The Future of Polycystic Kidney Disease Research As Seen By the 12 Kaplan Awardees

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1 The Future of Polyysti Kidney Disease Researh As Seen By the 12 Kaplan Awardees Corinne Antigna,* James P. Calvet, Gregory G. Germino, Jared J. Grantham, Lisa M. Guay-Woodford, Peter C. Harris,** Friedhelm Hildebrandt, Dorien J.M. Peters, Stefan Somlo, Viente E. Torres,** Gerd Walz, Jing Zhou, and Alan S.L. Yu *National Institute of Health and Medial Researh, Laboratory of Inherited Kidney Diseases, Paris Desartes-Sorbonne Paris Cité University, Imagine Institute, and The Department of Genetis, Neker Hospital, Paris, Frane; The Kidney Institute, University of Kansas Medial Center, Kansas City, Kansas; Kidney Disease Branh, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; Center for Translational Siene, Children s National Health System, Washington, DC; **Division of Nephrology and Hypertension, Mayo Clini, Rohester, Minnesota; Howard Hughes Medial Institute and Boston Children s Hospital, Harvard Medial Shool, Boston, Massahusetts; Department of Human Genetis, Leiden University Medial Center, Leiden, The Netherlands; Departments of Internal Mediine and Genetis, Yale University Shool of Mediine, New Haven, Connetiut; Renal Division, Department of Mediine, University Medial Center Freiburg, Freiburg, Germany; and Harvard Center for Polyysti Kidney Disease Researh, Renal Division, Department of Mediine, Brigham and Women s Hospital, Harvard Medial Shool, Boston, Massahusetts ABSTRACT Polyysti kidney disease (PKD) is one of the most ommon life-threatening geneti diseases. Jared J. Grantham, M.D., has done more than any other individual to promote PKD researh around the world. However, despite deades of investigation there is still no approved therapy for PKD in the United States. In May 2014, the University of Kansas Medial Center hosted a symposium in Kansas City honoring the oasion of Dr. Grantham s retirement and invited all the awardees of the Lillian Jean Kaplan International Prize for Advanement in the Understanding of Polyysti Kidney Disease to partiipate in a forward-thinking and interative forum foused on future diretions and innovations in PKD researh. This artile summarizes the ontributions of the 12 Kaplan awardees and their vision for the future of PKD researh. J Am So Nephrol 26: , doi: /ASN In 2013, Jared J. Grantham, M.D. retired from the University of Kansas Medial Center (KUMC) after a long and distinguished areer devoted to the searh for a ure for polyysti kidney disease (PKD). Dr. Grantham s interest in kidney disease developed in the sixth grade in Johnson, Kansas while trading omi books with his neighbor. His neighbor asually mentioned that he had ysts in his kidneys, his mother had them and his grandmother had reently died of kidney failure. That remained a vivid memory for Dr. Grantham and he reonneted with kidney ysts in 1970 when he was asked to lead the Nephrology Division at the KUMC and disovered in his laboratory new renal funtions in tubule fluid seretion that opened the door to innovative researh on PKD. Dr. Grantham was a member of the National Institutes of Health (NIH) researh team that developed the method to disset and perfuse isolated segments of renal tubules. His work emphasized the mehanisms of ation of vasopressin on olleting duts, a theme that was arried forward to his work on PKD, whih was ontinuously funded by the NIH until his retirement. With Kansas City businessman Joseph Bruening, Dr. Grantham founded in 1982 the Polyysti Kidney Disease Foundation, whih today is the leading organization worldwide supporting researh and eduation about the disease. Dr. Grantham, a die-hard native Kansan, direted the Division of Nephrology at Kansas for 25 years, was o-founder of the Kidney Institute whih he direted for 5 years, and was founding editor of the Journal of the Amerian Soiety of Nephrology.Without question, Jared Grantham has been a towering figure in PKD researh and has done more for this field than any other single individual. In Dr. Grantham s honor, the Kidney Institute at KUMC hosted the Jared J. Grantham Symposium, The Future of Published online ahead of print. Publiation date available at. Correspondene: Dr.AlanS.L.YuorDr.JamesP. Calvet, The Kidney Institute, WHE 6018, University of Kansas Medial Center, 3901 Rainbow Boulevard, Kansas City, KS ayu@kum.edu or JCALVET@kum.edu Copyright 2015 by the Amerian Soiety of Nephrology J Am So Nephrol 26: , 2015 ISSN : /

2 Polyysti Kidney Disease Researh on May 7 9, All reipients of the Lillian Jean Kaplan Prize were invited to Kansas City to lead a forward-thinking and interative forum foused on future diretions and innovations in PKD researh. The Lillian Jean Kaplan International Prize for the Advanement in the Understanding of Polyysti Kidney Disease was established by the PKD Foundation and the International Soiety of Nephrology (ISN) through the generosity of the family of Thomas and Dafna Kaplan, in honor of Mr. Kaplan s late mother, Lillian Jean Kaplan, who died of PKD. The prize was reated to stimulate interest in advaning PKD researh and to reognize those who have inreased our understanding and treatment of PKD through basi or linial sientifi researh, leading to new treatments and a ure for PKD. The Prize is awarded every 2 years at the ISN s World Congress of Nephrology and so far there have been 13 awardees. Vinent Gattone, Ph.D., who was awarded the prize in 2013, unfortunately passed away on January 26, He was highly respeted in the PKD field and had a major impat. Among his many ahievements, he developed the onept of using vasopressin antagonists to treat PKD and arried out the initial testing in rodent models of ysti disease that eventually led to linial trials of tolvaptan. The Grantham Symposium was a huge suess. All 12 living Kaplan awardees were able to partiipate, along with nearly 200 attendees who ame from all over the world to elebrate Dr. Grantham s areer and partiipate in shaping the future of PKD researh. The many outstanding talks and disussion forums stimulated onsiderable lively debate and exhange of ideas. These shed muh-needed light on the most important diretions that now need to be pursued to advane the field and move effetive treatments for this disease as quikly as possible into the lini. In this artile, eah Kaplan awardee was invited to ontribute a synopsis of his or her talk, fousing on their vision for the future. From the juxtaposition of their talks and these reviews, several ommon themes emerged. A number of fators, suh as alleli effets of PKD genes (Harris) and the funtion of iliary traffiking proteins (Zhou), onverge to determine the dosage of PKD1 protein that is delivered to the ell and iliary surfae, whih is seen as playing a ritial role in determining disease severity. Modifier genes have long been suspeted of influening the severity and natural history of the disease. Guay-Woodford illustrates how this an be effetively investigated in autosomal reessive PKD (ARPKD) by using mouse quantitative trait loi mapping and analogous studies in a unique, genetially isolated, human population. Muh reent evidene suggests that the ell and tissue ontext within whih PKD mutations our is a key determinant of the disease ourse. The developmental stage in early life, or superimposed renal injury in adulthood are well-established ontextual fators. To identify other ontextual elements, Germino used an agnosti transriptomi method and showed that the metaboli pathways assoiated with developmental age are important. Peters argues that loal pararine or mehanial signals may also play a role and that this perhaps resembles more the mehanism of human disease. Although the many ulprit genes in ysti diseases share a ommon loalization to the ilia, the true role of this organelle remains poorly understood. Somlo disusses observations that support the radial assertion that signals emanating from the intat ilia atually promote ystogenesis. The idea that ysti disease is aused by abnormal epithelial ell proliferation and onstitutes a neoplasti proess is well aepted, but Calvet provides epidemiologi data suggesting that patients with PKD may atually be proteted from aner, raising intriguing questions about how the proliferation of yst epithelial ells is regulated. Novel downstream yst-promoting mehanisms that have reently been unovered inlude the role of the atin ytoskeleton and diretional ell motility (Zhou) and the role of DNA damage repair in the regulation of ell yle hekpoint genes and hene epithelial ell proliferation (Hildebrandt). Hildebrandt and Antigna both make a strong ase for ontinued efforts to identify novel ysti disease genes as a rih soure for unovering new biology. With the identifiation of so many signaling pathways, ysti diseases are ripe for the development of pharmaologi therapies. Torres reviews the therapeuti landsape and urrent and future prospets. One of the more promising of these is the mammalian target of rapamyin (mtor) pathway, yet reent linial trials of mtor inhibitors have failed. Torres argues that the dosagethat ould beusedin humans was insuffiient, while Walz disusses evidene to suggest that ompensatory growth-promoting pathways may have beome ativated. The development of this type of resistane to targeted therapies is a growing onern, so for this and other reasons, several individuals expressed enthusiasm for the onept of ombinatorial drug approahes. Finally, Grantham revisited the question of how PKD leads to kidney failure and onludes that the yst burden is entral to this proess. Following this argument leads to the logial onlusion that treatment, if it is to be suessful, must begin as early in life as possible. WHAT ARE THE ALLELIC DETERMINANTS OF AUTOSOMAL DOMINANT PKD DISEASE SEVERITY? Peter C. Harris Autosomal dominant PKD (ADPKD) shows onsiderable phenotypi variability in terms of severity of renal disease and the ourrene of extrarenal manifestations (Figure 1). The gene involved, PKD1 (approximately 85% of ases) or PKD2 (approximately 15%), is strongly assoiated with renal disease severity, with ESRD ourring on average approximately 20 years earlier in PKD1 than PKD2 (approximately 58 years versus approximately 79 years). 1 Geneti bakground, along with environmental effets, also influene the phenotype, manifesting as signifiant intrafamilial phenotypi variability. Analysis of ommon single nuleotide polymorphisms 2082 Journal of the Amerian Soiety of Nephrology J Am So Nephrol 26: , 2015

3 SPECIAL ARTICLE Figure 1. Inherited and aquired determinants of ystogenesis. The formation of ysts in ADPKD is viewed as being dependent on the balane between ADPKD gene dosage (and hene protein expression, partiularly of PC1 at the plasma membrane) and the suseptibility of the renal tubule epithelium to its effets, the ystogeni threshold. The gene dosage is dependent on the nature of germline mutations in PKD1 and PKD2, other variants at the disease lous, modifier genes inluding PKHD1, HNF1B, and other iliopathy genes, and somati mutations (the lassi seond hit ), and other fators affeting the expression levels of these genes. 2 The ystogeni threshold seems to be strongly dependent on ell autonomous ontextual fators suh as the developmental stage and superimposition of environmental agents suh as renal injury and infetion, and an also potentially be affeted by modifier genes. Cell nonautonomous loal effets may alter the ystogeni threshold and aount for a snowball effet in yst appearane. In the graph, as gene dosage (solid blue line) dips below the ystogeni threshold (dashed red line), ysts will form (green area). As suh, the interplay between dereasing gene dosage and inreasing ystogeni threshold determines the onset, progression, and severity of ysti disease. (genome-wide assoiation studies) and rare variants (whole exome analysis) is likely to shed light on the importane of speifi modifier variants/genes. There has been a reent fous on alleli effets with several lines of evidene indiatingthat the PKD1 genotype is signifiantly assoiated with renal disease severity, and an explain some extreme phenotypes. Viable patients homozygous or ompound heterozygous for pathogeni PKD1 missense variants indiate that some alleles are inompletely penetrant (hypomorphi); hypomorphi heterozygotes develop very mild ysti disease without ESRD. 3,4 Furthermore, the in trans ombinationofaninativating and hypomorphi PKD1 allele an explain rare, very early onset ADPKD (phenotypially similar to ARPKD). 3,5,6 An in trans ombination of PKD1 hypomorphi alleles an also result in ARPKD-like disease with a negative family history, but without ongenital hepati fibrosis. 4 A mouse model mimiking the best haraterized PKD1 hypomorphi allele, p.r3277c, reapitulates the human phenotypes with slowly progressive disease in homozygotes, generating a good model for prelinial testing, and rapidly progressive disease in ompound heterozygotes with a null allele. 7,8 Detailed analysis of the model indiates that the level of the mature glyoform of the PKD1 protein (polyystin 1; PC1) is assoiated with disease severity, strongly supporting a dosage model of pathogenesis. Consistent with related mehanisms in ysti diseases, a ombination of a PKD1 allele and mutations in a seond ystogene an also result in severe PKD. 5 Analysis of large ADPKD populations indiates that a signifiant proportion of nontrunating mutations are hypomorphi with an average age at ESRD of approximately 67 years in nontrunating mutation patients ompared with 55 years in those with trunating hanges. 1 Moleular sreening in ADPKD an, hene, be of prognosti as well as diagnosti value, and an be employed to identify patients with rapidly progressive disease, suitable for linial trials and future treatments 9 ; although, trunating PKD1 mutation mosais an have mild disease However, a signifiant proportion of nontrunating mutations are fully inativating and so presently the prognosti value of knowing the mutation for the individual patient is limited. Short-term goals inlude determining the strength of PKD1 mutations bioinformatially, from family studies, through in vitro assays and by animal studies to enhane moleular diagnostis/prognostis. Cataloging variants beyond the ADPKD gene that influene the phenotype will also be of prognosti value. In the longer term, evidene that the level of PC1 is related to disease severity provides a potential therapeuti opportunity through modulating the level of funtional PC1, by inreasing the expression level, via targeting mirornas, for instane. 13 Chaperone treatment for missense hanges 14 and nonsense mutation read-through agents should also be onsidered as mutation tailored treatments as we start to apply personalized mediine to ADPKD. 15,16 BREAKING BAD WHAT MAKES GOOD TUBULES TURN CYSTIC? Gregory G. Germino What makes ysts form? We know that genetis plays a key role. Although the disease is inherited as an autosomal dominant trait, numerous lines of evidene indiate that it is reessive on a ellular level, with ysts arising when the total funtional ativity of the two alleles J Am So Nephrol 26: , 2015 Future of PKD Researh 2083

4 of either PKD1 or PKD2 falls below an ill-defined threshold (Figure 1). This most ommonly arises as a result of aquired mutations in renal epithelial ells that have a germline mutation of either lous The nature of the germline mutation likely plays a dominant role in determining the severity of disease. 1 Reent studies suggest that some individuals instead may have two germline, hypomorphi alleles whose ombined ativity falls below the threshold. 3,7 Thereisalsoevidenetosuggestthat the threshold may be different in different ell types and life stages. 7,22 Geneti modifiers are likely important in three steps of this proess: determining the rate of somati mutation, setting the ystogeni threshold, and in downstream effetor pathways. There are several other lines of evidene, however, that suggest other, nongeneti fators may also be important. For example, we have shown that adult mouse kidneys take months to develop ysts after inativation of Pkd1. 23 Metanephri Pkd null kidneys develop ysts in vivo and yet do not when ultured in vitro. 24 We have generated multiple mouse ell lines laking either Pkd1 or Pkd2 and find that they make tubules in three-dimensional ultures regardless of geneti status (L. Menezes, unpublished observation). We have applied network strategies and gene expression analyses to better understand the proesses responsible for yst formation. 25 We ompared gene expression patterns of a test set of mutant (Pkd1 ond/ond Cre + )and ontrol (Pkd1 ond/ond Cre )mieindued at around P7 and harvested at P12 P24 and identified a mutant gene signature that properly lustered an independent validation set of mouse samples by genotype. Comparing the network strutures of P12 and P14 ontrol and mutant samples, we found that they were highly similar, arguing against a developmental blok in ysts. There was, however, a hange in the network arhiteture of P12 versus P14 samples for both ontrols and mutants. The genes in this luster were highly enrihed for metaboli pathways, suggesting that metaboli ontext ould be an important disease modifier. Perturbation of the metaboli state seems to alter disease severity. In summary, geneti fators are a neessary initiating step for ysti disease but other, inompletely defined downstream proesses are required for yst growth. Identifiation of these may offer a unique opportunity for intervention. An agnosti, system-based approah has identified metaboli ontext as one of the proesses that affets yst growth in early-onset PKD. Future studies are required to determine the funtional relationship between PKD proteins and ellular metabolism. It also will be important to determine if the same proesses affet disease in the adult-onset mouse model. The field also would benefit from having a robust ell-based system that faithfully reapitulates the tube yst phenotypi swith aording to genotype. Suh a system ould be used to define immediate effetor pathways downstream of PKD proteins, to link these to other ellular signaling systems, to assess missense PKD gene variants and ultimately to sreen for drugs that an orret the ysti phenotype. HOW DO TISSUE CONTEXTS SUCH AS INJURY AND REPAIR AFFECT CYSTOGENESIS? Dorien J.M. Peters Most patients with ADPKD arry a heterozygous mutation in either the PKD1 or the PKD2 gene. However, the phenotype is also influened by additional geneti and environmental fators (Figure 1). In ADPKD, ysts develop in a minority of nephrons, suggesting that a heterozygous mutation in PKD1 or PKD2 is not suffiient to indue yst formation. In fat, the mutation primes the kidneys for yst formation, but the likelihood of yst formation strongly inreases when the level of funtional PKD1 or PKD2 gene produt drops below a ritial threshold. 3,17,26 A variety of data suggest that the rate of yst formation depends on the ontext of renal tissue. For example, both in mie and in humans, yst growth is muh faster during renal development than it is during adulthood. 23,27,28 It is likely that growth promoting onditions during renal development aelerate the proess. 23,27 Furthermore, the relatively slow proess of yst formation and progression in adult mie an be aelerated by aute renal injury treatments Renal injury is aompanied by a ombination of proesses inluding repair assoiated proliferation, seretion of growth fators, and inflammation. Aelerated yst formation seems to be the result of inreased suseptibility to yst-promoting stimuli, e.g., ytokines or growth fators, due to altered integrity of the ells, rather than the result of unontrolled ell proliferation. 30,32 In addition, the ysts themselves an have a profound impat on the surrounding tissue by induing mehanial stress on neighboring nephrons, whih loally inreases the likelihood of yst formation. 31 In patients, reurrent urinary trat infetions and toxi (waste) produts may ause loal injury. The effets of these may aumulate over years and ontribute to impaired renal funtion and renal aging. Individual variation in exposure to environmental fators that may ause renal injury, as well as renal aging, may ontribute to the variations in severity of renal ysti disease among patients. During renal development, injury/ repair, or normal homeostasis, the PKD proteins modulate a omplex network of signaling pathways (Figure 2) needed to establish and maintain the tubular arhiteture. Espeially during phases in whih the full potential of these pathways is required, the likelihood that this signaling network gets out of balane may inrease, making renal tissue more prone to yst formation, partiularly when the PKD proteins are absent. More insight into these proesses and the exat signaling pathways that underlie the key ellular transitions may reveal interesting targets for therapeuti intervention. Over all, future researh should inlude identifiation of the triggers and unraveling of the exat pathways that 2084 Journal of the Amerian Soiety of Nephrology J Am So Nephrol 26: , 2015

5 SPECIAL ARTICLE Figure 2. Diagram depiting signaling pathways that have been found to be inreased or dereased in PKD, and potential drugs that at on targets in these pathways and that might ameliorate PKD. underlie the key ellular transitions. In addition, obtaining more insight into the stimuli that promote yst growth may reveal interesting targets for therapeuti interventions. WHAT ROLE IS PLAYED BY MODIFIER GENES IN ARPKD AND WHAT ARE THE STRATEGIES TO IDENTIFY THEM? Lisa M. Guay-Woodford ARPKD is a single gene disorder in whih the renal and biliary lesions an be quite variable. This phenotypi variability is typial even among family members who share idential PKHD1 mutations, indiating modulating effets of other geneti (i.e., o-inherited modifier genes or quantitative trait loi [QTL]), epigeneti, or environmental fators (summarized previously 33 ). Efforts to identify QTL in human ARPKD ohorts are onfounded by several fators, most notably: (1) ARPKD is a relatively rare disorder; (2) the PKHD1 gene is transriptionally omplex 34 ;and(3) most patients are ompound heterozygotes for different PKHD1 mutations. 35 Geneti studies in experimental models would be a reasonable alternative, but of the eight urrently available mouse Pkhd1 models, none expresses a renal lesion that phenoopies the human disease. In omparison, the renal and biliary disease in the ongenital polyysti kidney (pk)model losely phenoopies human ARPKD. As an alternative experimental model, the pk mouse has several advantages: (1) it is the most extensively haraterized PKD model; (2) thecys1 pk mutation disrupts the protein ystin, whih is normally expressed in the primary ilium, as is the PKHD1-enoded protein; and (3) in ontrast to Pkhd1, Cys1 is a small, transriptionally unompliated gene. 36 In previous studies, we generated a ohort of F 2 affeted mie, haraterized the renal ysti disease as a series of quantitative traits, and used statistial analyses to identify a highly signifiant QTL omplex on hromosome 4 that exerts a dominant effet of the mus musulus astaneus (CAST) haplotype on renal disease severity. 33 This interval omprises a omplex of at least three QTL (Mpkd1 3), and ontains approximately 1300 genes. We have identified Kif12, a novel kinesin-enoding gene, as the major Mpkd2 andidate using a ombination of gene expression profiling, omputational analyses, single nuleotide polymorphism-based haplotype mapping, and ongeni strain generation. In addition, we have determined that Kif12 oloalizes with ystin in the primary ilium and that its expression pattern is highly orrelated with Cys1 and J Am So Nephrol 26: , 2015 Future of PKD Researh 2085

6 Pkhd1 in the GUDMAP kidney gene expression atlas database ( html). In related studies, we have ollaborated with Greg Germino s group and identified a geneti interation between Pkhd1 and Pkd1 that exaerbates the renal ysti phenotype in the Pkhd1 del3 4 model. 37 Therefore, these mouse studies have identified Kif12 and Pkd1 as putative geneti modifiers of the ARPKD phenotype. In parallel studies we have identified a PKHD1 founder mutation (.T1880A; p.m627k) in a South Afrian Afrikaner ohort. 38 Varied disease expression was doumented among the 27 affeted individuals who were homozygous for this founder mutation. Therefore, this Afrikaner ohort represents a unique, foundational resoure for direted testing of andidate reessive PKD QTL identified in our mouse model studies. Future efforts will need to fous on identifying tagsnps (tag single nuleotide polymorphisms) for KIF12 and PKD1, whih will be resolved into unique, ommon haplotypes to test for assoiation with disease severity in the Afrikaner ohort. In a reiproal strategy, we are using CRISPR tehnology to generate a p.m625k mutation in B6 mie that is orthologous to the human founder mutation. Mutant mie will be phenotypially haraterized. If this humanized mouse model phenoopies ARPKD, then we will generate a mutant F 2 ohort using the newly desribed resoure, Diversity Outbred mie. 39 The Diversity Outbred stok is derived from eight inbred mouse lines and is an ideal resoure for high-resolution geneti mapping, obviating the need for ostly fine-mapping studies. Taken together, these efforts will provide omplementary strategies for identifying ARPKD-related QTL. NEOPLASTIC CYST GROWTH BUT WHY NOT CANCER? James P. Calvet One of the more perplexing questions about polyysti kidneys is the nature of the abnormal ell proliferation required to form the growing yst. There is no doubt that yst growth involves inreased ell proliferation. However, this proess proeeds over months and years, and although perhaps unrelenting, it annot be haraterized as runaway ell growth. However, neither are most anerous tumors, whih probably take many years of growth before they reah a size that an be deteted by sreening. Cyst growth in PKD has many parallels with aner, inluding elevated proto-onogene ativation, Ras/B-Raf/ mitogen-ativated protein kinase, mtor, and EGF reeptor ativation, elevated ell yle ativity, marophage infiltration, elevated ytokines inluding TNF-a,altered basement membrane deposition, inreased integrin-linked kinase ativity andellmigration,elevatedperiostin, inreased angiogenesis, and tumor-like defetive gluose metabolism (Figure 2). 40,41 Uremia assoiated with ESRD an often ause aquired PKD and renal ell arinoma (RCC). Furthermore, there is reent evidene indiating that even less severe CKD an inrease the inidene of RCC and urothelial aner. 42 Yet, in PKD, numerous studies have shown that very little RCC is seen 43 unless the kidneys are removed and examined histologially. 44,45 This very low level of diagnosed RCC in ADPKD suggested that there may be a degree of protetion from aner assoiated with PKD mutations. As suh, we arried out an epidemiologi study to answer this question. 46 Data from the Sientifi Registry of Transplant Reipients, whih ontains information on all solid organ transplant reipients in the United States, were linked to 15 populationbased United States aner registries that inluded data on 50 different aners. Caner inidene was ompared in PKD versus non-pkd renal transplant reipients, and inidene rate ratios adjusted for age, sex, rae/ethniity, dialysis duration, and time sine transplantation were determined. The study inluded.10,000 kidney reipients with PKD and.100,000 without PKD. After multivariable adjustment, overall aner inidene was found to be signifiantly lower in patients with ADPKD by about 16% 17%, suggesting that there is aner protetion assoiated with PKD. 46 This was also found to be true for ARPKD, in a study that examined the inidene of olon aner in unaffeted arriers of a reessive PKHD1 mutation. 47 The reason for the lower aner risk in patients with ADPKD is not known, but may relate to biologi harateristis of ADPKD itself or to better aner risk behaviors with these patients. However, the fat that there was a dereased inidene in ARPKD arriers, who would not know their gene status, makes it more likely that an underlying ellular mehanism is protetive. Suh a mehanism might lie in the unique biology of PKD ells, in whih there is loss of intraellular alium homeostasis and dereased basal alium. 48,49 This lower alium allows AMP ativation of the Ras/B-Raf/mitogen-ativated protein kinase pathway and would ontribute to high levels of -my expression, a harateristi PKD hallmark. We have also shown that lower alium in PKD ells leads to lower levels of Cox-2 expression. How might these observations explain the aner protetive mehanism? The answer may lie in reent studies demonstrating that high My levels 50 and Cox-2 inhibition 51 prevent aner metastasis. As suh, the unusual PKD biology may paradoxially promote neoplasti yst growth while preventing progression to metastasis, leading to lower aner inidene. It will be important in the future to better understand these aner-related signaling mehanisms for the design of PKDspeifi antiproliferative therapies that will slow yst growth and kidney enlargement without inadvertently opening a Pandora s box of metastati aner. If the very therapy that suppresses neoplasti yst growth also allows rogue ells to establish themselves as metastati tumors, the treatment might end up harming the patient. On the other hand, if there is indeed a PKD-related mehanism that protets ells from beoming malignant, 2086 Journal of the Amerian Soiety of Nephrology J Am So Nephrol 26: , 2015

7 SPECIAL ARTICLE suh a disovery should be of signifiant interest to the aner researh ommunity. KNOWING WHAT WE DO NOT KNOW: POLYCYSTIN FUNCTION IN CILIA Stefan Somlo The past deade has seen a remarkable evolution in the understanding of the importane of primary ilia in the pathogenesis of strutural kidney diseases that manifest with ysts and fibrosis. Primary ilia are single nonmotile hairlike projetions on many mammalian ells, inluding most renal tubular epithelial ells where they reside on the apial surfae. They are highly privileged subellular ompartments omposed of a mirotubular saffold with overlying plasma membrane that is devoid of other subellular organelles. All omponent proteins must be synthesized in the ell body and the ilia are able to selet whih of these enter and leave the ilia ompartment and the respetive rates for eah of these proesses. The importane of ilia to the pathogenesis of PKD was brought to the fore by studies that show that mutations affeting ilia struture and omposition produe ysts in mammalian kidney tubules and liver bile duts and that many of the protein produts assoiated with human and mouse fibroysti diseases are expressed in and around the ilia ompartment. 52,53 Most notably, both PC1 and PC2 are expressed in ilia. 54,55 This led to the hypothesis that polyystin funtion is ritial to ilia funtion and that defets in ilia funtion are ritial to the pathogenesis of PKD. Although suh a unifying hypothesis is both appealing and reasonable, ertain features of the respetive ilia mutant and polyystin mutant phenotypes are inonveniently inonsistent. Most notably, loss of ilia resultsinmarkedlylesspronouned polyysti disease than is seen in ADPKD. 56,57 This apparent paradox led us to investigate the geneti interrelationship between inativation of polyystin proteins alone, of ilia alone, and of polyystin proteins and ilia together. 58 We found that, as previously noted, inativation of polyystins alone resulted in severe PKD whereas inativation of ilia alone resulted in mild disease. Surprisingly, simultaneous inativation of polyystins and ilia together resulted in a marked derease in severity of PKD when ompared with polyystin-only inativation. These findings proved universal they were appliable to Pkd1 and Pkd2, toallsegmentsofthe nephron, and to the bile dut and were independent of the timing of gene inativation (developmental or adult). We were able to show that the severity of PKD was diretly related to the period of time for whih intat ilia persisted following loss of polyystins. In aggregate, these data suggest that mehanistially polyystins are inhibitory signals that normally modulate a pathway whih is yet to be identified, but that requires intat ilia to funtion. When this pathway is de-repressed due to loss of polyystins, ADPKD ensues. These findings really should shift our thinking to ask the following two questions. First, why has evolution kept this omplex interplay of polyystin proteins and ilia in plae? In other words, what normal physiologi funtion is important enough to warrant suh a relatively omplex biologi pathway in the kidney? Seond, what is the ilia-dependent ystativating pathway that is normally inhibited by the presene of polyystins? Identifying the moleular omponents of these pathways an be expeted to identify novel and effetive moleular targets for therapy and ADPKD. The geneti evidene strongly suggests that if we an therapeutially repress the ilia-dependent pathway s ativity in the absene of its normal regulatory omponents, the polyystins, then we will be able to target ADPKD in a manner that will be speifitoadpkddiseaseandlikelyeffetive. Identifiation of this ilia-dependent yst-ativating pathway should beome one of the top priorities for ADPKD researh. POLYCYSTIC KIDNEY DISEASE: CILIA AND MORE Jing Zhou Polyystin-1 and PC2 are integral membrane proteins forming a reeptor hannel omplex. 59 Over a deade ago, PC1 andpc2werefoundontheprimary ilia. 54,55,60 Kidney tubular epithelial ells respond to flow shear stress with a alium signal. Cells without funtional PC1orPC2areunabletorespondto flow-indued shear stress with a alium signal. 60,61 This alium signal, visualized by ratiometri alium imaging, is detetable several seonds after flow stimulation and is amplified by a aliumindued alium release mehanism involving a ryanodine reeptor. 61 Using a reently developed alium indiator, the alium signal indued by flow shear stress an now be deteted in the primary ilia. 61,62 This PC2-dependent alium signal preedes an inrease in ytosoli alium level via a ryanodine reeptor, supporting the previous findings. 61,62 This PC1- and PC2-dependent alium signaling may modulate a number of ellular ativities (Figure 2). The iliary ompartment appears to be a separate ompartment for alium signaling. 62,63 Although the iliary funtions of PC1 and PC2 are likely not restrited to alium signaling, studies defining the loal as well as global impat of the alium signal mediated by iliary PC1 and PC2 will be instrutive in understanding polyystin funtion. ADPKD is now known as a iliopathy. Ciliopathies refer to a group of diseases aused by strutural or funtional defets of the primary ilia. Ciliopathies have a wide range ofphenotypes with ysti kidneys as a ommon feature. What are the proximal events of iliary polyystins? We searhed for polyystin interation partners. We found that PC2 interats with the ARPKD protein, fibroystin/polydutin, 64 and nek8 (namely nephronophthisis 9). 65 Reently we found that PC1 interats with several Bardet Biedl syndrome (BBS) proteins that are part of the BBSome protein omplex known to funtion in the transport of a set of proteins to the ilia. 66 Defiieny of BBS1 and BBS3 affets the iliary traffiking of PC1. Hene, the J Am So Nephrol 26: , 2015 Future of PKD Researh 2087

8 polyystins are argos of the BBSome and physial interations between the polyystins and BBS proteins may underlie the overlapping renal phenotypes in these two diseases. Other omponents of the traffiking pathways may well be important for polyystin targeting to the ilia. Polyystins are present at other subellular ompartments besides the primary ilia. 59 We reently found that PC1 is present in the lamellipodia in migrating kidney tubular epithelial ells. 67 binds to an F-bar protein Pasin 2 and regulates Pasin 2 interation with N- Wasp, an ativator of the atin nuleator Arp2/3 protein omplex. We found that disorganization of the atin ytoskeleton is a feature of PKD in vivo and that a novel PC1-Pasin 2-N-Wasp omplex is required for the atin remodeling and diretional ell migration. 67 Diretional ell migration is essential for the regeneration and maintenane of the epithelium and kidney development. 68 We propose that a defetive atin ytoskeleton and diretional ell migration ontributes yst formation. Further understanding the traffiking route and identifiation of modulators promoting polyystin targeting to the ilia may provide new therapeutis for patients with traffiking defetive mutations. In addition, studies to eluidate the role of the ytoskeleton and ell migration in yst formation may identify novel pathogeni mehanisms and therapeuti targets. DNA DAMAGE RESPONSE SIGNALING IS A NOVEL PATHOGENIC MECHANISM IN CILIOPATHIES Friedhelm Hildebrandt Nephronophthisis-related iliopathies (NPHP-RC) are reessive multisystem disorders that affet kidney, retina, liver, and erebellum either by prenatal-onset dysplasia or by hildhood-onset degeneration and fibrosis. Identifiation of.19 disease-ausing genes (NPHP1 to NPHP19) revealed that their produts are loated at primary ilia and entrosomes. However, the proximal disease mehanisms remain poorly understood. We identified, by whole exome resequening, mutations that affet the entrosomal proteins, namely, FAN1, MRE11, ZNF423, and CEP164, as novel auses of NPHP-RC. Surprisingly, these iliopathy genes serve funtions within the DNA damage response (DDR) pathway. 69 (1) ZNF423 interats with the DNA dsbreak sensor PARP1, whih reruits MRE11 (MRN) and ATM to sites of DNA damage. ATM, in turn, is ativated by MRE11 (MRN) and the TIP60 omplex. We demonstrate oloalization to TIP60-positive (and SC35-positive) nulear foi or protein protein interations for the following produts of genes mutated in NPHP-RC: SDCCAG8/NPHP10, ZNF423, CEP164, OFD1, RUVBL1, RUVBL2, NPHP5, NPHP1, and ATXN10. OFD1, RUVBL1, RUVBL2, are known to play a role in DDR. (2) In addition, in four different families with NPHP-RC, we identify reessive mutations of CEP164 as a novel ause of NPHP-RC. CEP164 ats in the ATR-Chk1-related arm of DDR, where it is neessary for ATR-dependent Chk1 ativation upon indued repliation stress. 70 Furthermore, to identify single-gene auses of renal fibrosis/ckd we performed homozygosity mapping and whole exome resequening in a model disorder for renal fibrosis known as karyomegali interstitial nephritis (KIN). 71 We identified reessive mutations of the Fanoni anemia-assoiated nulease 1gene(FAN1) as ausing the NPHP-like renal phenotype of karyomegali interstitial nephritis (KIN) in nine of 10 families asertained. KIN auses CKD with renal histology indistinguishable from NPHP, exept for the presene of karyomegaly. FAN1 has nulease ativity and ats in DNA interstrand rosslinking (ICL) repair within the Fanoni anemia (FA) pathway of DNA damage response (DDR). Interestingly, ICL-ausing genotoxins generate a KIN-like phenotype. We demonstrate that ells from individuals with FAN1 mutations exhibit sensitivity to the ICL agent mitomyin C. We omplement ICL sensitivity with wild-type FAN1 but not mutant DNA from individuals with KIN. The FAN1 defet was not epistati with the Fanoni anemia pathway. By depletion of fan1 in zebrafish we reapitulated inreased DDR, apoptosis, and kidney ysts akin to NPHP. 72 We suggest a working hypothesis for the pathogenesis of ertain forms of NPHP-RC proposing the following asade of events: defets of DDR lead to a lak of Chk1 (Chk2) ativation, thereby ausing inadequate G 2 /M ell yle arrest. This would lead in high proliferation states (high repliation stress) during morphogenesis to dysplasti phenotypes (Mekel syndrome) and in low proliferation states (low repliation stress) during tissue maintenane and repair to tissue degeneration and fibrosis (nephronophthisis). It is beoming inreasingly likely that disruption of ell yle regulation is entral to most forms of renal iliopathies. This mehanism an be targeted by drugs, as has been shown in models of renal ysti disease using rosovitine and other drugs that interfere with ell yle regulation. 73 INTRAFLAGELLAR TRANSPORT PROTEIN FUNCTION IS NOT RESTRICTED TO THE PRIMARY CILIUM Corinne Antigna Virtually all epithelial ells display primary ilia. However, whereas in immature rat glomeruli, podoytes express ilia, these tend to disappear during development. 74 Hene, our identifiation of a missense mutation (p.p209l) in the TTC21B gene enoding the intraflagellar transport (IFT) 139 protein in seven families with hereditary foal-segmental glomeruloslerosis, disovered during late adolesene or early adulthood, raised the question of a role of IFT139 (and potentially other IFT proteins) beyond the 2088 Journal of the Amerian Soiety of Nephrology J Am So Nephrol 26: , 2015

9 SPECIAL ARTICLE primary ilium. 75 Mutations in TTC21B had previously been reported in patients with nephronophthisis, 76 but areful reanalysis of the linial and histologi features of all patients bearing the p.p209l mutation learly showed that they presentwithbothglomerularandtubulointerstitial involvement. In agreement, we found that IFT139 was predominantly expressed in distal tubules, as expeted for a nephronophthisis-ausing gene, but was also strongly expressed in glomerular podoytes. IFT139 was mainly loalized at the base of the primary ilium in developing podoytes from human fetal tissue and in undifferentiated ultured podoytes. In ontrast, in noniliated adult podoytes and differentiated ultured ells, IFT139 reloalized along the extended mirotubule network. Our funtional studies in undifferentiated podoytes showed that the p.p209l mutation has a hypomorphi effet on podoyte iliogenesis, but was not suffiient to inhibit podoyte differentiation. These data suggested that the glomerular defets observed in patients with this mutation were, rather, due to a noniliary alteration of IFT139 in mature podoytes, unovered by the hypomorphi p.p209l mutation. As atin and mirotubule ytoskeletons are key regulators of the deliate arhiteture, plastiity, and ontratility of podoytes, 77 andasift139isredistributedalong the mirotubule network in mature podoytes, we studied the effet of the p.p209l mutant on ytoskeleton organization in differentiated podoytes. Interestingly, the depletion of IFT139 led to inreased alterations to the ell surfae assoiated with atin ytoskeleton, suh as short and misorganized stress fibers and mirotubule rearrangement into bike-wheel-like shape. All these defets were fully resued by the wild-type protein. In ontrast, p.p209l re-expression led to the resue of the ell-size defet, but atin and mirotubule networks remained severely altered. In addition, mirotubule repolymerization after noodazole treatment resulted in multiple nuleation sites dispersed within the ytoplasm in IFT139-depleted differentiated podoytes and these abnormalities were partially resued upon p.p209 mutant expression. These data show one again the power of geneti studies in families affeted with rare Mendelian disorders. Here, they allow the unovering of an unexpeted role of a iliary protein in the regulation of podoyte ytoskeleton arhiteture, and open a new field of investigations into the role of IFT proteins in the mature podoyte, a ell devoid, at this stage, of a primary ilium. We antiipate that future geneti studies in new families with iliary syndromes will ontinue the disovery of new gene mutations and be a rih soure for unovering new biology. SIGNALING PATHWAYS AND THERAPEUTIC MOLECULES IN PKD Viente E. Torres Understanding the pathogenesis of inreased fluid seretion and epithelial ell proliferation downstream from PKD1 or PKD2 mutations is important to identify therapies (Figure 2). 40,41 Those targeting proximal mehanisms are more likely to be effetive than those targeting distal mehanisms. Aberrant ross-talk between intraellular alium and AMP signaling likely is one of the first effets of PKD mutations. Disrupted alium may enhane AMP and protein kinase A signaling through ativation of alium-inhibitable adenylyl ylases and inhibition of alium-dependent phosphodiesterases (PDE1 and indiretly GMP-inhibited PDE3). 78 Enhaned protein kinase A ativity may in turn disrupt intraellular alium homeostasis through hyperphosphorylation of alium yling proteins in the endoplasmi retiulum. Prelinial studies have targeted alium signaling with some suess. Only triptolide (an ativator of the polyystin-2 hannel) is being linially investigated. Gas proteinoupled reeptor antagonists (i.e., vasopressin V2 reeptor antagonists) and Gai protein-oupled reeptor agonists (i.e., somatostatin analogs) have demonstrated enouraging results in prelinial and linial trials. Protein kinase A-indued phosphorylation of ysti fibrosis transmembrane ondutane regulator (CFTR) allows hloride and fluid seretion into the ysts. Anotamin-1, a alium-ativated hloride hannel, may synergistially interat with CFTR. 79 CFTR inhibitors inhibit ystogenesis in vitro and in kidney-speifi Pkd1 knokout mie, whereas anotamin-1 inhibitors have been effetive in vitro. Protein kinase A ativation inhibits ell proliferation in wild-type ells, but has a stimulatory effet in PKD ells. Calium deprivation in wild-type ells and delivery of alium in PKD ells reverse these effets. A proposed mehanism for the proliferative response in PKD and alium-deprived wild-type ells is inhibition of phosphoinositide 3-kinase and AKT releasing B-Raf from AKT inhibition. 49 This leads to dysregulation of signaling pathways (B-Raf/ MEK/ extraellular signal-regulated kinase [ERK]; AMP-ativated protein kinase [AMPK]/mTOR; arguably Wnt/ b-atenin) and transription fators (hypoxia-induible fator 1, My, P53, signal transduer and ativator of transription 3) that ontrol ell yle progression, energy metabolism, and the miroenvironment. Overexpression of growth fators, ytokines, hemokines, and their reeptors further ontributes to disease progression. Sr is a nonreeptor tyrosine kinase ativated downstream from G-protein oupled and growth fator reeptors. SKI-606 (bosutinib) is an Sr/Abl inhibitor. KD019 inhibits Sr and reeptor tyrosine kinases (EGF reeptor, ERBB2, vasular endothelial growth fator reeptor). Both have shown benefit in prelinial studies. Clinial trials are ongoing. On the other hand, targeting of B-Raf/MEK/ERK has given inonsistent results. Overwhelming evidene indiates that mtor signaling is enhaned in ysti tissues. Prelinial studies of rapalogs and mtor antisense oligonuleotides 80 have been enouraging. Clinial trials have been disappointing likely beause linially ahievable blood levels do not inhibit mtor in the kidney. J Am So Nephrol 26: , 2015 Future of PKD Researh 2089

10 Strategies to overome systemi toxiity and limited renal bioavailability are being investigated. Pan-sirtuin (niotinamide) and sirtuin-1 speifi inhibitors, AMPK ativators (metformin), thiazolidinediones, and signal transduer and ativator of transription 3 inhibitors have been effetive in animal models. 84 Niotinamide is being tested in an unontrolled, open label linial trial. Reent studies suggest that PKD ells, like aner ells, reprogram their energy metabolism from oxidative phosphorylation to aerobi glyolysis. 85 Whether drugs interfering withglyolysiswillbesafeandeffetive linially is unertain. Signifiant progress has been made toward identifiation of effetive therapies for PKD. As disussed above, many have been shown to be effetive in rodent models, but their safety and effiay in patients is diffiult to predit with auray, and only rigorous linial trials will answer this satisfatorily. One approah that should be onsidered is the use of ombination therapies, whih an potentially inrease effiay and redue toxiity. 8,86 THE ROLE OF MTOR SIGNALING IN CYSTIC KIDNEY DISEASE Gerd Walz The mtorc1 kinase asade is almost universally ativated in ysti kidney disease independent of the underlying disease-ausing gene mutation 87,88 (Figure 2). Although the moleular basis for this ativation remains inompletely understood, primary ilia appear to urtail mtorc1 ativity through a flowdependent ativation of the LKB1/AMPK signaling pathway, involving stabilization of the tuberous slerosis omplex 1 (TSC1)/TSC2 omplex and inhibition of Rheb. 89 In addition, PC1 might influene the ativity of this small GTPase through interation with the TSC omplex, 90,91 and inhibit MEK/ERK-mediated phosphorylation of TSC2 to ontrol mtorc1 ativity, 92 whereas -Cbl is sequestered in the Golgi apparatus in the absene of polyystin-1, allowing the HGF reeptor -Met to esape degradation after stimulation. 93 First showninthe Cy/+ ratmodel, 94 the effiay of mtorc1 inhibitors to redue yst growth and to ameliorate disease progression was subsequently demonstrated in several animal models of ysti kidney disease, inluding Pkd1-defiient mie. 90, Despite these enouraging prelinial data, mtor inhibitors did not yield the expeted benefits in patients with ADPKD. While everolimus slowed yst growth in ADPKD patients, this inhibition did not translate into an improvement of renal funtion. However, other pilot studies yielded more promising results, 101 leaving open the role of mtor inhibition as an ADPKD target. 102,103 To determine the involvement of mtorc1 in yst growth and ADPKD disease progression, we abrogated iliogenesisinthethikasendinglimbofhenleand distal tubular segments, generating Kif3a fl/fl*kspcre mie. These mie rapidly developed ysti kidney disease, and died before 12 weeks of age (median survival 7.3 weeks). In ontrast, the additional elimination of Raptor (Raptor fl/fl*kif3a fl/ fl*kspcre) delayed yst growth and dramatially inreased the survival by more than three times (median survival 24.0 weeks), revealing the importane of mtorc1 in promoting yst growth. However, despite the remarkable delay, the Raptor-defiient mie nevertheless developed ysti kidney disease. Gene expression profiling revealed that yst formation anddiseaseprogressionintheabseneof funtional mtorc1 were driven by other growth-promoting pathways, inluding AKT and ERK. The rapid development of mtorc1- independent yst growth in ADPKD resembles the resistane against targeted tumor therapies, where de novo somati mutations or bypass signaling pathways permit tumor ell proliferation despite effetive inhibition of the original onogeni driver. Reminisent of the neoplasia in disguise oined many years ago to apture the tumor-like harateristis of ysts in ADPKD, 104 amulti-targetapproah will likely replae the searh for a single magi bullet. Several novel onepts that blok ell yle progression and inflammation, or promote apoptosis and epigeneti hanges, have been suessfully tested in animal models of ysti kidney disease, holding great promise for future linial trials. TARGETING CYSTS TO PREVENT RENAL INSUFFICIENCY IN PKD Jared J. Grantham Cysts originate in tubules and are distinguished from simple tubule dilations by having inreased numbers of ells expanding the wall beyond normal boundaries. Cysts are the primary suspets ausing the deline of GFR (Figure 3). This is supported by the following findings: (1) yst enlargement ompresses the interstitial matrix and surrounding tubules entraining, distorting, and destroying arterioles, venules, apillaries, and lymphatis; (2) most ysts separate from the tubules from whih they formed; (3) an isolated olleting dut yst an blok urine flow in the branhing arade upstream of it that would ordinarily drain hundreds of filtering nephrons 105,106 ;(4) the glomerulo tubule juntions of bloked nephrons beome thinned and eventually the tubules separate reating atubular glomeruli and apoptoti proximal tubules ;(5) ysts lead to hypertension and impair urine onentrating apaity before GFR delines 110 ;(6) morphologi studies of human and animal kidneys reveal that ysts an form in the absene of interstitial pathology 105 ;(7) in ADPKD, ortial fibrosis in the absene of ysts nearby is likely seondary to the downstream blokade of olleting duts 109 ;(8)aseminal study in PKHD1-null rats unable to synthesize vasopressin proved that fibrosis and renal insuffiieny do not develop in the absene of yst formation, but do when the hormone is diretly administered ausing ysts to form. 111 What auses renal failure in PKD? The yst did it! Future therapy should fous on treatment in early hildhood. In most heritable ysti 2090 Journal of the Amerian Soiety of Nephrology J Am So Nephrol 26: , 2015