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

†‡ | Corinne Antignac,* James P. Calvet,§ Gregory G. Germino, Jared J. Grantham,§ †† ‡‡ Lisa M. Guay-Woodford,¶ Peter C. Harris,** Friedhelm Hildebrandt, Dorien J.M. Peters, || Stefan Somlo,§§ Vicente E. Torres,** Gerd Walz, Jing Zhou,¶¶ and Alan S.L. Yu§

*National Institute of Health and Medical Research, Laboratory of Inherited Kidney Diseases, Paris Descartes-Sorbonne Paris Cité University, †Imagine Institute, and ‡The Department of Genetics, Necker Hospital, Paris, France; §The Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas; |Kidney Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; ¶Center for Translational Science, Children’s National Health System, Washington, DC; **Division of Nephrology and Hypertension, Mayo Clinic, Rochester, Minnesota; ††Howard Hughes Medical Institute and Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts; ‡‡Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands; §§Departments of Internal Medicine and Genetics, Yale University School of Medicine, New Haven, Connecticut; ||Renal Division, Department of Medicine, University Medical Center Freiburg, Freiburg, Germany; and ¶¶Harvard Center for Polycystic Kidney Disease Research, Renal Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts

ABSTRACT Polycystic kidney disease (PKD) is one of the most common life-threatening genetic Joseph Bruening, Dr. Grantham foun- diseases. Jared J. Grantham, M.D., has done more than any other individual to ded in 1982 the Polycystic Kidney Dis- promote PKD research around the world. However, despite decades of investiga- ease Foundation, which today is the tion there is still no approved therapy for PKD in the United States. In May 2014, the leading organization worldwide sup- University of Kansas Medical Center hosted a symposium in Kansas City honoring porting research and education about the occasion of Dr. Grantham’s retirement and invited all the awardees of the Lillian the disease. Dr. Grantham, a die-hard Jean Kaplan International Prize for Advancement in the Understanding of Polycystic native Kansan, directed the Division of Kidney Disease to participate in a forward-thinking and interactive forum focused on Nephrology at Kansas for 25 years, was future directions and innovations in PKD research. This article summarizes the con- co-founder of the Kidney Institute tributions of the 12 Kaplan awardees and their vision for the future of PKD research. which he directed for 5 years, and was founding editor of the Journal of the J Am Soc Nephrol 26: 2081–2095, 2015. doi: 10.1681/ASN.2014121192 American Society of Nephrology.Without question, Jared Grantham has been a towering figure in PKD research and fi In 2013, Jared J. Grantham, M.D. retired asked to lead the Nephrology Division at has done more for this eld than any from the University of Kansas Medical the KUMC and discovered in his labora- other single individual. ’ Center (KUMC) after a long and distin- tory new renal functions in tubule fluid In Dr. Grantham s honor, the Kidney guished career devoted to the search for a secretion that opened the door to inno- Institute at KUMC hosted the Jared J. “ cure for polycystic kidney disease (PKD). vative research on PKD. Dr. Grantham Grantham Symposium, The Future of Dr. Grantham’s interest in kidney disease was a member of the National Institutes developed in the sixth grade in Johnson, of Health (NIH) research team that de- Kansas while trading comic books with veloped the method to dissect and per- Published online ahead of print. Publication date his neighbor. His neighbor casually fuse isolated segments of renal tubules. available at www.jasn.org. mentioned that he had cysts in his kid- His work emphasized the mechanisms of Correspondence: Dr.AlanS.L.YuorDr.JamesP. neys, his mother had them and his action of vasopressin on collecting Calvet, The Kidney Institute, WHE 6018, University of Kansas Medical Center, 3901 Rainbow Boule- grandmother had recently died of kidney ducts, a theme that was carried forward vard, Kansas City, KS 66160. Email: [email protected] failure. That remained a vivid memory to his work on PKD, which was contin- or [email protected] for Dr. Grantham and he reconnected uously funded by the NIH until his re- Copyright © 2015 by the American Society of with kidney cysts in 1970 when he was tirement. With Kansas City businessman Nephrology

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Polycystic Kidney Disease Research” on common themes emerged. A number brandt). Hildebrandt and Antignac both May 7–9, 2014. All recipients of the of factors, such as allelic effects of PKD makeastrongcaseforcontinuedeffortsto Lillian Jean Kaplan Prize were invited genes (Harris) and the function of ciliary identify novel cystic disease genes as a rich to Kansas City to lead a forward-thinking trafficking proteins (Zhou), converge to source for uncovering new biology. and interactive forum focused on future determine the dosage of PKD1 protein With the identification of so many directions and innovations in PKD re- that is delivered to the cell and ciliary signaling pathways, cystic diseases are search. The Lillian Jean Kaplan Interna- surface, which is seen as playing a critical ripeforthedevelopmentofpharmacologic tional Prize for the Advancement in the role in determining disease severity. Mod- therapies. Torres reviews the therapeutic Understanding of Polycystic Kidney Dis- ifier genes have long been suspected of landscape and current and future pros- ease was established by the PKD Founda- influencing the severity and natural pects. One of the more promising of these tion and the International Society of history of the disease. Guay-Woodford is the mammalian target of rapamycin Nephrology (ISN) through the generos- illustrates how this can be effectively in- (mTOR) pathway, yet recent clinical trials ity of the family of Thomas and Dafna vestigated in autosomal recessive PKD of mTOR inhibitors have failed. Torres Kaplan, in honor of Mr. Kaplan’slate (ARPKD) by using mouse quantitative arguesthat the dosagethat couldbeusedin mother, Lillian Jean Kaplan, who died trait loci mapping and analogous studies humans was insufficient, while Walz dis- of PKD. The prize was created to stimu- in a unique, genetically isolated, human cusses evidence to suggest that compen- late interest in advancing PKD research population. satory growth-promoting pathways and to recognize those who have in- Much recent evidence suggests that may have become activated. The develop- creased our understanding and treat- the cell and tissue context within which ment of this type of resistance to targeted ment of PKD through basic or clinical PKD mutations occur is a keydeterminant therapies is a growing concern, so for this scientific research, leading to new treat- of the disease course. The developmental and other reasons, several individuals ments and a cure for PKD. The Prize is stage in early life, or superimposed renal expressed enthusiasm for the concept of awarded every 2 years at the ISN’s World injury in adulthood are well-established combinatorial drug approaches. Finally, Congress of Nephrology and so far there contextual factors. To identify other con- Grantham revisited the question of how have been 13 awardees. Vincent Gattone, textual elements, Germino used an agnos- PKD leads to kidney failure and concludes Ph.D., who was awarded the prize in tic transcriptomic method and showed that the cyst burden is central to this 2013, unfortunately passed away on Jan- that the metabolic pathways associated process. Following this argument leads to uary 26, 2014. He was highly respected in with developmental age are important. the logical conclusion that treatment, if it the PKD field and had a major impact. Peters argues that local paracrine or me- is to be successful, must begin as early in Among his many achievements, he de- chanical signals may also play a role and life as possible. veloped the concept of using vasopressin that this perhaps resembles more the antagonists to treat PKD and carried out mechanism of human disease. the initial testing in rodent models of Although the many culprit genes in WHAT ARE THE ALLELIC cystic disease that eventually led to clin- cystic diseases share a common locali- DETERMINANTS OF AUTOSOMAL ical trials of tolvaptan. zation to the cilia, the true role of this DOMINANT PKD DISEASE The Grantham Symposium was a huge organelle remains poorly understood. SEVERITY? success. All 12 living Kaplan awardees Somlo discusses observations that sup- were able to participate, along with nearly port the radical assertion that signals Peter C. Harris 200 attendees who came from all over the emanating from the intact cilia actually Autosomal dominant PKD (ADPKD) world to celebrate Dr. Grantham’s career promote cystogenesis. The idea that cystic shows considerable phenotypic variabil- and participate in shaping the future of disease is caused by abnormal epithelial ity in terms of severity of renal disease PKD research. The many outstanding cell proliferation and constitutes a neo- and the occurrence of extrarenal mani- talks and discussion forums stimulated plastic process is well accepted, but Calvet festations (Figure 1). The gene involved, considerable lively debate and exchange provides epidemiologic data suggesting PKD1 (approximately 85% of cases) or of ideas. These shed much-needed light that patients with PKD may actually be PKD2 (approximately 15%), is strongly on the most important directions that protected from cancer, raising intriguing associated with renal disease severity, now need to be pursued to advance the questions about how the proliferation of with ESRD occurring on average ap- field and move effective treatments for cyst epithelial cells is regulated. Novel proximately 20 years earlier in PKD1 this disease as quickly as possible into downstream cyst-promoting mecha- than PKD2 (approximately 58 years ver- the clinic. nisms that have recently been uncovered sus approximately 79 years).1 Genetic In this article, each Kaplan awardee include the role of the actin cytoskeleton background, along with environmental was invited to contribute a synopsis of and directional cell motility (Zhou) and effects, also influence the phenotype, his or her talk, focusing on their vision the role of DNA damage repair in the reg- manifesting as significant intrafamilial for the future. From the juxtaposition of ulation of cell cycle checkpoint genes and phenotypic variability. Analysis of com- their talks and these reviews, several hence epithelial cell proliferation (Hilde- mon single nucleotide polymorphisms

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in a second cystogene can also result in severe PKD.5 Analysis of large ADPKD populations indicates that a significant proportion of nontruncating mutations are hypo- morphic with an average age at ESRD of approximately 67 years in nontruncating mutation patients compared with 55 years in those with truncating changes.1 Molecular screening in ADPKD can, hence, be of prognostic as well as diag- nostic value, and can be employed to identify patients with rapidly progressive disease, suitable for clinical trials and fu- ture treatments9; although, truncating PKD1 mutation mosaics can have mild disease.10–12 However, a significant pro- portion of nontruncating mutations are fully inactivating and so presently the prog- Figure 1. Inherited and acquired determinants of cystogenesis. The formation of cysts in ADPKD is viewed as being dependent on the balance between ADPKD gene dosage (and nostic value of knowing the mutation for hence protein expression, particularly of PC1 at the plasma membrane) and the suscep- the individual patient is limited. tibility of the renal tubule epithelium to its effects, the “cystogenic threshold.” The gene dosage is dependent on the nature of germline mutations in PKD1 and PKD2,other Future Directions variants at the disease locus, modifier genes including PKHD1, HNF1B, and other Short-term goals include determining the ciliopathy genes, and somatic mutations (the classic “second hit”), and other factors af- strength of PKD1 mutations bioinformat- fecting the expression levels of these genes.2 The cystogenic threshold seems to be ically, from family studies, through in vitro strongly dependent on cell autonomous contextual factors such as the developmental assays and by animal studies to enhance stage and superimposition of environmental agents such as renal injury and infection, and molecular diagnostics/prognostics. Cata- fi can also potentially be affected by modi er genes. Cell nonautonomous local effects may loging variants beyond the ADPKD gene alter the cystogenic threshold and account for a “snowball effect” in cyst appearance. In that influence the phenotype will also be the graph, as gene dosage (solid blue line) dips below the cystogenic threshold (dashed red line), cysts will form (green area). As such, the interplay between decreasing gene of prognostic value. In the longer term, dosage and increasing cystogenic threshold determines the onset, progression, and se- evidence that the level of PC1 is related verity of cystic disease. to disease severity provides a potential therapeutic opportunity through modu- lating the level of functional PC1, by in- (genome-wide association studies) and in trans combination of PKD1 hypomorphic creasing the expression level, via targeting rare variants (whole exome analysis) is alleles can also result in ARPKD-like dis- microRNAs, for instance.13 Chaperone likely to shed light on the importance of ease with a negative family history, but treatment for missense changes14 and specificmodifier variants/genes. without congenital hepatic fibrosis.4 nonsense mutation read-through agents There has been a recent focus on allelic A mouse model mimicking the best should also be considered as mutation tai- effects with several lines of evidence characterized PKD1 hypomorphic allele, lored treatments as we start to apply per- indicating that the PKD1 genotype is sig- p.R3277C, recapitulates the human phe- sonalized medicine to ADPKD.15,16 nificantly associated with renal disease notypes with slowly progressive disease severity, and can explain some extreme in homozygotes, generating a good phenotypes. Viable patients homozy- model for preclinical testing, and rapid- BREAKING BAD—WHAT MAKES gous or compound heterozygous for ly progressive disease in compound GOOD TUBULES TURN CYSTIC? pathogenic PKD1 missense variants in- heterozygotes with a null allele.7,8 De- dicate that some alleles are incompletely tailed analysis of the model indicates Gregory G. Germino penetrant (hypomorphic); hypomorphic that the level of the mature glycoform What makes cysts form? We know that heterozygotes develop very mild cystic of the PKD1 protein (polycystin 1; genetics plays a key role. Although the disease without ESRD.3,4 Furthermore, PC1) is associated with disease severity, disease is inherited as an autosomal the in trans combinationofaninacti- strongly supporting a dosage model of dominant trait, numerous lines of evi- vating and hypomorphic PKD1 allele pathogenesis. Consistent with related dence indicate that it is recessive on a can explain rare, very early onset ADPKD mechanisms in cystic diseases, a combi- cellular level, with cysts arising when the (phenotypically similar to ARPKD).3,5,6 An nation of a PKD1 allele and mutations total functional activity of the two alleles

J Am Soc Nephrol 26: 2081–2095, 2015 Future of PKD Research 2083 SPECIAL ARTICLE www.jasn.org of either PKD1 or PKD2 falls below an important disease modifier. Perturba- example, both in mice and in humans, ill-defined threshold (Figure 1). This tion of the metabolic state seems to alter cyst growth is much faster during renal most commonly arises as a result of ac- disease severity. development than it is during adult- quired mutations in renal epithelial cells hood.23,27,28 It is likely that growth that have a germline mutation of either Future Directions promoting conditions during renal devel- locus.17–21 The nature of the germline In summary, genetic factors are a neces- opment accelerate the process.23,27 Fur- mutation likely plays a dominant role sary initiating step for cystic disease but thermore, the relatively slow process of in determining the severity of disease.1 other, incompletely defined downstream cyst formation and progression in adult Recent studies suggest that some indi- processes are required for cyst growth. mice can be accelerated by acute renal in- viduals instead may have two germline, Identification of these may offer a unique jury treatments.29–31 Renal injury is ac- hypomorphic alleles whose combined opportunity for intervention. An agnos- companied by a combination of processes activity falls below the threshold.3,7 tic, system-based approach has identified including repair associated proliferation, Thereisalsoevidencetosuggestthat metabolic context as one of the processes secretion of growth factors, and inflam- the threshold may be different in differ- that affects cyst growth in early-onset mation. Accelerated cyst formation seems ent cell types and life stages.7,22 Genetic PKD. Future studies are required to to be the result of increased susceptibility modifiers are likely important in three determine the functional relationship to cyst-promoting stimuli, e.g., cytokines steps of this process: determining the between PKD proteins and cellular me- or growth factors, due to altered integrity rate of somatic mutation, setting the tabolism. It also will be important to of the cells, rather than the result of cystogenic threshold, and in down- determine if the same processes affect uncontrolled cell proliferation.30,32 In ad- stream effector pathways. disease in the adult-onset mouse model. dition, the cysts themselves can have a There are several other lines of evi- The field also would benefit from having a profound impact on the surrounding tissue dence, however, that suggest other, non- robust cell-based system that faithfully by inducing mechanical stress on neigh- genetic factors may also be important. recapitulates the tube–cyst phenotypic boring nephrons, which locally increases For example, we have shown that adult switch according to genotype. Such a sys- the likelihood of cyst formation.31 mouse kidneys take months to develop tem could be used to define immediate In patients, recurrent urinary tract cysts after inactivation of Pkd1.23 Meta- effector pathways downstream of PKD infections and toxic (waste) products nephric Pkd null kidneys develop cysts proteins, to link these to other cellular may cause local injury. The effects of these in vivo and yet do not when cultured in signaling systems, to assess missense may accumulate over years and contribute vitro.24 We have generated multiple PKD gene variants and ultimately to to impaired renal function and renal ag- mouse cell lines lacking either Pkd1 or screen for drugs that can correct the cys- ing. Individual variation in exposure to Pkd2 and find that they make tubules in tic phenotype. environmental factors that may cause three-dimensional cultures regardless of renal injury, as well as renal aging, may genetic status (L. Menezes, unpublished contribute to the variations in severity of observation). We have applied network HOW DO TISSUE CONTEXTS SUCH renal cystic disease among patients. strategies and gene expression analyses AS INJURY AND REPAIR AFFECT During renal development, injury/ to better understand the processes re- CYSTOGENESIS? repair, or normal homeostasis, the PKD sponsible for cyst formation.25 We com- proteins modulate a complex network of pared gene expression patterns of a test Dorien J.M. Peters signaling pathways (Figure 2) needed to set of mutant (Pkd1cond/cond Cre+)and Most patients with ADPKD carry a het- establish and maintain the tubular archi- – control (Pkd1cond/cond Cre )micein- erozygous mutation in either the PKD1 or tecture. Especially during phases in which duced at around P7 and harvested at the PKD2 gene. However, the phenotype the full potential of these pathways is re- P12–P24 and identified a mutant gene is also influenced by additional genetic quired, the likelihood that this signaling signature that properly clustered an and environmental factors (Figure 1). network gets out of balance may increase, independent validation set of mouse In ADPKD, cysts develop in a minor- making renal tissue more prone to cyst samples by genotype. Comparing the ity of nephrons, suggesting that a het- formation, particularly when the PKD network structures of P12 and P14 con- erozygous mutation in PKD1 or PKD2 is proteins are absent. trol and mutant samples, we found that not sufficient to induce cyst formation. they were highly similar, arguing In fact, the mutation primes the kidneys Future Directions against a developmental block in cysts. for cyst formation, but the likelihood of More insight into these processes and the There was, however, a change in the net- cyst formation strongly increases when exact signaling pathways that underlie work architecture of P12 versus P14 the level of functional PKD1 or PKD2 the key cellular transitions may reveal samples for both controls and mutants. gene product drops below a critical interesting targets for therapeutic inter- The genes in this cluster were highly en- threshold.3,17,26 A variety of data suggest vention. Over all, future research should riched for metabolic pathways, suggesting that the rate of cyst formation depends include identification of the triggers and that metabolic context could be an on the context of renal tissue. For unraveling of the exact pathways that

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Figure 2. Diagram depicting signaling pathways that have been found to be increased or decreased in PKD, and potential drugs that act on targets in these pathways and that might ameliorate PKD. underlie the key cellular transitions. In Efforts to identify QTL in human in contrast to Pkhd1, Cys1 is a small, addition, obtaining more insight into the ARPKD cohorts are confounded by sev- transcriptionally uncomplicated gene.36 stimuli that promote cyst growth may eral factors, most notably: (1)ARPKD In previous studies, we generated a reveal interesting targets for therapeutic is a relatively rare disorder; (2)the cohort of F2 affected mice, characterized interventions. PKHD1 gene is transcriptionally com- the renal cystic disease as a series of quan- plex34;and(3) most patients are com- titative traits, and used statistical analyses pound heterozygotes for different to identify a highly significant QTL com- WHAT ROLE IS PLAYED BY PKHD1 mutations.35 Genetic studies in plex on chromosome 4 that exerts a dom- MODIFIER GENES IN ARPKD AND experimental models would be a reason- inant effect of the mus musculus castaneus WHAT ARE THE STRATEGIES TO able alternative, but of the eight cur- (CAST) haplotype on renal disease sever- IDENTIFY THEM? rently available mouse Pkhd1 models, ity.33 This interval comprises a complex of none expresses a renal lesion that pheno- at least three QTL (Mpkd1–3), and con- Lisa M. Guay-Woodford copies the human disease. In compari- tains approximately 1300 genes. We have ARPKD is a single gene disorder in which son, the renal and biliary disease in the identified Kif12, a novel kinesin-encoding the renal and biliary lesions can be quite congenital polycystic kidney (cpk)model gene, as the major Mpkd2 candidate variable. This phenotypic variability is closely phenocopies human ARPKD. As using a combination of gene expression typical even among family members who an alternative experimental model, the profiling, computational analyses, single share identical PKHD1 mutations, indicat- cpk mouse has several advantages: (1)it nucleotide polymorphism-based haplo- ing modulating effects of other genetic is the most extensively characterized type mapping, and congenic strain gener- (i.e., co-inherited modifier genes or PKD model; (2)theCys1cpk mutation ation. In addition, we have determined quantitative trait loci [QTL]), epigenetic, disrupts the protein cystin, which is nor- that Kif12 colocalizes with cystin in the or environmental factors (summarized mally expressed in the primary cilium, as primary cilium and that its expression pat- previously33). is the PKHD1-encoded protein; and (3) tern is highly correlated with Cys1 and

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Pkhd1 in the GUDMAP kidney gene ex- the abnormal cell proliferation required ADPKD by about 16%–17%, suggesting pression atlas database (http://www.gud- to form the growing cyst. There is no that there is cancer protection associated map.org/Menu_Index/Gene_Expression. doubt that cyst growth involves increased with PKD.46 This was also found to be html). In related studies, we have collabo- cell proliferation. However, this process true for ARPKD, in a study that exam- rated with Greg Germino’s group and proceeds over months and years, and ined the incidence of colon cancer in identified a genetic interaction between although perhaps unrelenting, it cannot unaffected carriers of a recessive PKHD1 Pkhd1 and Pkd1 that exacerbates the renal be characterized as runaway cell growth. mutation.47 – cystic phenotype in the Pkhd1del3 4 However, neither are most cancerous The reason for the lower cancer risk in model.37 Therefore, these mouse studies tumors, which probably take many years patients with ADPKD is not known, but have identified Kif12 and Pkd1 as putative of growth before they reach a size that may relate to biologic characteristics of genetic modifiers of the ARPKD pheno- can be detected by screening. ADPKD itself or to better cancer risk type. Cyst growth in PKD has many paral- behaviors with these patients. However, In parallel studies we have identified lels with cancer, including elevated the fact that there was a decreased in- a PKHD1 founder mutation (c.T1880A; proto-oncogene activation, Ras/B-Raf/ cidence in ARPKD carriers, who would p.M627K) in a South African Afrikaner mitogen-activated protein kinase, mTOR, not know their gene status, makes it more cohort.38 Varied disease expression was and EGF receptor activation, elevated cell likely that an underlying cellular mech- documented among the 27 affected in- cycle activity, macrophage infiltration, el- anism is protective. Such a mechanism dividuals who were homozygous for this evated cytokines including TNF-a,altered might lie in the unique biology of PKD founder mutation. Therefore, this Afri- basement membrane deposition, in- cells, in which there is loss of intra- kaner cohort represents a unique, foun- creased integrin-linked kinase activity cellular calcium homeostasis and de- dational resource for directed testing of andcellmigration,elevatedperiostin, creased basal calcium.48,49 This lower candidate recessive PKD QTL identified increased angiogenesis, and tumor-like calcium allows cAMP activation of the in our mouse model studies. defective glucose metabolism (Figure Ras/B-Raf/mitogen-activated protein 2).40,41 Uremia associated with ESRD kinase pathway and would contribute Future Directions can often cause acquired PKD and renal to high levels of c-myc expression, a Future efforts will need to focus on iden- cell carcinoma (RCC). Furthermore, characteristic PKD hallmark. We have tifying tagSNPs (tag single nucleotide poly- there is recent evidence indicating that also shown that lower calcium in PKD morphisms) for KIF12 and PKD1,which even less severe CKD can increase the in- cells leads to lower levels of Cox-2 will be resolved into unique, common hap- cidence of RCC and urothelial cancer.42 expression. lotypes to test for association with disease Yet, in PKD, numerous studies have How might these observations ex- severity in the Afrikaner cohort. In a recip- shown that very little RCC is seen43 un- plain the cancer protective mechanism? rocal strategy, we are using CRISPR tech- less the kidneys are removed and exam- The answer may lie in recent studies nology to generate a p.M625K mutation in ined histologically.44,45 demonstrating that high Myc levels50 B6 mice that is orthologous to the human This very low level of diagnosed RCC and Cox-2 inhibition51 prevent cancer founder mutation. Mutant mice will be in ADPKD suggested that there may be a metastasis. As such, the unusual PKD phenotypically characterized. If this “hu- degree of protection from cancer associ- biology may paradoxically promote manized” mouse model phenocopies ated with PKD mutations. As such, we neoplastic cyst growth while preventing ARPKD, then we will generate a mutant carried out an epidemiologic study to progression to metastasis, leading to 46 F2 cohort using the newly described re- answer this question. Data from the lower cancer incidence. source, Diversity Outbred mice.39 The Di- Scientific Registry of Transplant Recipi- versity Outbred stock is derived from eight ents, which contains information on all Future Directions inbred mouse lines and is an ideal resource solid organ transplant recipients in the It will be important in the future to better for high-resolution genetic mapping, ob- United States, were linked to 15 population- understand these cancer-related signal- viating the need for costly fine-mapping based United States cancer registries that ing mechanisms for the design of PKD- studies. Taken together, these efforts will included data on 50 different cancers. specific antiproliferative therapies that provide complementary strategies for Cancer incidence was compared in PKD will slow cyst growth and kidney enlarge- identifying ARPKD-related QTL. versus non-PKD renal transplant recipi- ment without inadvertently opening a ents, and incidence rate ratios adjusted Pandora’s box of metastatic cancer. If the for age, sex, race/ethnicity, dialysis dura- very therapy that suppresses neoplastic NEOPLASTIC CYST GROWTH— tion, and time since transplantation were cyst growth also allows rogue cells to es- BUT WHY NOT CANCER? determined. The study included .10,000 tablish themselves as metastatic tumors, kidney recipients with PKD and .100,000 the treatment might end up harming the James P. Calvet without PKD. After multivariable adjust- patient. On the other hand, if there is One of the more perplexing questions ment, overall cancer incidence was found indeed a PKD-related mechanism that about polycystic kidneys is the nature of to be significantly lower in patients with protects cells from becoming malignant,

2086 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2081–2095, 2015 www.jasn.org SPECIAL ARTICLE such a discovery should be of significant teins alone, of cilia alone, and of poly- POLYCYSTIC KIDNEY DISEASE: interest to the cancer research commu- cystin proteins and cilia together.58 We CILIA AND MORE nity. found that, as previously noted, inacti- vation of polycystins alone resulted in Jing Zhou severe PKD whereas inactivation of cilia Polycystin-1 and PC2 are integral mem- KNOWING WHAT WE DO NOT alone resulted in mild disease. Surpris- brane proteins forming a receptor chan- KNOW: POLYCYSTIN FUNCTION ingly, simultaneous inactivation of poly- nel complex.59 Over a decade ago, PC1 IN CILIA cystins and cilia together resulted in a andPC2werefoundontheprimary marked decrease in severity of PKD cilia.54,55,60 Kidney tubular epithelial Stefan Somlo when compared with polycystin-only cells respond to flow shear stress with a The past decade has seen a remarkable inactivation. These findings proved calcium signal. Cells without functional evolution in the understanding of the universal—they were applicable to PC1orPC2areunabletorespondto importance of primary cilia in the path- Pkd1 and Pkd2,toallsegmentsofthe flow-induced shear stress with a calcium ogenesis of structural kidney diseases nephron, and to the bile duct and were signal.60,61 This calcium signal, visual- that manifest with cysts and fibrosis. independent of the timing of gene in- ized by ratiometric calcium imaging, is Primary cilia are single nonmotile hair- activation (developmental or adult). detectable several seconds after flow stim- like projections on many mammalian We were able to show that the severity ulation and is amplified by a calcium- cells, including most renal tubular epi- of PKD was directly related to the period induced calcium release mechanism thelial cells where they reside on the api- of time for which intact cilia persisted involving a ryanodine receptor.61 Using a cal surface. They are highly privileged following loss of polycystins. In aggre- recently developed calcium indicator, the subcellular compartments composed of gate, these data suggest that mechanisti- calcium signal induced by flow shear a microtubular scaffold with overlying cally polycystins are inhibitory signals stress can now be detected in the primary plasma membrane that is devoid of that normally modulate a pathway cilia.61,62 This PC2-dependent calcium other subcellular organelles. All compon- which is yet to be identified, but that re- signal precedes an increase in cytosolic ent proteins must be synthesized in the quires intact cilia to function. When this calcium level via a ryanodine receptor, cell body and the cilia are able to select pathway is de-repressed due to loss of supporting the previous findings.61,62 This which of these enter and leave the cilia polycystins, ADPKD ensues. PC1- and PC2-dependent calcium signal- compartment and the respective rates for ing may modulate a number of cellular ac- each of these processes. The importance Future Directions tivities (Figure 2). The ciliary compartment of cilia to the pathogenesis of PKD was These findings really should shift our appears to be a separate compartment for brought to the fore by studies that show thinking to ask the following two ques- calcium signaling.62,63 Although the ciliary that mutations affecting cilia structure tions. First, why has evolution kept this functions of PC1 and PC2 are likely not and composition produce cysts in mam- complex interplay of polycystin proteins restricted to calcium signaling, studies de- malian kidney tubules and liver bile ducts and cilia in place? In other words, what fining the local as well as global impact of and that many of the protein products normal physiologic function is impor- the calcium signal mediated by ciliary PC1 associated with human and mouse fibro- tant enough to warrant such a relatively and PC2 will be instructive in understand- cystic diseases are expressed in and complex biologic pathway in the kidney? ing polycystin function. around the cilia compartment.52,53 Second, what is the cilia-dependent cyst- ADPKD is now known as a ciliopathy. Most notably, both PC1 and PC2 are activating pathway that is normally in- Ciliopathies refer to a group of diseases expressed in cilia.54,55 This led to the hibited by the presence of polycystins? causedbystructuralorfunctionaldefectsof hypothesis that polycystin function is Identifying the molecular components the primary cilia. Ciliopathies have a wide critical to cilia function and that defects of these pathways can be expected to rangeofphenotypeswithcystickidneysasa in cilia function are critical to the path- identify novel and effective molecular tar- common feature. What are the proximal ogenesis of PKD. Although such a unify- gets for therapy and ADPKD. The genetic events of ciliary polycystins? We searched ing hypothesis is both appealing and evidence strongly suggests that if we can for polycystin interaction partners. We reasonable, certain features of the therapeutically repress the cilia-dependent found that PC2 interacts with the ARPKD respective cilia mutant and polycystin pathway’s activity in the absence of its protein, fibrocystin/polyductin,64 and mutant phenotypes are inconveniently normal regulatory components, the nek8 (namely nephronophthisis 9).65 Re- inconsistent. Most notably, loss of cilia polycystins, then we will be able to tar- cently we found that PC1 interacts with resultsinmarkedlylesspronounced get ADPKD in a manner that will be several Bardet–Biedl syndrome (BBS) pro- polycystic disease than is seen in specifictoADPKDdiseaseandlikelyeffec- teins that are part of the BBSome protein ADPKD.56,57 tive. Identification of this cilia-dependent complex known to function in the trans- This apparent paradox led us to in- cyst-activating pathway should become port of a set of proteins to the cilia.66 vestigate the genetic interrelationship one of the top priorities for ADPKD Deficiency of BBS1 and BBS3 affects the between inactivation of polycystin pro- research. ciliary trafficking of PC1. Hence, the

J Am Soc Nephrol 26: 2081–2095, 2015 Future of PKD Research 2087 SPECIAL ARTICLE www.jasn.org polycystins are cargos of the BBSome and are located at primary cilia and centro- KIN-like phenotype. We demonstrate physical interactions between the polycys- somes. However, the proximal disease that cells from individuals with FAN1 mu- tins and BBS proteins may underlie the mechanisms remain poorly understood. tations exhibit sensitivity to the ICL agent overlapping renal phenotypes in these We identified, by whole exome rese- mitomycin C. We complement ICL sensi- two diseases. Other components of the quencing, mutations that affect the cen- tivity with wild-type FAN1 but not mutant trafficking pathways may well be impor- trosomal proteins, namely, FAN1, MRE11, cDNA from individuals with KIN. The tant for polycystin targeting to the cilia. ZNF423,andCEP164, as novel causes of FAN1 defect was not epistatic with the Polycystins are present at other sub- NPHP-RC. Surprisingly, these ciliopathy Fanconi anemia pathway. By depletion of cellular compartments besides the pri- genes serve functions within the DNA fan1 in zebrafish we recapitulated in- mary cilia.59 We recently found that PC1 damage response (DDR) pathway.69 creased DDR, apoptosis, and kidney cysts is present in the lamellipodia in migrat- akin to NPHP.72 ing kidney tubular epithelial cells.67 (1) ZNF423 interacts with the DNA ds- We suggest a working hypothesis for binds to an F-bar protein Pacsin 2 and break sensor PARP1, which recruits the pathogenesis of certain forms of regulates Pacsin 2 interaction with N- MRE11 (MRN) and ATM to sites NPHP-RC proposing the following cas- Wasp, an activator of the actin nucleator of DNA damage. ATM, in turn, is cade of events: defects of DDR lead to a Arp2/3 protein complex. We found that activated by MRE11 (MRN) and the lack of Chk1 (Chk2) activation, thereby disorganization of the actin cytoskeleton ‘TIP60 complex’.Wedemonstrate causing inadequate G2/M cell cycle is a feature of PKD in vivo and that a colocalization to TIP60-positive arrest. This would lead in high prolifer- novel PC1-Pacsin 2-N-Wasp complex is (and SC35-positive) nuclear foci or ation states (high replication stress) during required for the actin remodeling and protein–protein interactions for the morphogenesis to dysplastic phenotypes directional cell migration.67 Directional following products of genes mutated (Meckel syndrome) and in low proliferation cell migration is essential for the regen- in NPHP-RC: SDCCAG8/NPHP10, states (low replication stress) during tissue eration and maintenance of the epithe- ZNF423, CEP164, OFD1, RUVBL1, maintenance and repair to tissue degenera- lium and kidney development.68 We RUVBL2, NPHP5, NPHP1, and tion and fibrosis (nephronophthisis). propose that a defective actin cytoskele- ATXN10. OFD1, RUVBL1, RUVBL2, ton and directional cell migration con- are known to play a role in DDR. Future Directions tributes cyst formation. It is becoming increasingly likely that (2) In addition, in four different families disruption of cell cycle regulation is cen- Future Directions with NPHP-RC, we identify recessive tral to most forms of renal ciliopathies. Further understanding the trafficking mutations of CEP164 as a novel cause This mechanism can be targeted by drugs, route and identification of modulators of NPHP-RC. CEP164 acts in the as has been shown in models of renal promoting polycystin targeting to the cilia ATR-Chk1-related arm of DDR, where cystic disease using roscovitine and may provide new therapeutics for pa- it is necessary for ATR-dependent other drugs that interfere with cell cycle tients with trafficking defective muta- Chk1 activation upon induced repli- regulation.73 tions. In addition, studies to elucidate the cation stress.70 role of the cytoskeleton and cell migration in cyst formation may identify novel Furthermore, to identify single-gene INTRAFLAGELLAR TRANSPORT pathogenic mechanisms and therapeutic causes of renal fibrosis/CKD we per- PROTEIN FUNCTION IS NOT targets. formed homozygosity mapping and RESTRICTED TO THE PRIMARY whole exome resequencing in a model CILIUM disorder for renal fibrosis known as DNA DAMAGE RESPONSE karyomegalic interstitial nephritis (KIN).71 Corinne Antignac SIGNALING IS A NOVEL We identified recessive mutations of Virtually all epithelial cells display primary PATHOGENIC MECHANISM IN the Fanconi anemia-associated nuclease cilia. However, whereas in immature rat CILIOPATHIES 1gene(FAN1) as causing the NPHP-like glomeruli, podocytes express cilia, these renal phenotype of karyomegalic intersti- tend to disappear during development.74 Friedhelm Hildebrandt tial nephritis (KIN) in nine of 10 families Hence, our identification of a missense Nephronophthisis-related ciliopathies ascertained. KIN causes CKD with renal mutation (p.P209L) in the TTC21B gene (NPHP-RC)arerecessivemultisystemdis- histology indistinguishable from NPHP, encoding the intraflagellar transport orders that affect kidney, retina, liver, except for the presence of karyomegaly. (IFT) 139 protein in seven families with and cerebellum either by prenatal-onset FAN1 has nuclease activity and acts in hereditary focal-segmental glomerulo- dysplasia or by childhood-onset degen- DNA interstrand crosslinking (ICL) repair sclerosis, discovered during late adoles- eration and fibrosis. Identification of within the Fanconi anemia (FA) pathway cence or early adulthood, raised the .19 disease-causing genes (NPHP1 to of DNA damage response (DDR). Interest- question of a role of IFT139 (and poten- NPHP19) revealed that their products ingly, ICL-causing genotoxins generate a tially other IFT proteins) beyond the

2088 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2081–2095, 2015 www.jasn.org SPECIAL ARTICLE primary cilium.75 Mutations in TTC21B were partially rescued upon p.P209 mu- encouraging results in preclinical and had previously been reported in patients tant expression. clinical trials. Protein kinase A-induced with nephronophthisis,76 but careful re- phosphorylation of cystic fibrosis trans- analysis of the clinical and histologic fea- Future Directions membrane conductance regulator (CFTR) tures of all patients bearing the p.P209L These data show once again the power of allows chloride and fluid secretion into the mutation clearly showed that they pres- genetic studies in families affected with cysts. Anoctamin-1, a calcium-activated entwithbothglomerularandtubulo- rare Mendelian disorders. Here, they chloride channel, may synergistically interstitial involvement. In agreement, allow the uncovering of an unexpected interact with CFTR.79 CFTR inhibitors we found that IFT139 was predomi- role of a ciliary protein in the regulation inhibit cystogenesis in vitro and in nantly expressed in distal tubules, as ex- of podocyte cytoskeleton architecture, kidney-specific Pkd1 knockout mice, pected for a nephronophthisis-causing and open a new field of investigations whereas anoctamin-1 inhibitors have gene, but was also strongly expressed in into the role of IFT proteins in the mature been effective in vitro. glomerular podocytes. IFT139 was podocyte, a cell devoid, at this stage, of a Protein kinase A activation inhibits mainly localized at the base of the pri- primary cilium. We anticipate that future cell proliferation in wild-type cells, but mary cilium in developing podocytes genetic studies in new families with has a stimulatory effect in PKD cells. from human fetal tissue and in undiffer- ciliary syndromes will continue the dis- Calcium deprivation in wild-type cells entiated cultured podocytes. In contrast, covery of new gene mutations and be a and delivery of calcium in PKD cells in nonciliated adult podocytes and differ- rich source for uncovering new biology. reverse these effects. A proposed mech- entiated cultured cells, IFT139 relocalized anism for the proliferative response in along the extended microtubule network. PKD and calcium-deprived wild-type Our functional studies in undiffer- SIGNALING PATHWAYS AND cells is inhibition of phosphoinositide entiated podocytes showed that the THERAPEUTIC MOLECULES 3-kinase and AKT releasing B-Raf from p.P209L mutation has a hypomorphic IN PKD AKT inhibition.49 This leads to dysregu- effect on podocyte ciliogenesis, but was lation of signaling pathways (B-Raf/ not sufficient to inhibit podocyte differ- Vicente E. Torres MEK/ extracellular signal-regulated ki- entiation. These data suggested that the Understanding the pathogenesis of in- nase [ERK]; AMP-activated protein ki- glomerular defects observed in patients creased fluid secretion and epithelial cell nase [AMPK]/mTOR; arguably Wnt/ with this mutation were, rather, due to a proliferation downstream from PKD1 or b-catenin) and transcription factors nonciliary alteration of IFT139 in ma- PKD2 mutations is important to identify (hypoxia-inducible factor 1, Myc, P53, ture podocytes, uncovered by the hypo- therapies (Figure 2).40,41 Those targeting signal transducer and activator of tran- morphic p.P209L mutation. As actin and proximal mechanisms are more likely to scription 3) that control cell cycle pro- microtubule cytoskeletons are key regu- be effective than those targeting distal gression, energy metabolism, and the lators of the delicate architecture, plas- mechanisms. microenvironment. Overexpression of ticity, and contractility of podocytes,77 Aberrant cross-talk between intracel- growth factors, cytokines, chemokines, andasIFT139isredistributedalong lular calcium and cAMP signaling likely and their receptors further contributes the microtubule network in mature po- is one of the first effects of PKD muta- to disease progression. Src is a nonre- docytes, we studied the effect of the tions. Disrupted calcium may enhance ceptor tyrosine kinase activated down- p.P209L mutant on cytoskeleton organi- cAMP and protein kinase A signaling stream from G-protein–coupled and zation in differentiated podocytes. Inter- through activation of calcium-inhibitable growth factor receptors. SKI-606 (bosutinib) estingly, the depletion of IFT139 led to adenylyl cyclases and inhibition of is an Src/Abl inhibitor. KD019 inhibits Src increased alterations to the cell surface calcium-dependent phosphodiesterases and receptor tyrosine kinases (EGF recep- associated with actin cytoskeleton, such (PDE1 and indirectly cGMP-inhibited tor, ERBB2, vascular endothelial growth as short and misorganized stress fibers PDE3).78 Enhanced protein kinase A factor receptor). Both have shown benefit and microtubule rearrangement into activity may in turn disrupt intracellular in preclinical studies. Clinical trials are bike-wheel-like shape. All these defects calcium homeostasis through hyperphos- ongoing. On the other hand, targeting of were fully rescued by the wild-type pro- phorylation of calcium cycling proteins in B-Raf/MEK/ERK has given inconsistent tein. In contrast, p.P209L re-expression the endoplasmic reticulum. Preclinical results. led to the rescue of the cell-size defect, studies have targeted calcium signaling Overwhelming evidence indicates but actin and microtubule networks with some success. Only triptolide (an that mTOR signaling is enhanced in remained severely altered. In addition, activator of the polycystin-2 channel) is cystic tissues. Preclinical studies of rapa- microtubule repolymerization after being clinically investigated. Gasprotein- logs and mTOR antisense oligonucleo- nocodazole treatment resulted in multi- coupled receptor antagonists (i.e., vaso- tides80 have been encouraging. Clinical ple nucleation sites dispersed within the pressin V2 receptor antagonists) and Gai trials have been disappointing81–83 likely cytoplasm in IFT139-depleted differen- protein-coupled receptor agonists (i.e., because clinically achievable blood levels tiated podocytes and these abnormalities somatostatin analogs) have demonstrated do not inhibit mTOR in the kidney.

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Strategies to overcome systemic toxicity c-Met to escape degradation after stimu- for a single magic bullet. Several novel and limited renal bioavailability are being lation.93 concepts that block cell cycle progres- investigated. Pan-sirtuin (nicotinamide) First showninthe Cy/+ratmodel,94 the sion and inflammation, or promote ap- and sirtuin-1–specific inhibitors, AMPK efficacy of mTORC1 inhibitors to reduce optosis and epigenetic changes, have activators (metformin), thiazolidinediones, cyst growth and to ameliorate disease pro- been successfully tested in animal mod- and signal transducer and activator of gression was subsequently demonstrated els of cystic kidney disease, holding great transcription 3 inhibitors have been effec- in several animal models of cystic kid- promise for future clinical trials. tive in animal models.84 Nicotinamide is ney disease, including Pkd1-deficient being tested in an uncontrolled, open la- mice.90,95–100 Despite these encouraging bel clinical trial. Recent studies suggest preclinical data, mTOR inhibitors did TARGETING CYSTS TO PREVENT that PKD cells, like cancer cells, repro- not yield the expected benefits in patients RENAL INSUFFICIENCY IN PKD gram their energy metabolism from with ADPKD. While everolimus slowed oxidative phosphorylation to aerobic cyst growth in ADPKD patients, this in- Jared J. Grantham glycolysis.85 Whether drugs interfering hibition did not translate into an improve- Cysts originate in tubules and are distin- withglycolysiswillbesafeandeffective ment of renal function. However, other guished from simple tubule dilations by clinically is uncertain. pilot studies yielded more promising re- having increased numbers of cells ex- sults,101 leaving open the role of mTOR panding the wall beyond normal bound- Future Directions inhibition as an ADPKD target.102,103 aries. Cysts are the primary suspects Significant progress has been made to- To determine the involvement of causing the decline of GFR (Figure 3). ward identification of effective therapies mTORC1 in cyst growth and ADPKD dis- This is supported by the following find- for PKD. As discussed above, many have ease progression, we abrogated ciliogene- ings: (1) cyst enlargement compresses been shown to be effective in rodent sisinthethickascendinglimbofHenleand the interstitial matrix and surrounding models, but their safety and efficacy in pa- distal tubular segments, generating Kif3a tubules entraining, distorting, and de- tients is difficult to predict with accuracy, fl/fl*KspCre mice. These mice rapidly de- stroying arterioles, venules, capillaries, and only rigorous clinical trials will answer veloped cystic kidney disease, and died be- and lymphatics; (2) most cysts separate this satisfactorily. One approach that fore 12 weeks of age (median survival 7.3 from the tubules from which they should be considered is the use of combi- weeks). In contrast, the additional elimi- formed; (3) an isolated collecting duct nation therapies, which can potentially nation of Raptor (Raptor fl/fl*Kif3a fl/ cyst can block urine flow in the branch- increase efficacy and reduce toxicity.8,86 fl*KspCre) delayed cyst growth and dra- ing arcade upstream of it that would or- matically increased the survival by more dinarily drain hundreds of filtering than three times (median survival 24.0 nephrons105,106;(4) the glomerulo– THE ROLE OF MTOR SIGNALING weeks), revealing the importance of tubule junctions of blocked nephrons IN CYSTIC KIDNEY DISEASE mTORC1 in promoting cyst growth. become thinned and eventually the tu- However, despite the remarkable delay, bules separate creating atubular glomeruli Gerd Walz the Raptor-deficient mice nevertheless de- and apoptotic proximal tubules107–109;(5) The mTORC1 kinase cascade is almost veloped cystic kidney disease. Gene expres- cysts lead to hypertension and impair universally activated in cystic kidney sion profiling revealed that cyst formation urine concentrating capacity before GFR disease independent of the underlying anddiseaseprogressionintheabsenceof declines110;(6) morphologic studies of disease-causing gene mutation87,88 (Fig- functional mTORC1 were driven by other human and animal kidneys reveal that ure 2). Although the molecular basis for growth-promoting pathways, including cysts can form in the absence of interstitial this activation remains incompletely AKT and ERK. pathology105;(7) in ADPKD, cortical fi- understood, primary cilia appear to cur- brosis in the absence of cysts nearby is tail mTORC1 activity through a flow- Future Directions likely secondary to the downstream dependent activation of the LKB1/AMPK The rapid development of mTORC1- blockade of collecting ducts109;(8)asem- signaling pathway, involving stabilization independentcystgrowthinADPKD inal study in PKHD1-null rats unable to of the tuberous sclerosis complex 1 resembles the resistance against targeted synthesize vasopressin proved that fibrosis (TSC1)/TSC2 complex and inhibition of tumor therapies, where de novo somatic and renal insufficiency do not develop in Rheb.89 In addition, PC1 might influence mutations or bypass signaling pathways the absence of cyst formation, but do the activity of this small GTPase through permit tumor cell proliferation despite when the hormone is directly adminis- interaction with the TSC complex,90,91 effective inhibition of the original onco- tered causing cysts to form.111 What causes and inhibit MEK/ERK-mediated phos- genic driver. Reminiscent of the neopla- renal failure in PKD? The cyst did it! phorylation of TSC2 to control mTORC1 sia in disguise coined many years ago to activity,92 whereas c-Cbl is sequestered in capture the tumor-like characteristics of Future Directions the Golgi apparatus in the absence of cysts in ADPKD,104 amulti-targetap- Future therapy should focus on treatment polycystin-1, allowing the HGF receptor proach will likely replace the search in earlychildhood. In most heritable cystic

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disorders affecting humans the cysts begin that more closely mimic the human con- affected or at risk members, develop suit- to form in utero and usually remain below dition with respect to cyst formation in able life-long diets that target protagonists the limits of radiologic detection for a few utero and cyst formation and growth of cyst formation and growth. years postpartum. Intrarenal injury by postpartum; (2)define in molecular de- cysts is clinically announced by hyperten- tail the initial steps between local synthe- CONCLUSIONS sion, abdominal pain and hematuria. Early sis and in situ malfunction of polycystin, childhood is the optimum time to admin- polyductin/fibrocystin and NPHP pro- The advances that have come from PKD ister therapies that temper the formation teins; (3) determine intermediate steps researchhavebeen impressive.Neverthe- and growth of cysts and preserve long-term in the pathway from mutated protein to less there are many unanswered ques- function. Evidence indicates that if the for- cell cycle activation; (4) find molecules tions that need to be addressed in the mation of cysts can be stopped, long-term that block or enhance key steps in cysto- future if we are to move the field forward function can be preserved indefinitely.111 genic pathways; (5) establish ethical and in terms of both a complete understand- The major goals of future research moral guidelines for treating the very ing of the pathogenesis of the disease, and should be to: (1) develop animal models young; and (6) for families comprised of the development and clinical testing of viable therapies:

c How do we determine the clinical significance of individual mutations in PKD genes?

c What genes modify ADPKD and ARPKD disease severity?

c What are the specific developmental and contextual factors that seem to determine the susceptibility to cysto- genesis?

c What is the relationship between cyst epithelial proliferation and cancer?

c What exactly is the role of cilia and ciliary proteins in cyst growth?

c How does DNA damage and its re- sponse influence renal development?

c How are different signaling pathways in PKD related and are there key step (s) that would be particularly advan- tageous to target for therapy?

c What is the most predictive animal model for preclinical testing of PKD therapies? Figure 3. Natural history and pathogenesis of PKD. Total kidney volume (red line) exhibits exponential growth at an average rate of 5% per year, presumably due to cyst epithelial cell c Is there a scientific, moral, ethical, fl proliferation and uid secretion, although this rate can range widely from patient-to-patient. and practical justification for treating This causes progressive compression of neighboring structures leading to nephron de- children with PKD? struction. Early in the disease, remaining intact nephrons exhibit compensatory hyper- filtration sufficient to preserve GFR (blue line). Further renal injury is mediated by multiple It is our hope that this symposium will additional mechanisms (purple block arrows) including inflammation and fibrosis. Late in the disease, nephron loss exceeds renal compensatory capacity and GFR starts to decline, inspire the next generation of PKD reaching a rapid phase generally when the total kidney volume reaches 1500 ml (black investigators to tackle these and other arrow), with an inexorable descent toward ESRD (58 years for PKD1, 79 years for PKD2). challenging questions in creative new Advancing uremia is likely to be accompanied by superimposed acquired cystic disease, ways, and bring hope to patients and but apparently without significant increase in the risk of metastatic RCC. families suffering from PKD.

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ACKNOWLEDGMENTS 7. Hopp K, Ward CJ, Hommerding CJ, Nasr 16. Ong T, Ramsey BW: Modifying disease in SH, Tuan HF, Gainullin VG, Rossetti S, Torres cystic fibrosis: current and future therapies VE, Harris PC: Functional polycystin-1 dosage on the horizon. Curr Opin Pulm Med 19: We wish to thank the sponsors of the 2014 governs autosomal dominant polycystic kid- 645–651, 2013 Grantham Symposium for their generous sup- ney disease severity. JClinInvest122: 4257– 17. Qian F, Watnick TJ, Onuchic LF, Germino port: The PKD Foundation, Otsuka Pharma- 4273, 2012 GG: The molecular basis of focal cyst ceuticals, and the Recanati-Kaplan Foundation. 8. Hopp K, Hommerding CJ, Wang X, Ye H, formation in human autosomal dominant Harris PC, Torres VE: Tolvaptan plus polycystic kidney disease type I. Cell 87: pasireotide shows enhanced efficacy in a 979–987, 1996 PKD1 model. J Am Soc Nephrol 26: 39– 18. Watnick TJ, Torres VE, Gandolph MA, Qian DISCLOSURES 47, 2015 F, Onuchic LF, Klinger KW, Landes G, J.J.G., L.M.G.W., D.J.M.P., and G.W. have served 9. Harris PC, Hopp K: The mutation, a key Germino GG: Somatic mutation in individu- as consultants or on advisory boards for Otsuka determinant of phenotype in ADPKD. JAm al liver cysts supports a two-hit model of – Pharmaceuticals, and V.T. and P.H. have received Soc Nephrol 24: 868 870, 2013 cystogenesis in autosomal dominant poly- 10. Connor A, Lunt PW, Dolling C, Patel Y, cystic kidney disease. Mol Cell 2: 247–251, research support from Otsuka. Meredith AL, Gardner A, Hamilton NK, 1998 Dudley CR: Mosaicism in autosomal domi- 19. Pei Y, Watnick T, He N, Wang K, Liang Y, REFERENCES nant polycystic kidney disease revealed by Parfrey P, Germino G, St George-Hyslop P: genetic testing to enable living related re- Somatic PKD2 mutations in individual kidney nal transplantation. Am J Transplant 8: 232– and liver cysts support a “two-hit” model of 1. Cornec-Le Gall E, Audrézet MP, Chen JM, 237, 2008 cystogenesis in type 2 autosomal dominant Hourmant M, Morin MP, Perrichot R, 11. Consugar MB, Wong WC, Lundquist PA, polycystic kidney disease. J Am Soc Nephrol Charasse C, Whebe B, Renaudineau E, Rossetti S, Kubly VJ, Walker DL, Rangel 10: 1524–1529, 1999 Jousset P, Guillodo MP, Grall-Jezequel A, LJ, Aspinwall R, Niaudet WP, Ozen S, 20. Wu G, D’Agati V, Cai Y, Markowitz G, Park Saliou P, Férec C, Le Meur Y: Type of PKD1 David A, Velinov M, Bergstralh EJ, Bae JH, Reynolds DM, Maeda Y, Le TC, Hou H mutation influences renal outcome in KT, Chapman AB, Guay-Woodford LM, Jr, Kucherlapati R, Edelmann W, Somlo S: ADPKD. J Am Soc Nephrol 24: 1006–1013, Grantham JJ, Torres VE, Sampson JR, Somatic inactivation of Pkd2 results in poly- 2013 Dawson BD, Harris PC; CRISP Con- cystic kidney disease. Cell 93: 177–188, 2. Islam MR, Jimenez T, Pelham C, Rodova M, sortium: Characterization of large rearrange- 1998 Puri S, Magenheimer BS, Maser RL, Widmann ments in autosomal dominant polycystic 21. Piontek KB, Huso DL, Grinberg A, Liu L, C, Calvet JP: MAP/ERK kinase kinase 1 (MEKK1) kidney disease and the PKD1/TSC2 con- Bedja D, Zhao H, Gabrielson K, Qian F, Mei mediates transcriptional repression by in- tiguous gene syndrome. Kidney Int 74: C, Westphal H, Germino GG: A functional teracting with polycystic kidney disease-1 1468–1479, 2008 floxed allele of Pkd1 that can be conditionally  (PKD1) promoter-bound p53 tumor sup- 12. Reiterová J, Stekrová J, Merta M, Kotlas J, inactivated in vivo. J Am Soc Nephrol 15: pressor protein. JBiolChem285: 38818– Elišáková V, Lneni cka P, Korabecná M, 3035–3043, 2004 38831, 2010 Kohoutová M, Tesar V: Autosomal domi- 22. Yu S, Hackmann K, Gao J, He X, Piontek K, 3. Rossetti S, Kubly VJ, Consugar MB, Hopp K, nant polycystic kidney disease in a family García-González MA, Menezes LF, Xu H, Roy S, Horsley SW, Chauveau D, Rees L, with mosaicism and hypomorphic allele. Germino GG, Zuo J, Qian F: Essential role of Barratt TM, van’t Hoff WG, Niaudet P, BMC Nephrol 14: 59, 2013 cleavage of Polycystin-1 at G protein-coupled Torres VE, Harris PC: Incompletely pene- 13. Wang E, Hsieh-Li HM, Chiou YY, Chien YL, receptor proteolytic site for kidney tubular trant PKD1 alleles suggest a role for gene Ho HH, Chin HJ, Wang CK, Liang SC, Jiang structure. Proc Natl Acad Sci U S A 104: dosage in cyst initiation in polycystic kidney ST: Progressive renal distortion by multiple 18688–18693, 2007 disease. Kidney Int 75: 848–855, 2009 cysts in transgenic mice expressing artificial 23. Piontek K, Menezes LF, Garcia-Gonzalez 4. Vujic M, Heyer CM, Ars E, Hopp K, Markoff microRNAs against Pkd1. J Pathol 222: MA, Huso DL, Germino GG: A critical de- A, Orndal C, Rudenhed B, Nasr SH, Torres 238–248, 2010 velopmental switch defines the kinetics of VE, Torra R, Bogdanova N, Harris PC: In- 14. Zode GS, Kuehn MH, Nishimura DY, Searby kidney cyst formation after loss of Pkd1. Nat completely penetrant PKD1 alleles mimic CC, Mohan K, Grozdanic SD, Bugge K, Med 13: 1490–1495, 2007 the renal manifestations of ARPKD. JAm Anderson MG, Clark AF, Stone EM, Sheffield 24. Magenheimer BS, St John PL, Isom KS, Soc Nephrol 21: 1097–1102, 2010 VC: Reduction of ER stress via a chemical Abrahamson DR, De Lisle RC, Wallace DP, 5. Bergmann C, von Bothmer J, Ortiz Brüchle chaperone prevents disease phenotypes Maser RL, Grantham JJ, Calvet JP: Early N, Venghaus A, Frank V, Fehrenbach H, in a mouse model of primary open angle embryonic renal tubules of wild-type Hampel T, Pape L, Buske A, Jonsson J, glaucoma. JClinInvest121: 3542–3553, and polycystic kidney disease kidneys Sarioglu N, Santos A, Ferreira JC, Becker 2011 respond to cAMP stimulation with cys- JU, Cremer R, Hoefele J, Benz MR, Weber 15. Welch EM, Barton ER, Zhuo J, Tomizawa Y, tic fibrosis transmembrane conductance – LT, Buettner R, Zerres K: Mutations in mul- Friesen WJ, Trifillis P, Paushkin S, Patel M, regulator/Na+,K+,2Cl Co-transporter-dependent tiple PKD genes may explain early and se- Trotta CR, Hwang S, Wilde RG, Karp G, cystic dilation. J Am Soc Nephrol 17: 3424– vere polycystic kidney disease. JAmSoc Takasugi J, Chen G, Jones S, Ren H, Moon 3437, 2006 Nephrol 22: 2047–2056, 2011 YC,CorsonD,TurpoffAA,CampbellJA, 25. Menezes LF, Zhou F, Patterson AD, Piontek 6. Zerres K, Rudnik-Schöneborn S, Deget F: Conn MM, Khan A, Almstead NG, Hedrick KB, Krausz KW, Gonzalez FJ, Germino GG: Childhood onset autosomal dominant J, Mollin A, Risher N, Weetall M, Yeh S, Network analysis of a Pkd1-mouse model of polycystic kidney disease in sibs: clin- Branstrom AA, Colacino JM, Babiak J, Ju WD, autosomal dominant polycystic kidney dis- ical picture and recurrence risk. German Hirawat S, Northcutt VJ, Miller LL, Spatrick P, ease identifies HNF4a as a disease modi- Working Group on Paediatric Nephrol- He F, Kawana M, Feng H, Jacobson A, Peltz fier. PLoS Genet 8: e1003053, 2012 ogy (Arbeitsgemeinschaft für Pädiatrische SW, Sweeney HL: PTC124 targets genetic 26. Lantinga-van Leeuwen IS, Dauwerse JG, Nephrologie. J Med Genet 30: 583–588, disorders caused by nonsense mutations. Baelde HJ, Leonhard WN, van de Wal A, 1993 Nature 447: 87–91, 2007 Ward CJ, Verbeek S, Deruiter MC, Breuning

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MH, de Heer E, Peters DJ: Lowering of Pkd1 dominant and recessive polycystic kidney to a cAMP-dependent growth-stimulated expression is sufficient to cause polycystic disease in a common pathway. Hum Mol phenotype. JBiolChem279: 40419– kidney disease. Hum Mol Genet 13: 3069– Genet 16: 1940–1950, 2007 40430, 2004 3077, 2004 38. Lambie L, Amin R, Essop F, Krause A, Cnaan 50. Liu H, Radisky DC, Yang D, Xu R, Radisky ES, 27. Lantinga-van Leeuwen IS, Leonhard WN, A, Guay-Woodford L: Clinical and genetic Bissell MJ, Bishop JM: MYC suppresses van der Wal A, Breuning MH, de Heer E, characterization of a founder PKHD1 mutation cancer metastasis by direct transcriptional Peters DJ: Kidney-specific inactivation of in Afrikaners with ARPKD. Pediatr Nephrol 30: silencing of avandb3 integrin subunits. the Pkd1 gene induces rapid cyst formation 273–279, 2015 Nat Cell Biol 14: 567–574, 2012 in developing kidneys and a slow onset of 39. Logan RW, Robledo RF, Recla JM, Philip 51. Xu L, Stevens J, Hilton MB, Seaman S, disease in adult mice. Hum Mol Genet 16: VM, Bubier JA, Jay JJ, Harwood C, Wilcox Conrads TP, Veenstra TD, Logsdon D, 3188–3196, 2007 T, Gatti DM, Bult CJ, Churchill GA, Chesler Morris H, Swing DA, Patel NL, Kalen J, 28. Grantham JJ, Cook LT, Wetzel LH, EJ: High-precision genetic mapping of be- Haines DC, Zudaire E, St Croix B: COX-2 Cadnapaphornchai MA, Bae KT: Evidence of havioral traits in the diversity outbred mouse Inhibition Potentiates Antiangiogenic Cancer extraordinary growth in the progressive en- population. Genes Brain Behav 12: 424–437, Therapy and Prevents Metastasis in Pre- largement of renal cysts. Clin J Am Soc 2013 clinical Models. Sci Transl Med 6: 242ra284, Nephrol 5: 889–896, 2010 40. Harris PC, Torres VE: Genetic mechanisms 2014 29. Patel V, Li L, Cobo-Stark P, Shao X, Somlo S, and signaling pathways in autosomal dom- 52. Gallagher AR, Germino GG, Somlo S: Mo- Lin F, Igarashi P: Acute kidney injury and inant polycystic kidney disease. J Clin Invest lecular advances in autosomal dominant aberrant planar cell polarity induce cyst 124: 2315–2324, 2014 polycystic kidney disease. Adv Chronic formation in mice lacking renal cilia. Hum 41. Torres VE, Harris PC: Strategies targeting Kidney Dis 17: 118–130, 2010 Mol Genet 17: 1578–1590, 2008 cAMP signaling in the treatment of poly- 53. Hildebrandt F, Benzing T, Katsanis N: 30. Happé H, Leonhard WN, van der Wal A, van cystic kidney disease. JAmSocNephrol25: Ciliopathies. NEnglJMed364: 1533– de Water B, Lantinga-van Leeuwen IS, 18–32, 2014 1543, 2011 Breuning MH, de Heer E, Peters DJ: Toxic 42. Lowrance WT, Ordoñez J, Udaltsova N, Russo 54. Pazour GJ, San Agustin JT, Follit JA, tubular injury in kidneys from Pkd1-deletion P, Go AS: CKD and the risk of incident cancer. Rosenbaum JL, Witman GB: Polycystin-2 mice accelerates cystogenesis accompa- J Am Soc Nephrol 25: 2327–2334, 2014 localizes to kidney cilia and the ciliary level is nied by dysregulated planar cell polarity 43. Keith DS, Torres VE, King BF, Zincki H, elevated in orpk mice with polycystic kidney and canonical Wnt signaling pathways. Farrow GM: Renal cell carcinoma in auto- disease. Curr Biol 12: R378–R380, 2002 Hum Mol Genet 18: 2532–2542, 2009 somal dominant polycystic kidney disease. 55. Yoder BK, Hou X, Guay-Woodford LM: 31. Leonhard WN, Zandbergen M, Veraar K, JAmSocNephrol4: 1661–1669, 1994 The polycystic kidney disease proteins, van den Berg S, van der Weerd L, Breuning 44. Hajj P, Ferlicot S, Massoud W, Awad A, polycystin-1, polycystin-2, polaris, and cystin, M, de Heer E, Peters DJ: Scattered deletion Hammoudi Y, Charpentier B, Durrbach A, are co-localized in renal cilia. JAmSocNephrol of Pkd1 in mouse kidneys causes a cystic Droupy S, Benoît G: Prevalence of renal cell 13: 2508–2516, 2002 snowball effect and recapitulates human carcinoma in patients with autosomal domi- 56. Lin F, Hiesberger T, Cordes K, Sinclair AM, polycystic kidney disease. JAmSocNeph- nant polycystic kidney disease and chronic Goldstein LS, Somlo S, Igarashi P: Kidney- rol :2014(inpress) renal failure. Urology 74: 631–634, 2009 specific inactivation of the KIF3A subunit of 32. Wilson PD, Du J, Norman JT: Autocrine, 45. Jilg CA, Drendel V, Bacher J, Pisarski P, kinesin-II inhibits renal ciliogenesis and endocrine and paracrine regulation of growth Neeff H, Drognitz O, Schwardt M, Gläsker S, produces polycystic kidney disease. Proc abnormalities in autosomal dominant poly- Malinoc A, Erlic Z, Nunez M, Weber A, Natl Acad Sci U S A 100: 5286–5291, 2003 cystic kidney disease. Eur J Cell Biol 61: 131– Azurmendi P, Schultze-Seemann W, Werner 57. Shibazaki S, Yu Z, Nishio S, Tian X, Thomson 138, 1993 M, Neumann HP: Autosomal dominant RB, Mitobe M, Louvi A, Velazquez H, Ishibe 33. Mrug M, Li R, Cui X, Schoeb TR, Churchill polycystic kidney disease: prevalence of S, Cantley LG, Igarashi P, Somlo S: Cyst GA, Guay-Woodford LM: Kinesin family renal neoplasias in surgical kidney speci- formation and activation of the extracellular member 12 is a candidate polycystic kidney mens. Nephron Clin Pract 123: 13–21, 2013 regulated kinase pathway after kidney spe- disease modifier in the cpk mouse. JAm 46. Wetmore JB, Calvet JP, Yu AS, Lynch CF, cific inactivation of Pkd1. Hum Mol Genet Soc Nephrol 16: 905–916, 2005 Wang CJ, Kasiske BL, Engels EA: Polycystic 17: 1505–1516, 2008 34. Boddu R, Yang C, O’Connor AK, Hendrickson kidney disease and cancer after renal trans- 58. Ma M, Tian X, Igarashi P, Pazour GJ, Somlo RC, Boone B, Cui X, Garcia-Gonzalez M, plantation. J Am Soc Nephrol 25: 2335– S: Loss of cilia suppresses cyst growth in Igarashi P, Onuchic LF, Germino GG, Guay- 2341, 2014 genetic models of autosomal dominant poly- Woodford LM: Intragenic motifs regulate the 47. Ward CJ, Wu Y, Johnson RA, Woollard JR, cystic kidney disease. Nat Genet 45: 1004– transcriptional complexity of Pkhd1/PKHD1. Bergstralh EJ, Cicek MS, Bakeberg J, Rossetti 1012, 2013 JMolMed(Berl)92(10): 1045–1056, 2014 S, Heyer CM, Petersen GM, Lindor NM, 59. Zhou J: Polycystins and primary cilia: prim- 35. Rossetti S, Harris PC: Genotype-phenotype Thibodeau SN, Harris PC, Torres VE, Hogan ers for cell cycle progression. Annu Rev correlations in autosomal dominant and au- MC, Boardman LA: Germline PKHD1 muta- Physiol 71: 83–113, 2009 tosomal recessive polycystic kidney disease. tions are protective against colorectal cancer. 60. Delmas P, Nomura H, Li X, Lakkis M, Luo Y, J Am Soc Nephrol 18: 1374–1380, 2007 Hum Genet 129: 345–349, 2011 Segal Y, Fernández-Fernández JM, Harris P, 36. Hou X, Mrug M, Yoder BK, Lefkowitz EJ, 48. Nagao S, Nishii K, Yoshihara D, Kurahashi H, Frischauf AM, Brown DA, Zhou J: Constitu- Kremmidiotis G, D’Eustachio P, Beier DR, Nagaoka K, Yamashita T, Takahashi H, tive activation of G-proteins by polycystin-1 Guay-Woodford LM: Cystin, a novel cilia- Yamaguchi T, Calvet JP, Wallace DP: Calcium is antagonized by polycystin-2. JBiolChem associated protein, is disrupted in the cpk channel inhibition accelerates polycystic kid- 277: 11276–11283, 2002 mouse model of polycystic kidney disease. ney disease progression in the Cy/+ rat. Kid- 61. Nauli SM, Alenghat FJ, Luo Y, Williams E, JClinInvest109: 533–540, 2002 ney Int 73: 269–277, 2008 Vassilev P, Li X, Elia AE, Lu W, Brown EM, 37. Garcia-Gonzalez MA, Menezes LF, Piontek 49. Yamaguchi T, Wallace DP, Magenheimer Quinn SJ, Ingber DE, Zhou J: Polycystins 1 KB, Kaimori J, Huso DL, Watnick T, Onuchic BS, Hempson SJ, Grantham JJ, Calvet JP: and 2 mediate mechanosensation in the LF, Guay-Woodford LM, Germino GG: Ge- Calcium restriction allows cAMP activation primary cilium of kidney cells. Nat Genet 33: netic interaction studies link autosomal of the B-Raf/ERK pathway, switching cells 129–137, 2003

J Am Soc Nephrol 26: 2081–2095, 2015 Future of PKD Research 2093 SPECIAL ARTICLE www.jasn.org

62. Jin X, Mohieldin AM, Muntean BS, Green 71. Mihatsch MJ, Gudat F, Zollinger HU, Heierli 79. Buchholz B, Faria D, Schley G, Schreiber R, JA, Shah JV, Mykytyn K, Nauli SM: Cilioplasm C, Thölen H, Reutter FW: Systemic karyomegaly Eckardt KU, Kunzelmann K: Anoctamin 1 in- is a cellular compartment for calcium sig- associated with chronic interstitial nephri- duces calcium-activated chloride secretion naling in response to mechanical and chem- tis. A new disease entity? Clin Nephrol 12: and proliferation of renal cyst-forming ical stimuli. Cell Mol Life Sci 71: 2165–2178, 54–62, 1979 epithelial cells. Kidney Int 85: 1058–1067, 2014 72. Zhou W, Otto EA, Cluckey A, Airik R, Hurd 2014 63. Delling M, DeCaen PG, Doerner JF, Febvay TW, Chaki M, Diaz K, Lach FP, Bennett GR, 80. Ravichandran K, Zafar I, He Z, Doctor RB, S, Clapham DE: Primary cilia are specialized Gee HY, Ghosh AK, Natarajan S, Thongthip Moldovan R, Mullick AE, Edelstein CL: An calcium signalling organelles. Nature 504: S, Veturi U, Allen SJ, Janssen S, Ramaswami mTOR anti-sense oligonucleotide decreases 311–314, 2013 G, Dixon J, Burkhalter F, Spoendlin M, polycystic kidney disease in mice with a tar- 64. Wang S, Zhang J, Nauli SM, Li X, Starremans Moch H, Mihatsch MJ, Verine J, Reade R, geted mutation in Pkd2. Hum Mol Genet 23: PG, Luo Y, Roberts KA, Zhou J: Fibrocystin/ Soliman H, Godin M, Kiss D, Monga G, 4919–4931, 2014 polyductin, found in the same protein com- Mazzucco G, Amann K, Artunc F, Newland 81. Serra AL, Poster D, Kistler AD, Krauer F, Raina plex with polycystin-2, regulates calcium RC, Wiech T, Zschiedrich S, Huber TB, Friedl S, Young J, Rentsch KM, Spanaus KS, Senn O, responses in kidney epithelia. MolCellBiol A, Slaats GG, Joles JA, Goldschmeding R, Kristanto P, Scheffel H, Weishaupt D, Wüthrich 27: 3241–3252, 2007 Washburn J, Giles RH, Levy S, Smogorzewska RP: Sirolimus and kidney growth in autosomal 65. Sohara E, Luo Y, Zhang J, Manning DK, A, Hildebrandt F: FAN1 mutations cause dominant polycystic kidney disease. NEnglJ Beier DR, Zhou J: Nek8 regulates the ex- karyomegalic interstitial nephritis, linking Med 363: 820–829, 2010 pression and localization of polycystin-1 chronic kidney failure to defective DNA dam- 82. Perico N, Antiga L, Caroli A, Ruggenenti P, and polycystin-2. JAmSocNephrol19: age repair. Nat Genet 44: 910–915, 2012 Fasolini G, Cafaro M, Ondei P, Rubis N, 469–476, 2008 73. Bukanov NO, Moreno SE, Natoli TA, Rogers Diadei O, Gherardi G, Prandini S, Panozo A, 66. Su X, Driscoll K, Yao G, Raed A, Wu M, KA, Smith LA, Ledbetter SR, Oumata N, Bravo RF, Carminati S, De Leon FR, Gaspari Beales PL, Zhou J: Bardet-Biedl syndrome Galons H, Meijer L, Ibraghimov-Beskrovnaya F, Cortinovis M, Motterlini N, Ene-Iordache proteins 1 and 3 regulate the ciliary trafficking O: CDK inhibitors R-roscovitine and S-CR8 B, Remuzzi A, Remuzzi G: Sirolimus therapy of polycystic kidney disease 1 protein. Hum effectively block renal and hepatic cysto- to halt the progression of ADPKD. JAmSoc Mol Genet 23: 5441–5451, 2014 genesis in an orthologous model of ADPKD. Nephrol 21: 1031–1040, 2010 67. Yao G, Su X, Nguyen V, Roberts K, Li X, Cell Cycle 11: 4040–4046, 2012 83. Walz G, Budde K, Mannaa M, Nürnberger J, Takakura A, Plomann M, Zhou J: Polycystin- 74. Ichimura K, Kurihara H, Sakai T: Primary cilia Wanner C, Sommerer C, Kunzendorf U, 1 regulates actin cytoskeleton organization disappear in rat podocytes during glomer- Banas B, Hörl WH, Obermüller N, Arns W, and directional cell migration through a ular development. Cell Tissue Res 341: Pavenstädt H, Gaedeke J, Büchert M, May novel PC1-Pacsin 2-N-Wasp complex. Hum 197–209, 2010 C, Gschaidmeier H, Kramer S, Eckardt KU: Mol Genet 23: 2769–2779, 2014 75. Huynh Cong E, Bizet AA, Boyer O, Woerner S, Everolimus in patients with autosomal 68. Karner CM, Chirumamilla R, Aoki S, Igarashi GribouvalO,FilholE,ArrondelC,ThomasS, dominant polycystic kidney disease. NEngl P, Wallingford JB, Carroll TJ: Wnt9b sig- Silbermann F, Canaud G, Hachicha J, Ben Dhia JMed363: 830–840, 2010 naling regulates planar cell polarity and N, Peraldi MN, Harzallah K, Iftene D, Daniel L, 84. Zhou X, Fan LX, Sweeney WE Jr, Denu JM, kidney tubule morphogenesis. Nat Genet Willems M, Noel LH, Bole-Feysot C, Nitschké Avner ED, Li X: Sirtuin 1 inhibition delays 41: 793–799, 2009 P, Gubler MC, Mollet G, Saunier S, Antignac C: cyst formation in autosomal-dominant poly- 69. Chaki M, Airik R, Ghosh AK, Giles RH, Chen Ahomozygousmissensemutationintheciliary cystic kidney disease. J Clin Invest 123: 3084– R, Slaats GG, Wang H, Hurd TW, Zhou W, gene TTC21B causes familial FSGS. JAmSoc 3098, 2013 Cluckey A, Gee HY, Ramaswami G, Hong Nephrol 25: 2435–2443, 2014 85. Rowe I, Chiaravalli M, Mannella V, Ulisse V, CJ, Hamilton BA, Cervenka I, Ganji RS, Bryja 76. Davis EE, Zhang Q, Liu Q, Diplas BH, Davey Quilici G, Pema M, Song XW, Xu H, Mari S, V, Arts HH, van Reeuwijk J, Oud MM, LM, Hartley J, Stoetzel C, Szymanska K, Qian F, Pei Y, Musco G, Boletta A: Defective Letteboer SJ, Roepman R, Husson H, Ramaswami G, Logan CV, Muzny DM, Young glucose metabolism in polycystic kidney Ibraghimov-Beskrovnaya O, Yasunaga T, AC, Wheeler DA, Cruz P, Morgan M, Lewis LR, disease identifies a new therapeutic strat- Walz G, Eley L, Sayer JA, Schermer B, Cherukuri P, Maskeri B, Hansen NF, Mullikin egy. Nat Med 19: 488–493, 2013 Liebau MC, Benzing T, Le Corre S, JC, Blakesley RW, Bouffard GG, Gyapay G, 86. Sabbatini M, Russo L, Cappellaio F, Drummond I, Janssen S, Allen SJ, Natarajan Rieger S, Tönshoff B, Kern I, Soliman NA, Troncone G, Bellevicine C, De Falco V, S, O’Toole JF, Attanasio M, Saunier S, Neuhaus TJ, Swoboda KJ, Kayserili H, Buonocore P, Riccio E, Bisesti V, Federico S, Antignac C, Koenekoop RK, Ren H, Lopez I, Gallagher TE, Lewis RA, Bergmann C, Otto Pisani A: Effects of combined administration Nayir A, Stoetzel C, Dollfus H, Massoudi R, EA, Saunier S, Scambler PJ, Beales PL, of rapamycin, tolvaptan, and AEZ-131 on the Gleeson JG, Andreoli SP, Doherty DG, Gleeson JG, Maher ER, Attié-Bitach T, Dollfus progression of polycystic disease in PCK rats. Lindstrad A, Golzio C, Katsanis N, Pape L, H, Johnson CA, Green ED, Gibbs RA, Am J Physiol Renal Physiol 306: F1243– Abboud EB, Al-Rajhi AA, Lewis RA, Omran Hildebrandt F, Pierce EA, Katsanis N; NISC F1250, 2014 H, Lee EY, Wang S, Sekiguchi JM, Saunders Comparative Sequencing Program: TTC21B 87. Boletta A: Emerging evidence of a link be- R, Johnson CA, Garner E, Vanselow K, contributes both causal and modifying alleles tween the polycystins and the mTOR path- Andersen JS, Shlomai J, Nurnberg G, across the ciliopathy spectrum. Nat Genet 43: ways. Pathogenetics 2: 6, 2009 Nurnberg P, Levy S, Smogorzewska A, Otto 189–196, 2011 88. Ibraghimov-Beskrovnaya O, Natoli TA: mTOR EA, Hildebrandt F: Exome capture reveals 77. Welsh GI, Saleem MA: The podocyte cyto- signalinginpolycystickidneydisease.Trends ZNF423 and CEP164 mutations, linking renal skeleton—key to a functioning glomerulus Mol Med 17: 625–633, 2011 ciliopathies to DNA damage response sig- in health and disease. Nat Rev Nephrol 8: 89. Boehlke C, Kotsis F, Patel V, Braeg S, Voelker naling. Cell 150: 533–548, 2012 14–21, 2012 H, Bredt S, Beyer T, Janusch H, Hamann C, 70. Sivasubramaniam S, Sun X, Pan YR, Wang S, 78. Sussman CR, Ward CJ, Leightner AC, Smith Gödel M, Müller K, Herbst M, Hornung M, Lee EY: Cep164 is a mediator protein re- JL, Agarwal R, Harris PC, Torres VE: Phos- Doerken M, Köttgen M, Nitschke R, Igarashi quired for the maintenance of genomic phodiesterase 1A Modulates Cystogenesis P, Walz G, Kuehn EW: Primary cilia regulate stability through modulation of MDC1, RPA, in Zebrafish. JAmSocNephrol25: 2222– mTORC1 activity and cell size through Lkb1. and CHK1. Genes Dev 22: 587–600, 2008 2230, 2015 Nat Cell Biol 12: 1115–1122, 2010

2094 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2081–2095, 2015 www.jasn.org SPECIAL ARTICLE

90. Shillingford JM, Murcia NS, Larson CH, Low resulting from conditional inactivation of controlled pilot study. Clin J Am Soc Nephrol SH, Hedgepeth R, Brown N, Flask CA, Pkd1. JAmSocNephrol21: 489–497, 2010 9: 881–888, 2014 Novick AC, Goldfarb DA, Kramer-Zucker A, 97. Zafar I, Ravichandran K, Belibi FA, Doctor 104. Grantham JJ: Polycystic kidney disease: Walz G, Piontek KB, Germino GG, Weimbs RB, Edelstein CL: Sirolimus attenuates dis- neoplasia in disguise. Am J Kidney Dis 15: T: The mTOR pathway is regulated by ease progression in an orthologous mouse 110–116, 1990 polycystin-1, and its inhibition reverses renal model of human autosomal dominant poly- 105. Grantham JJ, Geiser JL, Evan AP: Cyst for- cystogenesis in polycystic kidney disease. cystic kidney disease. Kidney Int 78: 754–761, mation and growth in autosomal dominant Proc Natl Acad Sci U S A 103: 5466–5471, 2010 polycystic kidney disease. Kidney Int 31: 2006 98. Shillingford JM, Leamon CP, Vlahov IR, 1145–1152, 1987 91. Dere R, Wilson PD, Sandford RN, Walker CL: Weimbs T: Folate-conjugated rapamycin 106. Grantham JJ, Mulamalla S, Swenson-Fields Carboxy terminal tail of polycystin-1 regu- slows progression of polycystic kidney dis- KI: Why kidneys fail in autosomal dominant lates localization of TSC2 to repress mTOR. ease. JAmSocNephrol23: 1674–1681, polycystic kidney disease. Nat Rev Nephrol PLoS ONE 5: e9239, 2010 2012 7: 556–566, 2011 92. Distefano G, Boca M, Rowe I, Wodarczyk C, 99. Stayner C, Shields J, Slobbe L, Shillingford 107. Chevalier RL, Forbes MS: Generation and Ma L, Piontek KB, Germino GG, Pandolfi PP, JM, Weimbs T, Eccles MR: Rapamycin- evolution of atubular glomeruli in the pro- Boletta A: Polycystin-1 regulates extracellular mediated suppression of renal cyst expan- gression of renal disorders. JAmSoc signal-regulated kinase-dependent phos- sion in del34 Pkd1-/- mutant mouse embryos: Nephrol 19: 197–206, 2008 phorylation of tuberin to control cell size an investigation of the feasibility of renal cyst 108. Forbes MS, Thornhill BA, Chevalier RL: through mTOR and its downstream effec- prevention in the foetus. Nephrology (Carlton) Proximal tubular injury and rapid formation tors S6K and 4EBP1. MolCellBiol29: 17: 739–747, 2012 of atubular glomeruli in mice with unilateral 2359–2371, 2009 100. Novalic Z, van der Wal AM, Leonhard WN, ureteral obstruction: a new look at an old 93. Qin S, Taglienti M, Nauli SM, Contrino L, Koehl G, Breuning MH, Geissler EK, de model. Am J Physiol Renal Physiol 301: Takakura A, Zhou J, Kreidberg JA: Failure to Heer E, Peters DJ: Dose-dependent effects F110–F117, 2011 ubiquitinate c-Met leads to hyperactivation of sirolimus on mTOR signaling and poly- 109. Galarreta CI, Grantham JJ, Forbes MS, of mTOR signaling in a mouse model of cystic kidney disease. JAmSocNephrol23: Maser RL, Wallace DP, Chevalier RL: Tubu- autosomal dominant polycystic kidney dis- 842–853, 2012 lar obstruction leads to progressive proximal ease. J Clin Invest 120: 3617–3628, 2010 101. Soliman A, Zamil S, Lotfy A, Ismail E: Siroli- tubular injury and atubular glomeruli in 94. Tao Y, Kim J, Schrier RW, Edelstein CL: Ra- mus produced S-shaped effect on adult polycystic kidney disease. Am J Pathol 184: pamycin markedly slows disease progression polycystic kidneys after 2-year treatment. 1957–1966, 2014 in a rat model of polycystic kidney disease. Transplant Proc 44: 2936–2939, 2012 110. Fonseca JM, Bastos AP, Amaral AG, Sousa MF, J Am Soc Nephrol 16: 46–51, 2005 102. Jardine MJ, Liyanage T, Buxton E, Perkovic Souza LE, Malheiros DM, Piontek K, Irigoyen 95. Wahl PR, Serra AL, Le Hir M, Molle KD, Hall V: mTOR inhibition in autosomal-dominant MC, Watnick TJ, Onuchic LF: Renal cyst growth MN, Wüthrich RP: Inhibition of mTOR with polycystic kidney disease (ADPKD): the ques- is the main determinant for hypertension and sirolimus slows disease progression in Han: tion remains open. Nephrol Dial Transplant concentrating deficit in Pkd1-deficient mice. SPRD rats with autosomal dominant poly- 28: 242–244, 2013 Kidney Int 85: 1137–1150, 2014 cystic kidney disease (ADPKD). Nephrol 103. Braun WE, Schold JD, Stephany BR, Spirko 111. Wang X, Wu Y, Ward CJ, Harris PC, Torres VE: Dial Transplant 21: 598–604, 2006 RA, Herts BR: Low-dose rapamycin (sirolimus) Vasopressin directly regulates cyst growth in 96. Shillingford JM, Piontek KB, Germino GG, effects in autosomal dominant polycystic polycystic kidney disease. J Am Soc Nephrol Weimbs T: Rapamycin ameliorates PKD kidney disease: an open-label randomized 19: 102–108, 2008

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