Multiomics profiling of mouse polycystic kidney disease progression at a single-cell resolution.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to kidney failure. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single-nucleus multimodal atlas of an orthologous mouse PKD model at early, mid, and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalog differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single-nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution.

Monoallelic IFT140 pathogenic variants are an important cause of the autosomal dominant polycystic kidney-spectrum phenotype.

Autosomal dominant polycystic kidney disease (ADPKD), characterized by progressive cyst formation/expansion, results in enlarged kidneys and often end stage kidney disease. ADPKD is genetically heterogeneous; PKD1 and PKD2 are the common loci (∼78% and ∼15% of families) and GANAB, DNAJB11, and ALG9 are minor genes. PKD is a ciliary-associated disease, a ciliopathy, and many syndromic ciliopathies have a PKD phenotype. In a multi-cohort/-site collaboration, we screened ADPKD-diagnosed families that were naive to genetic testing (n = 834) or for whom no PKD1 and PKD2 pathogenic variants had been identified (n = 381) with a PKD targeted next-generation sequencing panel (tNGS; n = 1,186) or whole-exome sequencing (WES; n = 29). We identified monoallelic IFT140 loss-of-function (LoF) variants in 12 multiplex families and 26 singletons (1.9% of naive families). IFT140 is a core component of the intraflagellar transport-complex A, responsible for retrograde ciliary trafficking and ciliary entry of membrane proteins; bi-allelic IFT140 variants cause the syndromic ciliopathy, short-rib thoracic dysplasia (SRTD9). The distinctive monoallelic phenotype is mild PKD with large cysts, limited kidney insufficiency, and few liver cysts. Analyses of the cystic kidney disease probands of Genomics England 100K showed that 2.1% had IFT140 LoF variants. Analysis of the UK Biobank cystic kidney disease group showed probands with IFT140 LoF variants as the third most common group, after PKD1 and PKD2. The proximity of IFT140 to PKD1 (∼0.5 Mb) in 16p13.3 can cause diagnostic confusion, and PKD1 variants could modify the IFT140 phenotype. Importantly, our studies link a ciliary structural protein to the ADPKD spectrum.

Polycystin-2-dependent transcriptome reveals early response of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in polycystin genes, Pkd1 and Pkd2, but the underlying pathogenic mechanisms are poorly understood. To identify genes and pathways that operate downstream of polycystin-2 (PC2), a comprehensive gene expression database was created, cataloging changes in the transcriptome immediately following PC2 protein depletion. To explore cyst initiation processes, an immortalized mouse inner medullary collecting duct line was developed with the ability to knock out the Pkd2 gene conditionally. Genome-wide transcriptome profiling was performed using RNA sequencing in the cells immediately after PC2 was depleted and compared with isogenic control cells. Differentially expressed genes were identified, and a bioinformatic analysis pipeline was implemented. Altered expression of candidate cystogenic genes was validated in Pkd2 knockout mice. The expression of nearly 900 genes changed upon PC2 depletion. Differentially expressed genes were enriched for genes encoding components of the primary cilia, the canonical Wnt pathway, and MAPK signaling. Among the PC2-dependent ciliary genes, the transcription factor Glis3 was significantly downregulated. MAPK signaling formed a key node at the epicenter of PC2-dependent signaling networks. Activation of Wnt and MAPK signaling, concomitant with the downregulation of Glis3, was corroborated in Pkd2 knockout mice. The data identify a PC2 cilia-to-nucleus signaling axis and dysregulation of the Gli-similar subfamily of transcription factors as a potential initiator of cyst formation in ADPKD. The catalog of PC2-regulated genes should provide a valuable resource for future ADPKD research and new opportunities for drug development.NEW & NOTEWORTHY Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Mutations in polycystin genes cause the disease, but the underlying mechanisms of cystogenesis are unknown. To help fill this knowledge gap, we created an inducible cell model of ADPKD and assembled a catalog of genes that respond in immediate proximity to polycystin-2 depletion using transcriptomic profiling. The catalog unveils a ciliary signaling-to-nucleus axis proximal to polycystin-2 dysfunction, highlighting Glis, Wnt, and MAPK signaling.

A genetic screen in Drosophila reveals an unexpected role for the KIP1 ubiquitination-promoting complex in male fertility.

A unifying feature of polycystin-2 channels is their localization to both primary and motile cilia/flagella. In Drosophila melanogaster, the fly polycystin-2 homologue, Amo, is an ER protein early in sperm development but the protein must ultimately cluster at the flagellar tip in mature sperm to be fully functional. Male flies lacking appropriate Amo localization are sterile due to abnormal sperm motility and failure of sperm storage. We performed a forward genetic screen to identify additional proteins that mediate ciliary trafficking of Amo. Here we report that Drosophila homologues of KPC1 and KPC2, which comprise the mammalian KIP1 ubiquitination-promoting complex (KPC), form a conserved unit that is required for the sperm tail tip localization of Amo. Male flies lacking either KPC1 or KPC2 phenocopy amo mutants and are sterile due to a failure of sperm storage. KPC is a heterodimer composed of KPC1, an E3 ligase, and KPC2 (or UBAC1), an adaptor protein. Like their mammalian counterparts Drosophila KPC1 and KPC2 physically interact and they stabilize one another at the protein level. In flies, KPC2 is monoubiquitinated and phosphorylated and this modified form of the protein is located in mature sperm. Neither KPC1 nor KPC2 directly interact with Amo but they are detected in proximity to Amo at the tip of the sperm flagellum. In summary we have identified a new complex that is involved in male fertility in Drosophila melanogaster.

GDNF drives rapid tubule morphogenesis in a novel 3D in vitro model for ADPKD.

Cystogenesis is a morphological consequence of numerous genetic diseases of the epithelium. In the kidney, the pathogenic mechanisms underlying the program of altered cell and tubule morphology are obscured by secondary effects of cyst expansion. Here, we developed a new 3D tubuloid system to isolate the rapid changes in protein localization and gene expression that correlate with altered cell and tubule morphology during cyst initiation. Mouse renal tubule fragments were pulsed with a cell differentiation cocktail including glial-derived neurotrophic factor (GDNF) to yield collecting duct-like tubuloid structures with appropriate polarity, primary cilia, and gene expression. Using the 3D tubuloid model with an inducible Pkd2 knockout system allowed the tracking of morphological, protein, and genetic changes during cyst formation. Within hours of inactivation of Pkd2 and loss of polycystin-2, we observed significant progression in tubuloid to cyst morphology that correlated with 35 differentially expressed genes, many related to cell junctions, matrix interactions, and cell morphology previously implicated in cystogenesis.This article has an associated First Person interview with the first author of the paper.

Polycystin-1 dependent regulation of polycystin-2 via GRP94, a member of HSP90 family that resides in the endoplasmic reticulum.

Autosomal dominant polycystic kidney disease is a common inherited renal disorder that results from mutations in either PKD1 or PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Downregulation or overexpression of PKD1 or PKD2 in mouse models results in renal cyst formation, suggesting that the quantity of PC1 and PC2 needs to be maintained within a tight functional window to prevent cystogenesis. Here we show that enhanced PC2 expression is a common feature of PKD1 mutant tissues, in part due to an increase in Pkd2 mRNA. However, our data also suggest that more effective protein folding contributes to the augmented levels of PC2. We demonstrate that the unfolded protein response is activated in Pkd1 knockout kidneys and in Pkd1 mutant cells and that this is coupled with increased levels of GRP94, an endoplasmic reticulum protein that is a member of the HSP90 family of chaperones. GRP94 was found to physically interact with PC2 and depletion or chemical inhibition of GRP94 led to a decrease in PC2, suggesting that GRP94 serves as its chaperone. Moreover, GRP94 is acetylated and binds to histone deacetylase 6 (HDAC6), a known deacetylase and activator of HSP90 proteins. Inhibition of HDAC6 decreased PC2 suggesting that HDAC6 and GRP94 work together to regulate PC2 levels. Lastly, we showed that inhibition of GRP94 prevents cAMP-induced cyst formation in vitro. Taken together our data uncovered a novel HDAC6-GRP94-related axis that likely participates in maintaining elevated PC2 levels in Pkd1 mutant cells.

TWEAK Signaling Pathway Blockade Slows Cyst Growth and Disease Progression in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: In autosomal dominant polycystic kidney disease (ADPKD), cyst development and enlargement lead to ESKD. Macrophage recruitment and interstitial inflammation promote cyst growth. TWEAK is a TNF superfamily (TNFSF) cytokine that regulates inflammatory responses, cell proliferation, and cell death, and its receptor Fn14 (TNFRSF12a) is expressed in macrophage and nephron epithelia. METHODS: To evaluate the role of the TWEAK signaling pathway in cystic disease, we evaluated Fn14 expression in human and in an orthologous murine model of ADPKD. We also explored the cystic response to TWEAK signaling pathway activation and inhibition by peritoneal injection. RESULTS: Meta-analysis of published animal-model data of cystic disease reveals mRNA upregulation of several components of the TWEAK signaling pathway. We also observed that TWEAK and Fn14 were overexpressed in mouse ADPKD kidney cysts, and TWEAK was significantly high in urine and cystic fluid from patients with ADPKD. TWEAK administration induced cystogenesis and increased cystic growth, worsening the phenotype in a murine ADPKD model. Anti-TWEAK antibodies significantly slowed the progression of ADPKD, preserved renal function, and improved survival. Furthermore, the anti-TWEAK cystogenesis reduction is related to decreased cell proliferation-related MAPK signaling, decreased NF-κB pathway activation, a slight reduction of fibrosis and apoptosis, and an indirect decrease in macrophage recruitment. CONCLUSIONS: This study identifies the TWEAK signaling pathway as a new disease mechanism involved in cystogenesis and cystic growth and may lead to a new therapeutic approach in ADPKD.

A novel model of autosomal recessive polycystic kidney questions the role of the fibrocystin C-terminus in disease mechanism.

Autosomal recessive polycystic kidney disease (OMIM 263200) is a serious condition of the kidney and liver caused by mutations in a single gene, PKHD1. This gene encodes fibrocystin/polyductin (FPC, PD1), a large protein shown by in vitro studies to undergo Notch-like processing. Its cytoplasmic tail, reported to include a ciliary targeting sequence, a nuclear localization signal, and a polycystin-2 binding domain, is thought to traffic to the nucleus after cleavage. We now report a novel mouse line with a triple HA-epitope "knocked-in" to the C-terminus along with lox P sites flanking exon 67, which encodes most of the C-terminus (Pkhd1Flox67HA). The triple HA-epitope has no functional effect as assayed by phenotype and allows in vivo tracking of Fibrocystin. We used the HA tag to identify previously predicted Fibrocystin cleavage products in tissue. In addition, we found that Polycystin-2 fails to co-precipitate with Fibrocystin in kidney samples. Immunofluorescence studies with anti-HA antibodies demonstrate that Fibrocystin is primarily present in a sub-apical location the in kidney, biliary duct, and pancreatic ducts, partially overlapping with the Golgi. In contrast to previous studies, the endogenous protein in the primary cilia was not detectable in mouse tissues. After Cre-mediated deletion, homozygous Pkhd1Δ67 mice are completely normal. Thus, Pkhd1Flox67HA is a valid model to track Pkhd1-derived products containing the C-terminus. Significantly, exon 67 containing the nuclear localization signal and the polycystin-2 binding domain is not essential for Fibrocystin function in our model.

Inhibition of histone deacetylase 6 activity reduces cyst growth in polycystic kidney disease.

Abnormal proliferation of cyst-lining epithelium and increased intracystic fluid secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) are thought to contribute to cyst growth in autosomal dominant polycystic kidney disease (ADPKD). Histone deacetylase 6 (HDAC6) expression and activity are increased in certain cancers, neurodegenerative diseases, and in Pkd1-mutant renal epithelial cells. Inhibition of HDAC6 activity with specific inhibitors slows cancer growth. Here we studied the effect of tubacin, a specific HDAC6 inhibitor, on cyst growth in polycystic kidney disease. Treatment with tubacin prevented cyst formation in MDCK cells, an in vitro model of cystogenesis. Cyclic AMP stimulates cell proliferation and activates intracystic CFTR-mediated chloride secretion in ADPKD. Treatment with tubacin downregulated cyclic AMP levels, inhibited cell proliferation, and inhibited cyclic AMP-activated CFTR chloride currents in MDCK cells. We also found that tubacin reduced cyst growth by inhibiting proliferation of cyst-lining epithelial cells, downregulated cyclic AMP levels, and improved renal function in a Pkd1-conditional mouse model of ADPKD. Thus, HDAC6 could play a role in cyst formation and could serve as a potential therapeutic target in ADPKD.

Multi-omics profiling of mouse polycystic kidney disease progression at a single cell resolution.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to end-stage kidney disease. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single nucleus multimodal atlas of an orthologous mouse PKD model at early, mid and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalogue differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution.

Fibrocystin/Polyductin releases a C-terminal fragment that translocates into mitochondria and suppresses cystogenesis.

Fibrocystin/Polyductin (FPC), encoded by PKHD1, is associated with autosomal recessive polycystic kidney disease (ARPKD), yet its precise role in cystogenesis remains unclear. Here we show that FPC undergoes complex proteolytic processing in developing kidneys, generating three soluble C-terminal fragments (ICDs). Notably, ICD15, contains a novel mitochondrial targeting sequence at its N-terminus, facilitating its translocation into mitochondria. This enhances mitochondrial respiration in renal epithelial cells, partially restoring impaired mitochondrial function caused by FPC loss. FPC inactivation leads to abnormal ultrastructural morphology of mitochondria in kidney tubules without cyst formation. Moreover, FPC inactivation significantly exacerbates renal cystogenesis and triggers severe pancreatic cystogenesis in a Pkd1 mouse mutant Pkd1V/V in which cleavage of Pkd1-encoded Polycystin-1 at the GPCR Proteolysis Site is blocked. Deleting ICD15 enhances renal cystogenesis without inducing pancreatic cysts in Pkd1V/V mice. These findings reveal a direct link between FPC and a mitochondrial pathway through ICD15 cleavage, crucial for cystogenesis mechanisms.

Genomic organization and mutation screening of the human ortholog of Pkdr1 associated with polycystic kidney disease in the rat.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited disorders in humans. Although disease-causing mutations have been found in two genes, PKD1 and PKD2, a small number of ADPKD families exist that are unlinked to either of these genes, suggesting involvement of a third, as yet unidentified PKD3 gene. Susceptibility to renal cyst formation in the (cy/+) rat is caused by a missense mutation in Pkdr1 encoding the novel protein SamCystin. To initiate studies of the human orthologous gene, we determined the location and the organization of human PKDR1. We genotyped microsatellite markers flanking the human ortholog in PKD families that either are unlinked to known PKD genes, or in which mutations have not yet been identified and carried out mutation analysis in PKD patients. We identified eight novel single nucleotide polymorphisms, including three leading to amino acid changes. These variants are unlikely to account for PKD in these patients, yet the screening of other affected populations may provide information about the involvement of PKDR1 as a modifier gene in cystic kidney disease.

A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed.

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 Pkd1 k o /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 Pkd1 ko/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.

NEDD4-family E3 ligase dysfunction due to PKHD1/Pkhd1 defects suggests a mechanistic model for ARPKD pathobiology.

Autosomal recessive polycystic kidney disease (ARPKD) is an important childhood nephropathy, occurring 1 in 20,000 live births. The major clinical phenotypes are expressed in the kidney with dilatation of the collecting ducts, systemic hypertension, and progressive renal insufficiency, and in the liver with biliary dysgenesis, portal tract fibrosis, and portal hypertension. The systemic hypertension has been attributed to enhanced distal sodium reabsorption in the kidney, the structural defects have been ascribed to altered cellular morphology, and fibrosis to increased TGF-ß signaling in the kidney and biliary tract, respectively. The pathogenic mechanisms underlying these abnormalities have not been determined. In the current report, we find that disrupting PKHD1 results in altered sub-cellular localization and function of the C2-WWW-HECT domain E3 family of ligases regulating these processes. We also demonstrate altered activity of RhoA and increased TGF-ß signaling and ENaC activity. Linking these phenomena, we found that vesicles containing the PKHD1/Pkhd1 gene product, FPC, also contain the NEDD4 ubiquitin ligase interacting protein, NDFIP2, which interacts with multiple members of the C2-WWW-HECT domain E3 family of ligases. Our results provide a mechanistic explanation for both the cellular effects and in vivo phenotypic abnormalities in mice and humans that result from Pkhd1/PKHD1 mutation.

The polycystin complex mediates Wnt/Ca(2+) signalling.

WNT ligands induce Ca(2+) signalling on target cells. PKD1 (polycystin 1) is considered an orphan, atypical G-protein-coupled receptor complexed with TRPP2 (polycystin 2 or PKD2), a Ca(2+)-permeable ion channel. Inactivating mutations in their genes cause autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases. Here, we show that WNTs bind to the extracellular domain of PKD1 and induce whole-cell currents and Ca(2+) influx dependent on TRPP2. Pathogenic PKD1 or PKD2 mutations that abrogate complex formation, compromise cell surface expression of PKD1, or reduce TRPP2 channel activity suppress activation by WNTs. Pkd2(-/-) fibroblasts lack WNT-induced Ca(2+) currents and are unable to polarize during directed cell migration. In Xenopus embryos, pkd1, Dishevelled 2 (dvl2) and wnt9a act within the same pathway to preserve normal tubulogenesis. These data define PKD1 as a WNT (co)receptor and implicate defective WNT/Ca(2+) signalling as one of the causes of ADPKD.

A novel pattern of mutation in uromodulin disorders: autosomal dominant medullary cystic kidney disease type 2, familial juvenile hyperuricemic nephropathy, and autosomal dominant glomerulocystic kidney disease.

BACKGROUND: Autosomal dominant medullary cystic kidney disease type 2 (MCKD2), familial juvenile hyperuricemic nephropathy (FJHN), and autosomal dominant glomerulocystic kidney disease (GCKD) constitute a hereditary renal disease group that may lead to end-stage renal failure caused by mutations of the UMOD gene and its product, uromodulin or Tamm-Horsfall protein. Of 34 different UMOD mutations described to date, 28 were located in exon 4. Based on such mutation clustering, some investigators have proposed that the sequencing of UMOD exon 4 might become a preliminary diagnostic test for patients with this phenotype. METHODS: We performed linkage analysis and sequencing of the entire codifying region of the UMOD gene in 4 Spanish families with MCKD/FJHN/GCKD. RESULTS: All families were shown to present mutations in the UMOD gene. In 3 families, the detected mutations were located in exon 5. Although 1 novel mutation (Gln316Pro) was observed in 2 of these families, a previously reported mutation (Cys300Gly) was found in the other kindred. The Cys300Gly mutation was found in the family presenting with a GCKD phenotype. CONCLUSION: Our data show a novel mutation pattern in UMOD , suggesting that exon 5 mutations can be more frequent in some populations. Our results support that every exon of the UMOD gene must be included in molecular testing and provide additional evidence for the existence of a fourth calcium-binding epidermal growth factor-like domain in the structure of Tamm-Horsfall protein. A second family reported to date is described, confirming that the GCKD phenotype may be caused by a UMOD mutation.

Polycystin signaling is required for directed endothelial cell migration and lymphatic development.

Autosomal dominant polycystic kidney disease is a common form of inherited kidney disease that is caused by mutations in two genes, PKD1 (polycystin-1) and PKD2 (polycystin-2). Mice with germline deletion of either gene die in midgestation with a vascular phenotype that includes profound edema. Although an endothelial cell defect has been suspected, the basis of this phenotype remains poorly understood. Here, we demonstrate that edema in Pkd1- and Pkd2-null mice is likely to be caused by defects in lymphatic development. Pkd1 and Pkd2 mutant embryos exhibit reduced lymphatic vessel density and vascular branching along with aberrant migration of early lymphatic endothelial cell precursors. We used cell-based assays to confirm that PKD1- and PKD2-depleted endothelial cells have an intrinsic defect in directional migration that is associated with a failure to establish front-rear polarity. Our studies reveal a role for polycystin signaling in lymphatic development.

Ciliary membrane proteins traffic through the Golgi via a Rabep1/GGA1/Arl3-dependent mechanism.

Primary cilia contain specific receptors and channel proteins that sense the extracellular milieu. Defective ciliary function causes ciliopathies such as autosomal dominant polycystic kidney disease (ADPKD). However, little is known about how large ciliary transmembrane proteins traffic to the cilia. Polycystin-1 (PC1) and -2 (PC2), the two ADPKD gene products, are large transmembrane proteins that co-localize to cilia where they act to control proper tubular diameter. Here we describe that PC1 and PC2 must interact and form a complex to reach the trans-Golgi network (TGN) for subsequent ciliary targeting. PC1 must also be proteolytically cleaved at a GPS site for this to occur. Using yeast two-hybrid screening coupled with a candidate approach, we identify a Rabep1/GGA1/Arl3-dependent ciliary targeting mechanism, whereby Rabep1 couples the polycystin complex to a GGA1/Arl3-based ciliary trafficking module at the TGN. This study provides novel insights into the ciliary trafficking mechanism of membrane proteins.

Pkd1 and Pkd2 are required for normal placental development.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of inherited renal failure that results from mutations in PKD1 and PKD2. The disorder is characterized by focal cyst formation that involves somatic mutation of the wild type allele in a large fraction of cysts. Consistent with a two-hit mechanism, mice that are homozygous for inactivating mutations of either Pkd1 or Pkd2 develop cystic kidneys, edema and hemorrhage and typically die in midgestation. Cystic kidney disease is unlikely to be the cause of fetal loss since renal function is not required to complete gestation. One hypothesis is that embryonic demise is due to leaky vessels or cardiac pathology. METHODOLOGY/PRINCIPAL FINDINGS: In these studies we used a series of genetically modified Pkd1 and Pkd2 murine models to investigate the cause of embryonic lethality in mutant embryos. Since placental defects are a frequent cause of fetal loss, we conducted histopathologic analyses of placentas from Pkd1 null mice and detected abnormalities of the labyrinth layer beginning at E12.5. We performed placental rescue experiments using tetraploid aggregation and conditional inactivation of Pkd1 with the Meox2 Cre recombinase. We found that both strategies improved the viability of Pkd1 null embryos. Selective inactivation of Pkd1 and Pkd2 in endothelial cells resulted in polyhydramnios and abnormalities similar to those observed in Pkd1(-/-) placentas. However, endothelial cell specific deletion of Pkd1 or Pkd2 did not yield the dramatic vascular phenotypes observed in null animals. CONCLUSIONS/SIGNIFICANCE: Placental abnormalities contribute to the fetal demise of Pkd(-/-) embryos. Endothelial cell specific deletion of Pkd1 or Pkd2 recapitulates a subset of findings seen in Pkd null animals. Our studies reveal a complex role for polycystins in maintaining vascular integrity.