Autosomal dominant polycystic kidney disease is an important cause of end-stage renal disease, for which there is no proven therapy. Mutations in PKD1 (the gene encoding polycystin-1) are the principal cause of this disease. The disease begins in utero and is slowly progressive, but it is not known whether cystogenesis is an ongoing process during adult life. We now show that inactivation of Pkd1 in mice before postnatal day 13 results in severely cystic kidneys within 3 weeks, whereas inactivation at day 14 and later results in cysts only after 5 months. We found that cellular proliferation was not appreciably higher in cystic specimens than in age-matched controls, but the abrupt change in response to Pkd1 inactivation corresponded to a previously unrecognized brake point during renal growth and significant changes in gene expression. These findings suggest that the effects of Pkd1 inactivation are defined by a developmental switch that signals the end of the terminal renal maturation process. Our studies show that Pkd1 regulates tubular morphology in both developing and adult kidney, but the pathologic consequences of inactivation are defined by the organ's developmental status. These results have important implications for clinical understanding of the disease and therapeutic approaches.

Polycystin-1 (PC1) has an essential function in renal tubular morphogenesis and disruption of its function causes cystogenesis in human autosomal dominant polycystic kidney disease. We have previously shown that recombinant human PC1 is cis -autoproteolytically cleaved at the G protein-coupled receptor proteolytic site domain. To investigate the role of cleavage in vivo , we generated by gene targeting a Pkd1 knockin mouse ( Pkd1 V/V ) that expresses noncleavable PC1. The Pkd1 V/V mice show a hypomorphic phenotype, characterized by a delayed onset and distal nephron segment involvement of cystogenesis at postnatal maturation stage. We show that PC1 is ubiquitously and incompletely cleaved in wild-type mice, so that uncleaved and cleaved PC1 molecules coexist. Our study establishes a critical but restricted role of cleavage for PC1 function and suggests a differential function of the two types of PC1 molecules in vivo .

The results indicate that polyductin is part of the group of polycystic kidney disease (PKD)-related proteins expressed in primary apical cilia. Our data also suggest that, in addition to its likely involvement in cilia function, polyductin probably serves in other subcellular functional roles. The detection of three different products using two antisera, with evidence for distinct subcellular localizations, suggests that PKHD1 encodes membrane-bound and soluble isoforms.

BackgroundThe major gene mutated in autosomal dominant polycystic kidney disease was first identified over 20 years ago, yet its function remains poorly understood. We have used a systems-based approach to examine the effects of acquired loss of Pkd1 in adult mouse kidney as it transitions from normal to cystic state.MethodsWe performed transcriptional profiling of a large set of male and female kidneys, along with metabolomics and lipidomics analyses of a subset of male kidneys. We also assessed the effects of a modest diet change on cyst progression in young cystic mice. Fatty acid oxidation and glycolytic rates were measured in five control and mutant pairs of epithelial cells.ResultsWe find that females have a significantly less severe kidney phenotype and correlate this protection with differences in lipid metabolism. We show that sex is a major determinant of the transcriptional profile of mouse kidneys and that some of this difference is due to genes involved in lipid metabolism. Pkd1 mutant mice have transcriptional profiles consistent with changes in lipid metabolism and distinct metabolite and complex lipid profiles in kidneys. We also show that cells lacking Pkd1 have an intrinsic fatty acid oxidation defect and that manipulation of lipid content of mouse chow modifies cystic disease.InterpretationOur results suggest PKD could be a disease of altered cellular metabolism.

Polycystic kidney disease (PKD) describes a heterogeneous collection of disorders that differ significantly with respect to their etiology and clinical presentation. They share, however, abnormal tubular morphology as a common feature, leading to the hypothesis that their respective gene products may function cooperatively in a common pathway to maintain tubular integrity. To study the pathobiology of one major form of human PKD, we generated a mouse line with a floxed allele of Pkhd1, the orthologue of the gene mutated in human autosomal recessive PKD. Cre-mediated excision of exons 3-4 results in a probable hypomorphic allele. Pkhd1(del3-4/del3-4) developed a range of phenotypes that recapitulate key features of the human disease. Like in humans, abnormalities of the biliary tract were an invariant finding. Most mice 6 months or older also developed renal cysts. Subsets of animals presented with either perinatal respiratory failure or exhibited growth retardation that was not due to the renal disease. We then tested for genetic interaction between Pkhd1 and Pkd1, the mouse orthologue of the gene most commonly linked to human autosomal dominant PKD. Pkd1(+/-); Pkhd1(del3-4/del3-4) mice had markedly more severe disease than Pkd1(+/+); Pkhd1(del3-4/del3-4) littermates. These studies are the first to show genetic interaction between the major loci responsible for human renal cystic disease in a common PKD pathway.

Mutations at a single locus, PKHD1, are responsible for causing human autosomal recessive polycystic kidney disease (ARPKD). Recent studies suggest that the cystic disease might result from defects in planar cell polarity, but how the 4074 amino acid ciliary protein encoded by the longest open reading frame of this transcriptionally complex gene may regulate this process is unknown. Using novel in vitro expression systems, we show that the PKHD1 gene product polyductin/fibrocystin undergoes a complicated pattern of Notch-like proteolytic processing. Cleavage at a probable proprotein convertase site produces a large extracellular domain that is tethered to the C-terminal stalk by disulfide bridges. This fragment is then shed from the primary cilium by activation of a member of the ADAM metalloproteinase disintegrins family, resulting in concomitant release of an intra-cellular C-terminal fragment via a gamma-secretase-dependent process. The ectodomain of endogenous PD1 is similarly shed from the primary cilium upon activation of sheddases. This is the first known example of this process involving a protein of the primary cilium and suggests a novel mechanism whereby proteins that localize to this structure may function as bi-directional signaling molecules. Regulated release from the primary cilium into the lumen may be a mechanism to distribute signal to down-stream targets using flow.

Recent studies have reported intrinsic metabolic reprogramming in Pkd1 knock-out cells, implicating dysregulated cellular metabolism in the pathogenesis of polycystic kidney disease. However, the exact nature of the metabolic changes and their underlying cause remains controversial. We show herein that Pkd1ko/ko renal epithelial cells have impaired fatty acid utilization, abnormal mitochondrial morphology and function, and that mitochondria in kidneys of ADPKD patients have morphological alterations. We further show that a C-terminal cleavage product of polycystin-1 (CTT) translocates to the mitochondria matrix and that expression of CTT in Pkd1ko/ko cells rescues some of the mitochondrial phenotypes. Using Drosophila to model in vivo effects, we find that transgenic expression of mouse CTT results in decreased viability and exercise endurance but increased CO2 production, consistent with altered mitochondrial function. Our results suggest that PC1 may play a direct role in regulating mitochondrial function and cellular metabolism and provide a framework to understand how impaired mitochondrial function could be linked to the regulation of tubular diameter in both physiological and pathological conditions.

Mutations in PKD1 cause the majority of cases of autosomal dominant polycystic kidney disease (ADPKD). Because polycystin 1 modulates cell proliferation, cell differentiation, and apoptosis, its lower biologic activity observed in ADPKD might influence the degree of injury after renal ischemia/reperfusion. We induced renal ischemia/reperfusion in 10-to 12-wk-old male noncystic Pkd1 ϩ/Ϫ and wild-type mice. Compared with wild-type mice, heterozygous mice had higher fractional excretions of sodium and potassium and higher serum creatinine after 48 h. In addition, in heterozygous mice, also cortical damage, rates of apoptosis, and inflammatory infiltration into the interstitium at time points out to 14 d after injury all increased, as well as cell proliferation at 48 h and 7 d. The mRNA and protein expression of p21 was lower in heterozygous mice than wild-type mice at 48 h. After 6 wk, we observed dilated tubules, microcysts, and increased renal fibrosis in heterozygotes. The early mortality of heterozygotes was significantly higher than that of wild-type mice when we extended the duration of ischemia from 32 to 35 min. In conclusion, ischemia/reperfusion induces a more severe injury in kidneys of Pkd1-haploinsufficient mice, a process that apparently depends on a relative deficiency of p21 activity, tubular dilation, and microcyst formation. These data suggest the possibility that humans with ADPKD from PKD1 mutations may be at greater risk for damage from renal ischemia/reperfusion injury.