipid product of PI 3-kinase activity, PtdIns(3,4,5)P 3, is required for activation of PDK-1, and through binding to PKB exposes otherwise cryptic sites on PKB whose phosphorylation is required for PKB activation (30,31). The enzyme pp70 s6 kinase participates in the translational control of mrna transcripts encoding essential components of the protein synthetic apparatus. Several authors have tentatively implicated PI 3-kinase activity in the activation of pp70 s6 kinase (32,33) in some systems, and indeed it has recently been demonstrated that pp70 s6 kinase is phosphorylated and activated by PDK-1 (34). Our data clearly demonstrate that stimulation of PI 3-kinase by albumin results in downstream phosphorylation and hence activation of pp70 s6 kinase, and that increased activity of both of these enzymes is required for rhsa-induced cell proliferation. The findings of the current study not only establish the importance of this kinase cascade in mitogenic signaling, but they also provide for the first time a potential explanation for some of the reported effects of albumin on the function of a number of distinct cell types. Albumin was found to protect endothelial cells against serum starvation-induced apoptosis by Zoellner et al. (9), although the mechanism was not examined. A clue to the mechanism of albumin s capacity to act as a survival factor may be found in the actions of nerve growth factor and platelet-derived growth factor. These growth factors are able to prevent apoptosis of neurons and pheochromocytoma cells, respectively, through the activation of PI 3-kinase (35). Indeed, PI 3-kinase and its downstream kinase PKB have also been invoked as important mediators of cell survival in serum-starved neuronal cells (36). Therefore, albumin s capacity to act as a survival factor in endothelial cells could be mediated via PI 3-kinase activation in a manner akin to that described for nerve growth factor and platelet-derived growth factor. Several authors have sought a role for filtered urinary proteins in the induction of interstitial inflammation in renal disease. Wang et al. described increased production of MCP-1 by proximal tubular cells in response to albumin and postulated a novel pathway by which urinary proteins may trigger interstitial inflammation (14). Zoja et al. demonstrated NF- Bdependent stimulation of production of the chemoattractant chemokine RANTES by cultured proximal tubular cells treated with albumin (15). Although we have no evidence yet that albumin stimulation of PI 3-kinase/pp70 s6 kinase can directly provoke transcription factor activation, it is possible that the increased translational activity observed in response to stimulation of pp70 s6 kinase may ultimately result in increased translation of message coding for transcription factors or cytokines. Interestingly, Levine et al. (37) have implicated PI 3-kinase in lysophosphatidic acid signaling in proximal tubular cells, and postulated that this lipid signaling pathway may be important in tubular injury. Data have also implicated albumin in proximal tubular cell phenotypic changes related to expression of cell surface integrins. Human proximal tubular cells, when incubated with albumin, demonstrate upregulation of expression of v 5 integrin (16). The logical conclusion from this finding was that albuminuria may have the ability to modulate the relationship of proximal tubular cells with the interstitium in a potentially detrimental manner. Activation of integrin adhesion pathways in response to PI 3-kinase stimulation has been described in Jurkat cells (38). Again, the results of the present study therefore provide a potential explanation for these earlier observations in that PI 3-kinase activity stimulated by albumin in proximal tubular cells may impinge on the integrin signaling pathway. A key question arising from this current study is the identification of the receptor mediating these albumin effects. In endothelial cells, albumin binds to gp60, a specific albuminbinding protein, and thereby stimulates the phosphorylation of a number of intracellular substrates, including caveolin-1 and members of the Src family tyrosine kinases pp60 c-src and Fyn (10). However, gp60 is not detected in OK cells (17), and the albumin receptor responsible for signaling the currently described effects is not known. What are the physiologic, or indeed pathophysiologic, implications of these findings? Appreciable rhsa-induced activation of PI 3-kinase and pp70 s6 kinase activity was seen with incubated rhsa concentrations as low as 1 to 10 g/ml, although a significant effect of rhsa on OK cell proliferation was not seen until a concentration of 100 g/ml was attained.

10 1496 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 In health, albumin is not totally excluded from the proximal tubular fluid, and although in humans direct measurement is obviously not possible, micropuncture studies in rodents have indicated a tubular fluid albumin concentration around 30 g/ml in this nephron segment. This albumin concentration is considerably higher in nephrotic states (39 41). An intriguing possibility therefore is that in health, filtered albumin may exert an important tonic effect on proximal tubule growth homeostasis. Under conditions of increased and chronic albumin stimulation in nephrosis, this homeostatic balance may be upset, thus favoring the development of the typical renal histologic changes seen in progressive disease in which proximal tubule growth is so manifestly disordered. 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