Abstract
Polycystic kidney disease (PKD) is one of the most common life-threatening genetic diseases. Jared J. Grantham, M.D., has done more than any other individual to promote PKD research around the world. However, despite decades of investigation there is still no approved therapy for PKD in the United States. In May 2014, the University of Kansas Medical Center hosted a symposium in Kansas City honoring the occasion of Dr. Grantham's retirement and invited all the awardees of the Lillian Jean Kaplan International Prize for Advancement in the Understanding of Polycystic Kidney Disease to participate in a forward-thinking and interactive forum focused on future directions and innovations in PKD research. This article summarizes the contributions of the 12 Kaplan awardees and their vision for the future of PKD research.
In 2013, Jared J. Grantham, M.D. retired from the University of Kansas Medical Center (KUMC) after a long and distinguished career devoted to the search for a cure for polycystic kidney disease (PKD). Dr. Grantham’s interest in kidney disease developed in the sixth grade in Johnson, Kansas while trading comic books with his neighbor. His neighbor casually mentioned that he had cysts in his kidneys, his mother had them and his grandmother had recently died of kidney failure. That remained a vivid memory for Dr. Grantham and he reconnected with kidney cysts in 1970 when he was asked to lead the Nephrology Division at the KUMC and discovered in his laboratory new renal functions in tubule fluid secretion that opened the door to innovative research on PKD. Dr. Grantham was a member of the National Institutes of Health (NIH) research team that developed the method to dissect and perfuse isolated segments of renal tubules. His work emphasized the mechanisms of action of vasopressin on collecting ducts, a theme that was carried forward to his work on PKD, which was continuously funded by the NIH until his retirement. With Kansas City businessman Joseph Bruening, Dr. Grantham founded in 1982 the Polycystic Kidney Disease Foundation, which today is the leading organization worldwide supporting research and education about the disease. Dr. Grantham, a die-hard native Kansan, directed the Division of Nephrology at Kansas for 25 years, was co-founder of the Kidney Institute which he directed for 5 years, and was founding editor of the Journal of the American Society of Nephrology. Without question, Jared Grantham has been a towering figure in PKD research 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 Polycystic Kidney Disease Research” on May 7–9, 2014. All recipients of the Lillian Jean Kaplan Prize were invited to Kansas City to lead a forward-thinking and interactive forum focused on future directions and innovations in PKD research. The Lillian Jean Kaplan International Prize for the Advancement in the Understanding of Polycystic Kidney Disease was established by the PKD Foundation and the International Society 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 created to stimulate interest in advancing PKD research and to recognize those who have increased our understanding and treatment of PKD through basic or clinical scientific research, leading to new treatments and a cure 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. Vincent Gattone, Ph.D., who was awarded the prize in 2013, unfortunately passed away on January 26, 2014. He was highly respected in the PKD field and had a major impact. Among his many achievements, he developed the concept of using vasopressin antagonists to treat PKD and carried out the initial testing in rodent models of cystic disease that eventually led to clinical trials of tolvaptan.
The Grantham Symposium was a huge success. All 12 living Kaplan awardees were able to participate, along with nearly 200 attendees who came from all over the world to celebrate Dr. Grantham’s career and participate in shaping the future of PKD research. The many outstanding talks and discussion forums stimulated considerable lively debate and exchange of ideas. These shed much-needed light on the most important directions that now need to be pursued to advance the field and move effective treatments for this disease as quickly as possible into the clinic.
In this article, each Kaplan awardee was invited to contribute a synopsis of his or her talk, focusing on their vision for the future. From the juxtaposition of their talks and these reviews, several common themes emerged. A number of factors, such as allelic effects of PKD genes (Harris) and the function of ciliary trafficking proteins (Zhou), converge to determine the dosage of PKD1 protein that is delivered to the cell and ciliary surface, which is seen as playing a critical role in determining disease severity. Modifier genes have long been suspected of influencing the severity and natural history of the disease. Guay-Woodford illustrates how this can be effectively investigated in autosomal recessive PKD (ARPKD) by using mouse quantitative trait loci mapping and analogous studies in a unique, genetically isolated, human population.
Much recent evidence suggests that the cell and tissue context within which PKD mutations occur is a key determinant of the disease course. The developmental stage in early life, or superimposed renal injury in adulthood are well-established contextual factors. To identify other contextual elements, Germino used an agnostic transcriptomic method and showed that the metabolic pathways associated with developmental age are important. Peters argues that local paracrine or mechanical signals may also play a role and that this perhaps resembles more the mechanism of human disease.
Although the many culprit genes in cystic diseases share a common localization to the cilia, the true role of this organelle remains poorly understood. Somlo discusses observations that support the radical assertion that signals emanating from the intact cilia actually promote cystogenesis. The idea that cystic disease is caused by abnormal epithelial cell proliferation and constitutes a neoplastic process is well accepted, but Calvet provides epidemiologic data suggesting that patients with PKD may actually be protected from cancer, raising intriguing questions about how the proliferation of cyst epithelial cells is regulated. Novel downstream cyst-promoting mechanisms that have recently been uncovered include the role of the actin cytoskeleton and directional cell motility (Zhou) and the role of DNA damage repair in the regulation of cell cycle checkpoint genes and hence epithelial cell proliferation (Hildebrandt). Hildebrandt and Antignac both make a strong case for continued efforts to identify novel cystic disease genes as a rich source for uncovering new biology.
With the identification of so many signaling pathways, cystic diseases are ripe for the development of pharmacologic therapies. Torres reviews the therapeutic landscape and current and future prospects. One of the more promising of these is the mammalian target of rapamycin (mTOR) pathway, yet recent clinical trials of mTOR inhibitors have failed. Torres argues that the dosage that could be used in humans was insufficient, while Walz discusses evidence to suggest that compensatory growth-promoting pathways may have become activated. The development of this type of resistance to targeted therapies is a growing concern, so for this and other reasons, several individuals expressed enthusiasm for the concept of combinatorial drug approaches. Finally, Grantham revisited the question of how PKD leads to kidney failure and concludes that the cyst burden is central to this process. Following this argument leads to the logical conclusion that treatment, if it is to be successful, 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 considerable phenotypic variability in terms of severity of renal disease and the occurrence of extrarenal manifestations (Figure 1). The gene involved, PKD1 (approximately 85% of cases) or PKD2 (approximately 15%), is strongly associated with renal disease severity, with ESRD occurring on average approximately 20 years earlier in PKD1 than PKD2 (approximately 58 years versus approximately 79 years).1 Genetic background, along with environmental effects, also influence the phenotype, manifesting as significant intrafamilial phenotypic variability. Analysis of common single nucleotide polymorphisms (genome-wide association studies) and rare variants (whole exome analysis) is likely to shed light on the importance of specific modifier variants/genes.
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 hence protein expression, particularly of PC1 at the plasma membrane) and the susceptibility 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 variants at the disease locus, modifier genes including PKHD1, HNF1B, and other ciliopathy genes, and somatic mutations (the classic “second hit”), and other factors affecting the expression levels of these genes.2 The cystogenic threshold seems to be strongly dependent on cell autonomous contextual factors such as the developmental stage and superimposition of environmental agents such as renal injury and infection, and can also potentially be affected by modifier genes. Cell nonautonomous local effects may alter the cystogenic threshold and account for a “snowball effect” in cyst appearance. In 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 dosage and increasing cystogenic threshold determines the onset, progression, and severity of cystic disease.
There has been a recent focus on allelic effects with several lines of evidence indicating that the PKD1 genotype is significantly associated with renal disease severity, and can explain some extreme phenotypes. Viable patients homozygous or compound heterozygous for pathogenic PKD1 missense variants indicate that some alleles are incompletely penetrant (hypomorphic); hypomorphic heterozygotes develop very mild cystic disease without ESRD.3,4 Furthermore, the in trans combination of an inactivating and hypomorphic PKD1 allele can explain rare, very early onset ADPKD (phenotypically similar to ARPKD).3,5,6 An in trans combination of PKD1 hypomorphic alleles can also result in ARPKD-like disease with a negative family history, but without congenital hepatic fibrosis.4
A mouse model mimicking the best characterized PKD1 hypomorphic allele, p.R3277C, recapitulates the human phenotypes with slowly progressive disease in homozygotes, generating a good model for preclinical testing, and rapidly progressive disease in compound heterozygotes with a null allele.7,8 Detailed analysis of the model indicates that the level of the mature glycoform of the PKD1 protein (polycystin 1; PC1) is associated with disease severity, strongly supporting a dosage model of pathogenesis. Consistent with related mechanisms in cystic diseases, a combination of a PKD1 allele and mutations 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 hypomorphic 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 diagnostic value, and can be employed to identify patients with rapidly progressive disease, suitable for clinical trials and future treatments9; although, truncating PKD1 mutation mosaics can have mild disease.10–12 However, a significant proportion of nontruncating mutations are fully inactivating and so presently the prognostic value of knowing the mutation for the individual patient is limited.
Future Directions
Short-term goals include determining the strength of PKD1 mutations bioinformatically, from family studies, through in vitro assays and by animal studies to enhance molecular diagnostics/prognostics. Cataloging variants beyond the ADPKD gene that influence the phenotype will also be of prognostic value. In the longer term, evidence that the level of PC1 is related to disease severity provides a potential therapeutic opportunity through modulating the level of functional PC1, by increasing the expression level, via targeting microRNAs, for instance.13 Chaperone treatment for missense changes14 and nonsense mutation read-through agents should also be considered as mutation tailored treatments as we start to apply personalized medicine to ADPKD.15,16
Breaking Bad—What Makes Good Tubules Turn Cystic?
Gregory G. Germino
What makes cysts form? We know that genetics plays a key role. Although the disease is inherited as an autosomal dominant trait, numerous lines of evidence indicate that it is recessive on a cellular level, with cysts arising when the total functional activity of the two alleles of either PKD1 or PKD2 falls below an ill-defined threshold (Figure 1). This most commonly arises as a result of acquired mutations in renal epithelial cells that have a germline mutation of either locus.17–21 The nature of the germline mutation likely plays a dominant role in determining the severity of disease.1 Recent studies suggest that some individuals instead may have two germline, hypomorphic alleles whose combined activity falls below the threshold.3,7 There is also evidence to suggest that the threshold may be different in different cell types and life stages.7,22 Genetic modifiers are likely important in three steps of this process: determining the rate of somatic mutation, setting the cystogenic threshold, and in downstream effector pathways.
There are several other lines of evidence, however, that suggest other, nongenetic factors may also be important. For example, we have shown that adult mouse kidneys take months to develop cysts after inactivation of Pkd1.23 Metanephric Pkd null kidneys develop cysts in vivo and yet do not when cultured in vitro.24 We have generated multiple mouse cell lines lacking either Pkd1 or Pkd2 and find that they make tubules in three-dimensional cultures regardless of genetic status (L. Menezes, unpublished observation). We have applied network strategies and gene expression analyses to better understand the processes responsible for cyst formation.25 We compared gene expression patterns of a test set of mutant (Pkd1cond/cond Cre+) and control (Pkd1cond/cond Cre–) mice induced at around P7 and harvested at P12–P24 and identified a mutant gene signature that properly clustered an independent validation set of mouse samples by genotype. Comparing the network structures of P12 and P14 control and mutant samples, we found that they were highly similar, arguing against a developmental block in cysts. There was, however, a change in the network architecture of P12 versus P14 samples for both controls and mutants. The genes in this cluster were highly enriched for metabolic pathways, suggesting that metabolic context could be an important disease modifier. Perturbation of the metabolic state seems to alter disease severity.
Future Directions
In summary, genetic factors are a necessary initiating step for cystic disease but other, incompletely defined downstream processes are required for cyst growth. Identification of these may offer a unique opportunity for intervention. An agnostic, system-based approach has identified metabolic context as one of the processes that affects cyst growth in early-onset PKD. Future studies are required to determine the functional relationship between PKD proteins and cellular metabolism. It also will be important to determine if the same processes affect disease in the adult-onset mouse model. The field also would benefit from having a robust cell-based system that faithfully recapitulates the tube–cyst phenotypic switch according to genotype. Such a system could be used to define immediate effector pathways downstream of PKD proteins, to link these to other cellular signaling systems, to assess missense PKD gene variants and ultimately to screen for drugs that can correct the cystic phenotype.
HOW do tissue contexts such as injury and repair affect cystogenesis?
Dorien J.M. Peters
Most patients with ADPKD carry a heterozygous mutation in either the PKD1 or the PKD2 gene. However, the phenotype is also influenced by additional genetic and environmental factors (Figure 1).
In ADPKD, cysts develop in a minority of nephrons, suggesting that a heterozygous mutation in PKD1 or PKD2 is not sufficient to induce cyst formation. In fact, the mutation primes the kidneys for cyst formation, but the likelihood of cyst formation strongly increases when the level of functional PKD1 or PKD2 gene product drops below a critical threshold.3,17,26 A variety of data suggest that the rate of cyst formation depends on the context of renal tissue. For example, both in mice and in humans, cyst growth is much faster during renal development than it is during adulthood.23,27,28 It is likely that growth promoting conditions during renal development accelerate the process.23,27 Furthermore, the relatively slow process of cyst formation and progression in adult mice can be accelerated by acute renal injury treatments.29–31 Renal injury is accompanied by a combination of processes including repair associated proliferation, secretion of growth factors, and inflammation. Accelerated cyst formation seems to be the result of increased susceptibility to cyst-promoting stimuli, e.g., cytokines or growth factors, due to altered integrity of the cells, rather than the result of uncontrolled cell proliferation.30,32 In addition, the cysts themselves can have a profound impact on the surrounding tissue by inducing mechanical stress on neighboring nephrons, which locally increases the likelihood of cyst formation.31
In patients, recurrent urinary tract infections and toxic (waste) products may cause local injury. The effects of these may accumulate over years and contribute to impaired renal function and renal aging. Individual variation in exposure to environmental factors that may cause renal injury, as well as renal aging, may contribute to the variations in severity of renal cystic disease among patients.
During renal development, injury/repair, or normal homeostasis, the PKD proteins modulate a complex network of signaling pathways (Figure 2) needed to establish and maintain the tubular architecture. Especially during phases in which the full potential of these pathways is required, the likelihood that this signaling network gets out of balance may increase, making renal tissue more prone to cyst formation, particularly when the PKD proteins are absent.
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.
Future Directions
More insight into these processes and the exact signaling pathways that underlie the key cellular transitions may reveal interesting targets for therapeutic intervention. Over all, future research should include identification of the triggers and unraveling of the exact pathways that underlie the key cellular transitions. In addition, obtaining more insight into the stimuli that promote cyst growth may reveal interesting targets for therapeutic 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 which the renal and biliary lesions can be quite variable. This phenotypic variability is typical even among family members who share identical PKHD1 mutations, indicating modulating effects of other genetic (i.e., co-inherited modifier genes or quantitative trait loci [QTL]), epigenetic, or environmental factors (summarized previously33).
Efforts to identify QTL in human ARPKD cohorts are confounded by several factors, most notably: (1) ARPKD is a relatively rare disorder; (2) the PKHD1 gene is transcriptionally complex34; and (3) most patients are compound heterozygotes for different PKHD1 mutations.35 Genetic studies in experimental models would be a reasonable alternative, but of the eight currently available mouse Pkhd1 models, none expresses a renal lesion that phenocopies the human disease. In comparison, the renal and biliary disease in the congenital polycystic kidney (cpk) model closely phenocopies human ARPKD. As an alternative experimental model, the cpk mouse has several advantages: (1) it is the most extensively characterized PKD model; (2) the Cys1cpk mutation disrupts the protein cystin, which is normally expressed in the primary cilium, as is the PKHD1-encoded protein; and (3) in contrast to Pkhd1, Cys1 is a small, transcriptionally uncomplicated gene.36
In previous studies, we generated a cohort of F2 affected mice, characterized the renal cystic disease as a series of quantitative traits, and used statistical analyses to identify a highly significant QTL complex on chromosome 4 that exerts a dominant effect of the mus musculus castaneus (CAST) haplotype on renal disease severity.33 This interval comprises a complex of at least three QTL (Mpkd1–3), and contains approximately 1300 genes. We have identified Kif12, a novel kinesin-encoding gene, as the major Mpkd2 candidate using a combination of gene expression profiling, computational analyses, single nucleotide polymorphism-based haplotype mapping, and congenic strain generation. In addition, we have determined that Kif12 colocalizes with cystin in the primary cilium and that its expression pattern is highly correlated with Cys1 and Pkhd1 in the GUDMAP kidney gene expression atlas database (http://www.gudmap.org/Menu_Index/Gene_Expression.html). In related studies, we have collaborated with Greg Germino’s group and identified a genetic interaction between Pkhd1 and Pkd1 that exacerbates the renal cystic phenotype in the Pkhd1del3–4 model.37 Therefore, these mouse studies have identified Kif12 and Pkd1 as putative genetic modifiers of the ARPKD phenotype.
In parallel studies we have identified a PKHD1 founder mutation (c.T1880A; p.M627K) in a South African Afrikaner cohort.38 Varied disease expression was documented among the 27 affected individuals who were homozygous for this founder mutation. Therefore, this Afrikaner cohort represents a unique, foundational resource for directed testing of candidate recessive PKD QTL identified in our mouse model studies.
Future Directions
Future efforts will need to focus on identifying tagSNPs (tag single nucleotide polymorphisms) for KIF12 and PKD1, which will be resolved into unique, common haplotypes to test for association with disease severity in the Afrikaner cohort. In a reciprocal strategy, we are using CRISPR technology to generate a p.M625K mutation in B6 mice that is orthologous to the human founder mutation. Mutant mice will be phenotypically characterized. If this “humanized” mouse model phenocopies ARPKD, then we will generate a mutant F2 cohort using the newly described resource, Diversity Outbred mice.39 The Diversity Outbred stock is derived from eight inbred mouse lines and is an ideal resource for high-resolution genetic mapping, obviating the need for costly fine-mapping studies. Taken together, these efforts will provide complementary strategies for identifying ARPKD-related QTL.
Neoplastic Cyst Growth—But Why Not Cancer?
James P. Calvet
One of the more perplexing questions about polycystic kidneys is the nature of the abnormal cell proliferation required to form the growing cyst. There is no doubt that cyst growth involves increased cell proliferation. However, this process proceeds over months and years, and although perhaps unrelenting, it cannot be characterized as runaway cell growth. However, neither are most cancerous tumors, which probably take many years of growth before they reach a size that can be detected by screening.
Cyst growth in PKD has many parallels with cancer, including elevated proto-oncogene activation, Ras/B-Raf/mitogen-activated protein kinase, mTOR, and EGF receptor activation, elevated cell cycle activity, macrophage infiltration, elevated cytokines including TNF-α, altered basement membrane deposition, increased integrin-linked kinase activity and cell migration, elevated periostin, increased angiogenesis, and tumor-like defective glucose metabolism (Figure 2).40,41 Uremia associated with ESRD can often cause acquired PKD and renal cell carcinoma (RCC). Furthermore, there is recent evidence indicating that even less severe CKD can increase the incidence of RCC and urothelial cancer.42 Yet, in PKD, numerous studies have shown that very little RCC is seen43 unless the kidneys are removed and examined histologically.44,45
This very low level of diagnosed RCC in ADPKD suggested that there may be a degree of protection from cancer associated with PKD mutations. As such, we carried out an epidemiologic study to answer this question.46 Data from the Scientific Registry of Transplant Recipients, which contains information on all solid organ transplant recipients in the United States, were linked to 15 population-based United States cancer registries that included data on 50 different cancers. Cancer incidence was compared in PKD versus non-PKD renal transplant recipients, and incidence rate ratios adjusted for age, sex, race/ethnicity, dialysis duration, and time since transplantation were determined. The study included >10,000 kidney recipients with PKD and >100,000 without PKD. After multivariable adjustment, overall cancer incidence was found to be significantly lower in patients with ADPKD by about 16%–17%, suggesting that there is cancer protection associated with PKD.46 This was also found to be true for ARPKD, in a study that examined the incidence of colon cancer in unaffected carriers of a recessive PKHD1 mutation.47
The reason for the lower cancer risk in patients with ADPKD is not known, but may relate to biologic characteristics of ADPKD itself or to better cancer risk behaviors with these patients. However, the fact that there was a decreased incidence in ARPKD carriers, who would not know their gene status, makes it more likely that an underlying cellular mechanism is protective. Such a mechanism might lie in the unique biology of PKD cells, in which there is loss of intracellular calcium homeostasis and decreased basal calcium.48,49 This lower calcium allows cAMP activation of the Ras/B-Raf/mitogen-activated protein kinase pathway and would contribute to high levels of c-myc expression, a characteristic PKD hallmark. We have also shown that lower calcium in PKD cells leads to lower levels of Cox-2 expression.
How might these observations explain the cancer protective mechanism? The answer may lie in recent studies demonstrating that high Myc levels50 and Cox-2 inhibition51 prevent cancer metastasis. As such, the unusual PKD biology may paradoxically promote neoplastic cyst growth while preventing progression to metastasis, leading to lower cancer incidence.
Future Directions
It will be important in the future to better understand these cancer-related signaling mechanisms for the design of PKD-specific antiproliferative therapies that will slow cyst growth and kidney enlargement without inadvertently opening a Pandora’s box of metastatic cancer. If the very therapy that suppresses neoplastic cyst growth also allows rogue cells to establish themselves as metastatic tumors, the treatment might end up harming the patient. On the other hand, if there is indeed a PKD-related mechanism that protects cells from becoming malignant, such a discovery should be of significant interest to the cancer research community.
Knowing What We Do Not Know: Polycystin Function In Cilia
Stefan Somlo
The past decade has seen a remarkable evolution in the understanding of the importance of primary cilia in the pathogenesis of structural kidney diseases that manifest with cysts and fibrosis. Primary cilia are single nonmotile hair-like projections on many mammalian cells, including most renal tubular epithelial cells where they reside on the apical surface. They are highly privileged subcellular compartments composed of a microtubular scaffold with overlying plasma membrane that is devoid of other subcellular organelles. All component proteins must be synthesized in the cell body and the cilia are able to select which of these enter and leave the cilia compartment and the respective rates for each of these processes. The importance of cilia to the pathogenesis of PKD was brought to the fore by studies that show that mutations affecting cilia structure and composition produce cysts in mammalian kidney tubules and liver bile ducts and that many of the protein products associated with human and mouse fibrocystic diseases are expressed in and around the cilia compartment.52,53 Most notably, both PC1 and PC2 are expressed in cilia.54,55 This led to the hypothesis that polycystin function is critical to cilia function and that defects in cilia function are critical to the pathogenesis of PKD. Although such a unifying hypothesis is both appealing and reasonable, certain features of the respective cilia mutant and polycystin mutant phenotypes are inconveniently inconsistent. Most notably, loss of cilia results in markedly less pronounced polycystic disease than is seen in ADPKD.56,57
This apparent paradox led us to investigate the genetic interrelationship between inactivation of polycystin proteins alone, of cilia alone, and of polycystin proteins and cilia together.58 We found that, as previously noted, inactivation of polycystins alone resulted in severe PKD whereas inactivation of cilia alone resulted in mild disease. Surprisingly, simultaneous inactivation of polycystins and cilia together resulted in a marked decrease in severity of PKD when compared with polycystin-only inactivation. These findings proved universal—they were applicable to Pkd1 and Pkd2, to all segments of the nephron, and to the bile duct and were independent of the timing of gene inactivation (developmental or adult). We were able to show that the severity of PKD was directly related to the period of time for which intact cilia persisted following loss of polycystins. In aggregate, these data suggest that mechanistically polycystins are inhibitory signals that normally modulate a pathway which is yet to be identified, but that requires intact cilia to function. When this pathway is de-repressed due to loss of polycystins, ADPKD ensues.
Future Directions
These findings really should shift our thinking to ask the following two questions. First, why has evolution kept this complex interplay of polycystin proteins and cilia in place? In other words, what normal physiologic function is important enough to warrant such a relatively complex biologic pathway in the kidney? Second, what is the cilia-dependent cyst-activating pathway that is normally inhibited by the presence of polycystins? Identifying the molecular components of these pathways can be expected to identify novel and effective molecular targets for therapy and ADPKD. The genetic evidence strongly suggests that if we can therapeutically repress the cilia-dependent pathway’s activity in the absence of its normal regulatory components, the polycystins, then we will be able to target ADPKD in a manner that will be specific to ADPKD disease and likely effective. Identification of this cilia-dependent cyst-activating pathway should become one of the top priorities for ADPKD research.
Polycystic Kidney Disease: Cilia And More
Jing Zhou
Polycystin-1 and PC2 are integral membrane proteins forming a receptor channel complex.59 Over a decade ago, PC1 and PC2 were found on the primary cilia.54,55,60 Kidney tubular epithelial cells respond to flow shear stress with a calcium signal. Cells without functional PC1 or PC2 are unable to respond to flow-induced shear stress with a calcium signal.60,61 This calcium signal, visualized by ratiometric calcium imaging, is detectable several seconds after flow stimulation and is amplified by a calcium-induced calcium release mechanism involving a ryanodine receptor.61 Using a recently developed calcium indicator, the calcium signal induced by flow shear stress can now be detected in the primary cilia.61,62 This PC2-dependent calcium signal precedes an increase in cytosolic calcium level via a ryanodine receptor, supporting the previous findings.61,62 This PC1- and PC2-dependent calcium signaling may modulate a number of cellular activities (Figure 2). The ciliary compartment appears to be a separate compartment for calcium signaling.62,63 Although the ciliary functions of PC1 and PC2 are likely not restricted to calcium signaling, studies defining the local as well as global impact of the calcium signal mediated by ciliary PC1 and PC2 will be instructive in understanding polycystin function.
ADPKD is now known as a ciliopathy. Ciliopathies refer to a group of diseases caused by structural or functional defects of the primary cilia. Ciliopathies have a wide range of phenotypes with cystic kidneys as a common feature. What are the proximal events of ciliary polycystins? We searched for polycystin interaction partners. We found that PC2 interacts with the ARPKD protein, fibrocystin/polyductin,64 and nek8 (namely nephronophthisis 9).65 Recently we found that PC1 interacts with several Bardet–Biedl syndrome (BBS) proteins that are part of the BBSome protein complex known to function in the transport of a set of proteins to the cilia.66 Deficiency of BBS1 and BBS3 affects the ciliary trafficking of PC1. Hence, the polycystins are cargos of the BBSome and physical interactions between the polycystins and BBS proteins may underlie the overlapping renal phenotypes in these two diseases. Other components of the trafficking pathways may well be important for polycystin targeting to the cilia.
Polycystins are present at other subcellular compartments besides the primary cilia.59 We recently found that PC1 is present in the lamellipodia in migrating kidney tubular epithelial cells.67 binds to an F-bar protein Pacsin 2 and regulates Pacsin 2 interaction with N-Wasp, an activator of the actin nucleator Arp2/3 protein complex. We found that disorganization of the actin cytoskeleton is a feature of PKD in vivo and that a novel PC1-Pacsin 2-N-Wasp complex is required for the actin remodeling and directional cell migration.67 Directional cell migration is essential for the regeneration and maintenance of the epithelium and kidney development.68 We propose that a defective actin cytoskeleton and directional cell migration contributes cyst formation.
Future Directions
Further understanding the trafficking route and identification of modulators promoting polycystin targeting to the cilia may provide new therapeutics for patients with trafficking defective mutations. In addition, studies to elucidate the role of the cytoskeleton and cell migration in cyst formation may identify novel pathogenic mechanisms and therapeutic targets.
Dna Damage Response Signaling Is A Novel Pathogenic Mechanism In Ciliopathies
Friedhelm Hildebrandt
Nephronophthisis-related ciliopathies (NPHP-RC) are recessive multisystem disorders that affect kidney, retina, liver, and cerebellum either by prenatal-onset dysplasia or by childhood-onset degeneration and fibrosis. Identification of >19 disease-causing genes (NPHP1 to NPHP19) revealed that their products are located at primary cilia and centrosomes. However, the proximal disease mechanisms remain poorly understood.
We identified, by whole exome resequencing, mutations that affect the centrosomal proteins, namely, FAN1, MRE11, ZNF423, and CEP164, as novel causes of NPHP-RC. Surprisingly, these ciliopathy genes serve functions within the DNA damage response (DDR) pathway.69
(1) ZNF423 interacts with the DNA ds-break sensor PARP1, which recruits MRE11 (MRN) and ATM to sites of DNA damage. ATM, in turn, is activated by MRE11 (MRN) and the ‘TIP60 complex’. We demonstrate colocalization to TIP60-positive (and SC35-positive) nuclear foci or protein–protein interactions for the following products 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 recessive mutations of CEP164 as a novel cause of NPHP-RC. CEP164 acts in the ATR-Chk1-related arm of DDR, where it is necessary for ATR-dependent Chk1 activation upon induced replication stress.70
Furthermore, to identify single-gene causes of renal fibrosis/CKD we performed homozygosity mapping and whole exome resequencing in a model disorder for renal fibrosis known as karyomegalic interstitial nephritis (KIN).71
We identified recessive mutations of the Fanconi anemia-associated nuclease 1 gene (FAN1) as causing the NPHP-like renal phenotype of karyomegalic interstitial nephritis (KIN) in nine of 10 families ascertained. KIN causes CKD with renal histology indistinguishable from NPHP, except for the presence of karyomegaly. FAN1 has nuclease activity and acts in DNA interstrand crosslinking (ICL) repair within the Fanconi anemia (FA) pathway of DNA damage response (DDR). Interestingly, ICL-causing genotoxins generate a KIN-like phenotype. We demonstrate that cells from individuals with FAN1 mutations exhibit sensitivity to the ICL agent mitomycin C. We complement ICL sensitivity with wild-type FAN1 but not mutant cDNA from individuals with KIN. The FAN1 defect was not epistatic with the Fanconi anemia pathway. By depletion of fan1 in zebrafish we recapitulated increased DDR, apoptosis, and kidney cysts akin to NPHP.72
We suggest a working hypothesis for the pathogenesis of certain forms of NPHP-RC proposing the following cascade of events: defects of DDR lead to a lack of Chk1 (Chk2) activation, thereby causing inadequate G2/M cell cycle arrest. This would lead in high proliferation states (high replication stress) during morphogenesis to dysplastic phenotypes (Meckel syndrome) and in low proliferation states (low replication stress) during tissue maintenance and repair to tissue degeneration and fibrosis (nephronophthisis).
Future Directions
It is becoming increasingly likely that disruption of cell cycle regulation is central to most forms of renal ciliopathies. This mechanism can be targeted by drugs, as has been shown in models of renal cystic disease using roscovitine and other drugs that interfere with cell cycle regulation.73
Intraflagellar Transport Protein Function Is Not Restricted To The Primary Cilium
Corinne Antignac
Virtually all epithelial cells display primary cilia. However, whereas in immature rat glomeruli, podocytes express cilia, these tend to disappear during development.74 Hence, our identification of a missense mutation (p.P209L) in the TTC21B gene encoding the intraflagellar transport (IFT) 139 protein in seven families with hereditary focal-segmental glomerulosclerosis, discovered during late adolescence or early adulthood, raised the question of a role of IFT139 (and potentially other IFT proteins) beyond the primary cilium.75 Mutations in TTC21B had previously been reported in patients with nephronophthisis,76 but careful re-analysis of the clinical and histologic features of all patients bearing the p.P209L mutation clearly showed that they present with both glomerular and tubulo-interstitial involvement. In agreement, we found that IFT139 was predominantly expressed in distal tubules, as expected for a nephronophthisis-causing gene, but was also strongly expressed in glomerular podocytes. IFT139 was mainly localized at the base of the primary cilium in developing podocytes from human fetal tissue and in undifferentiated cultured podocytes. In contrast, in nonciliated adult podocytes and differentiated cultured cells, IFT139 relocalized along the extended microtubule network.
Our functional studies in undifferentiated podocytes showed that the p.P209L mutation has a hypomorphic effect on podocyte ciliogenesis, but was not sufficient to inhibit podocyte differentiation. These data suggested that the glomerular defects observed in patients with this mutation were, rather, due to a nonciliary alteration of IFT139 in mature podocytes, uncovered by the hypomorphic p.P209L mutation. As actin and microtubule cytoskeletons are key regulators of the delicate architecture, plasticity, and contractility of podocytes,77 and as IFT139 is redistributed along the microtubule network in mature podocytes, we studied the effect of the p.P209L mutant on cytoskeleton organization in differentiated podocytes. Interestingly, the depletion of IFT139 led to increased alterations to the cell surface associated with actin cytoskeleton, such as short and misorganized stress fibers and microtubule rearrangement into bike-wheel-like shape. All these defects were fully rescued by the wild-type protein. In contrast, p.P209L re-expression led to the rescue of the cell-size defect, but actin and microtubule networks remained severely altered. In addition, microtubule repolymerization after nocodazole treatment resulted in multiple nucleation sites dispersed within the cytoplasm in IFT139-depleted differentiated podocytes and these abnormalities were partially rescued upon p.P209 mutant expression.
Future Directions
These data show once again the power of genetic studies in families affected with rare Mendelian disorders. Here, they allow the uncovering of an unexpected role of a ciliary protein in the regulation of podocyte cytoskeleton architecture, and open a new field of investigations into the role of IFT proteins in the mature podocyte, a cell devoid, at this stage, of a primary cilium. We anticipate that future genetic studies in new families with ciliary syndromes will continue the discovery of new gene mutations and be a rich source for uncovering new biology.
Signaling Pathways And Therapeutic Molecules In Pkd
Vicente E. Torres
Understanding the pathogenesis of increased fluid secretion and epithelial cell proliferation downstream from PKD1 or PKD2 mutations is important to identify therapies (Figure 2).40,41 Those targeting proximal mechanisms are more likely to be effective than those targeting distal mechanisms.
Aberrant cross-talk between intracellular calcium and cAMP signaling likely is one of the first effects of PKD mutations. Disrupted calcium may enhance cAMP and protein kinase A signaling through activation of calcium-inhibitable adenylyl cyclases and inhibition of calcium-dependent phosphodiesterases (PDE1 and indirectly cGMP-inhibited PDE3).78 Enhanced protein kinase A activity may in turn disrupt intracellular calcium homeostasis through hyperphosphorylation of calcium cycling proteins in the endoplasmic reticulum. Preclinical studies have targeted calcium signaling with some success. Only triptolide (an activator of the polycystin-2 channel) is being clinically investigated. Gαs protein-coupled receptor antagonists (i.e., vasopressin V2 receptor antagonists) and Gαi protein-coupled receptor agonists (i.e., somatostatin analogs) have demonstrated encouraging results in preclinical and clinical trials. Protein kinase A-induced phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) allows chloride and fluid secretion into the cysts. Anoctamin-1, a calcium-activated chloride channel, may synergistically interact with CFTR.79 CFTR inhibitors inhibit cystogenesis in vitro and in kidney-specific Pkd1 knockout mice, whereas anoctamin-1 inhibitors have been effective in vitro.
Protein kinase A activation inhibits cell proliferation in wild-type cells, but has a stimulatory effect in PKD cells. Calcium deprivation in wild-type cells and delivery of calcium in PKD cells reverse these effects. A proposed mechanism for the proliferative response in PKD and calcium-deprived wild-type cells 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/ extracellular signal-regulated kinase [ERK]; AMP-activated protein kinase [AMPK]/mTOR; arguably Wnt/β-catenin) and transcription factors (hypoxia-inducible factor 1, Myc, P53, signal transducer and activator of transcription 3) that control cell cycle progression, energy metabolism, and the microenvironment. Overexpression of growth factors, cytokines, chemokines, and their receptors further contributes to disease progression. Src is a nonreceptor tyrosine kinase activated downstream from G-protein–coupled and growth factor receptors. SKI-606 (bosutinib) is an Src/Abl inhibitor. KD019 inhibits Src and receptor tyrosine kinases (EGF receptor, ERBB2, vascular endothelial growth factor receptor). Both have shown benefit in preclinical studies. Clinical trials are ongoing. On the other hand, targeting of B-Raf/MEK/ERK has given inconsistent results.
Overwhelming evidence indicates that mTOR signaling is enhanced in cystic tissues. Preclinical studies of rapalogs and mTOR antisense oligonucleotides80 have been encouraging. Clinical trials have been disappointing81–83 likely because clinically achievable blood levels do not inhibit mTOR in the kidney. Strategies to overcome systemic toxicity and limited renal bioavailability are being investigated. Pan-sirtuin (nicotinamide) and sirtuin-1–specific inhibitors, AMPK activators (metformin), thiazolidinediones, and signal transducer and activator of transcription 3 inhibitors have been effective in animal models.84 Nicotinamide is being tested in an uncontrolled, open label clinical trial. Recent studies suggest that PKD cells, like cancer cells, reprogram their energy metabolism from oxidative phosphorylation to aerobic glycolysis.85 Whether drugs interfering with glycolysis will be safe and effective clinically is uncertain.
Future Directions
Significant progress has been made toward identification of effective therapies for PKD. As discussed above, many have been shown to be effective in rodent models, but their safety and efficacy in patients is difficult to predict with accuracy, and only rigorous clinical trials will answer this satisfactorily. One approach that should be considered is the use of combination therapies, which can potentially increase efficacy and reduce toxicity.8,86
The Role Of Mtor Signaling In Cystic Kidney Disease
Gerd Walz
The mTORC1 kinase cascade is almost universally activated in cystic kidney disease independent of the underlying disease-causing gene mutation87,88 (Figure 2). Although the molecular basis for this activation remains incompletely understood, primary cilia appear to curtail mTORC1 activity through a flow-dependent activation of the LKB1/AMPK signaling pathway, involving stabilization of the tuberous sclerosis complex 1 (TSC1)/TSC2 complex and inhibition of Rheb.89 In addition, PC1 might influence the activity of this small GTPase through interaction with the TSC complex,90,91 and inhibit MEK/ERK-mediated phosphorylation of TSC2 to control mTORC1 activity,92 whereas c-Cbl is sequestered in the Golgi apparatus in the absence of polycystin-1, allowing the HGF receptor c-Met to escape degradation after stimulation.93
First shown in the Cy/+ rat model,94 the efficacy of mTORC1 inhibitors to reduce cyst growth and to ameliorate disease progression was subsequently demonstrated in several animal models of cystic kidney disease, including Pkd1-deficient mice.90,95–100 Despite these encouraging preclinical data, mTOR inhibitors did not yield the expected benefits in patients with ADPKD. While everolimus slowed cyst growth in ADPKD patients, this inhibition did not translate into an improvement of renal function. 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 cyst growth and ADPKD disease progression, we abrogated ciliogenesis in the thick ascending limb of Henle and distal tubular segments, generating Kif3a fl/fl*KspCre mice. These mice rapidly developed cystic kidney disease, and died before 12 weeks of age (median survival 7.3 weeks). In contrast, the additional elimination of Raptor (Raptor fl/fl*Kif3a fl/fl*KspCre) delayed cyst growth and dramatically increased the survival by more than three times (median survival 24.0 weeks), revealing the importance of mTORC1 in promoting cyst growth. However, despite the remarkable delay, the Raptor-deficient mice nevertheless developed cystic kidney disease. Gene expression profiling revealed that cyst formation and disease progression in the absence of functional mTORC1 were driven by other growth-promoting pathways, including AKT and ERK.
Future Directions
The rapid development of mTORC1-independent cyst growth in ADPKD resembles the resistance against targeted tumor therapies, where de novo somatic mutations or bypass signaling pathways permit tumor cell proliferation despite effective inhibition of the original oncogenic driver. Reminiscent of the neoplasia in disguise coined many years ago to capture the tumor-like characteristics of cysts in ADPKD,104 a multi-target approach will likely replace the search for a single magic bullet. Several novel concepts that block cell cycle progression and inflammation, or promote apoptosis and epigenetic changes, have been successfully tested in animal models of cystic kidney disease, holding great promise for future clinical 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 increased numbers of cells expanding the wall beyond normal boundaries. Cysts are the primary suspects causing the decline of GFR (Figure 3). This is supported by the following findings: (1) cyst enlargement compresses the interstitial matrix and surrounding tubules entraining, distorting, and destroying arterioles, venules, capillaries, and lymphatics; (2) most cysts separate from the tubules from which they formed; (3) an isolated collecting duct cyst can block urine flow in the branching arcade upstream of it that would ordinarily drain hundreds of filtering nephrons105,106; (4) the glomerulo–tubule junctions of blocked nephrons become thinned and eventually the tubules separate creating atubular glomeruli and apoptotic proximal tubules107–109; (5) cysts lead to hypertension and impair urine concentrating capacity before GFR declines110; (6) morphologic studies of human and animal kidneys reveal that cysts can form in the absence of interstitial pathology105; (7) in ADPKD, cortical fibrosis in the absence of cysts nearby is likely secondary to the downstream blockade of collecting ducts109; (8) a seminal study in PKHD1-null rats unable to synthesize vasopressin proved that fibrosis and renal insufficiency do not develop in the absence of cyst formation, but do when the hormone is directly administered causing cysts to form.111 What causes renal failure in PKD? The cyst did it!
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 proliferation and fluid secretion, although this rate can range widely from patient-to-patient. This causes progressive compression of neighboring structures leading to nephron destruction. Early in the disease, remaining intact nephrons exhibit compensatory hyperfiltration sufficient to preserve GFR (blue line). Further renal injury is mediated by multiple additional mechanisms (purple block arrows) including inflammation and fibrosis. Late in the disease, nephron loss exceeds renal compensatory capacity and GFR starts to decline, reaching a rapid phase generally when the total kidney volume reaches 1500 ml (black arrow), with an inexorable descent toward ESRD (58 years for PKD1, 79 years for PKD2). Advancing uremia is likely to be accompanied by superimposed acquired cystic disease, but apparently without significant increase in the risk of metastatic RCC.
Future Directions
Future therapy should focus on treatment in early childhood. In most heritable cystic disorders affecting humans the cysts begin to form in utero and usually remain below the limits of radiologic detection for a few years postpartum. Intrarenal injury by cysts is clinically announced by hypertension, abdominal pain and hematuria. Early childhood is the optimum time to administer therapies that temper the formation and growth of cysts and preserve long-term function. Evidence indicates that if the formation of cysts can be stopped, long-term function can be preserved indefinitely.111
The major goals of future research should be to: (1) develop animal models that more closely mimic the human condition with respect to cyst formation in utero and cyst formation and growth postpartum; (2) define in molecular detail the initial steps between local synthesis and in situ malfunction of polycystin, polyductin/fibrocystin and NPHP proteins; (3) determine intermediate steps in the pathway from mutated protein to cell cycle activation; (4) find molecules that block or enhance key steps in cystogenic pathways; (5) establish ethical and moral guidelines for treating the very young; and (6) for families comprised of affected or at risk members, develop suitable life-long diets that target protagonists of cyst formation and growth.
Conclusions
The advances that have come from PKD research have been impressive. Nevertheless there are many unanswered questions that need to be addressed in the future if we are to move the field forward in terms of both a complete understanding of the pathogenesis of the disease, and the development and clinical testing of viable therapies:
How do we determine the clinical significance of individual mutations in PKD genes?
What genes modify ADPKD and ARPKD disease severity?
What are the specific developmental and contextual factors that seem to determine the susceptibility to cystogenesis?
What is the relationship between cyst epithelial proliferation and cancer?
What exactly is the role of cilia and ciliary proteins in cyst growth?
How does DNA damage and its response influence renal development?
How are different signaling pathways in PKD related and are there key step(s) that would be particularly advantageous to target for therapy?
What is the most predictive animal model for preclinical testing of PKD therapies?
Is there a scientific, moral, ethical, and practical justification for treating children with PKD?
It is our hope that this symposium will inspire the next generation of PKD investigators to tackle these and other challenging questions in creative new ways, and bring hope to patients and families suffering from PKD.
Disclosures
J.J.G., L.M.G.W., D.J.M.P., and G.W. have served as consultants or on advisory boards for Otsuka Pharmaceuticals, and V.T. and P.H. have received research support from Otsuka.
Acknowledgments
We wish to thank the sponsors of the 2014 Grantham Symposium for their generous support: The PKD Foundation, Otsuka Pharmaceuticals, and the Recanati-Kaplan Foundation.
Footnotes
Published online ahead of print. Publication date available at www.jasn.org.
- Copyright © 2015 by the American Society of Nephrology