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Introduction Isolated polycystic liver disease (PCLD; also called ADPLD) presents in adult life with radiologically and pathologically identical liver cysts to those seen in autosomal dominant polycystic kidney disease (ADPKD), [...], Dominantly inherited isolated polycystic liver disease (PCLD) consists of liver cysts that are radiologically and pathologically identical to those seen in autosomal dominant polycystic kidney disease, but without clinically relevant kidney cysts. The causative genes are known for fewer than 40% of PCLD index cases. Here, we have used whole exome sequencing in a discovery cohort of 102 unrelated patients who were excluded for mutations in the 2 most common PCLD genes, PRKCSH and SEC63, to identify heterozygous loss-of-function mutations in 3 additional genes, ALC8, CANAB, and SEC61B. Similarly to PRKCSH and SEC63, these genes encode proteins that are integral to the protein biogenesis pathway in the endoplasmic reticulum. We inactivated these candidate genes in cell line models to show that loss of function of each results in defective maturation and trafficking of polycystin-1, the central determinant of cyst pathogenesis. Despite acting in a common pathway, each PCLD gene product demonstrated distinct effects on polycystin-1 biogenesis. We also found enrichment on a genome-wide basis of heterozygous mutations in the autosomal recessive polycystic kidney disease gene PKHD1, indicating that adult PKHD1 carriers can present with clinical PCLD. These findings define genetic and biochemical modulators of polycystin-1 function and provide a more complete definition of the spectrum of dominant human polycystic diseases.

Introduction Isolated autosomal dominant polycystic liver disease (ADPLD) is a slowly progressive genetic disorder characterized by overgrowth of the biliary epithelium and the surrounding connective tissue leading to the formation [...], The HSP40 cochaperone SEC63 is associated with the SEC61 translocon complex in the ER. Mutations in the gene encoding SEC63 cause polycystic liver disease in humans; however, it is not clear how altered SEC63 influences disease manifestations. In mice, loss of SEC63 induces cyst formation both in liver and kidney as the result of reduced polycystin-1 (PC1). Here we report that inactivation of SEC63 induces an unfolded protein response (UPR) pathway that is protective against cyst formation. Specifically, using murine genetic models, we determined that SEC63 deficiency selectively activates the IRE1α-XBP1 branch of UPR and that SEC63 exists in a complex with PC1. Concomitant inactivation of both SEC63 and XBP1 exacerbated the polycystic kidney phenotype in mice by markedly suppressing cleavage at the G protein-coupled receptor proteolysis site (GPS) in PC1. Enforced expression of spliced XBP1 (XBP1s) enhanced GPS cleavage of PC1 in SEC63- deficient cells, and XBP1 overexpression in vivo ameliorated cystic disease in a murine model with reduced PC1 function that is unrelated to SEC63 inactivation. Collectively, the findings show that SEC63 function regulates IRE1α/XBP1 activation, SEC63 and XBP1 are required for GPS cleavage and maturation of PC1, and activation of XBP1 can protect against polycystic disease in the setting of impaired biogenesis of PC1.

Introduction The most common and severe form of autosomal dominant polycystic kidney disease (ADPKD) results from mutations in PKD1. PKD1 encodes human polycystin-1 (PC1), a large, complex, low-abundance polytopic membrane [...], The most severe form of autosomal dominant polycystic kidney disease occurs in patients with mutations in the gene (PKD1) encoding polycystin-1 (PC1). PC1 is a complex polytopic membrane protein expressed in cilia that undergoes autoproteolytic cleavage at a G protein-coupled receptor proteolytic site (GPS). A quarter of PKD1 mutations are missense variants, though it is not clear how these mutations promote disease. Here, we established a cell-based system to evaluate these mutations and determined that GPS cleavage is required for PC1 trafficking to cilia. A common feature among a subset of pathogenic missense mutations is a resulting failure of PC1 to traffic to cilia regardless of GPS cleavage. The application of our system also identified a missense mutation in the gene encoding polycystin-2 (PC2) that prevented this protein from properly trafficking to cilia. Using a Pkd1-BAC recombineering approach, we developed murine models to study the effects of these mutations and confirmed that only the cleaved form of PC1 exits the ER and can rescue the embryonically lethal Pkd1-null mutation. Additionally, steady-state expression levels of the intramembranous COOH-terminal fragment of cleaved PC1 required an intact interaction with PC2. The results of this study demonstrate that PC1 trafficking and expression require GPS cleavage and PC2 interaction, respectively, and provide a framework for functional assays to categorize the effects of missense mutations in polycystins.

Introduction Epithelial cell polarity is an important process involved in renal tubule formation presenting itself in two dimensions: apical-basolateral polarity, established at the point the cell membrane differentiates into an [...], Epithelial cell polarity is essential for organ development; aberrations in this process have been implicated in various diseases, including polycystic kidney disease. Establishment and maintenance of cell polarity is governed by a number of molecular processes and how these processes operate remains an interesting question. Conserved protein complexes guide both apical-basolateral polarity and planar cell polarity. In this review we discuss the recent findings that provide insights into polarity mechanisms and the intriguing crosstalk between apical-basolateral polarity and planar cell polarity, and their relationship to cystic kidney disease. Keywords Cystic kidney disease * Cell polarity * Planar polarity * Apical junction

Nephronophthisis belongs to a family of recessive cystic kidney diseases and may arise from mutations in multiple genes. In this report we have used a spontaneous mouse mutant of type 3 nephronophthisis to examine whether the doxycycline-inducible synthesis of Timp-2, a natural inhibitor of matrix metalloproteinases, can influence renal cyst growth in transgenic mice. Metalloproteinases may exert either a negative or a positive effect on the progression of cystic kidney disease, and we reasoned that this may be most effectively examined by using a natural inhibitor. Surprisingly, already the application of doxycycline, which also inhibits matrix metalloproteinases, accelerated renal cyst growth and led to increased renal fibrosis, an additional effect of Timp-2 was not detected. The positive effect of doxycycline on kidney size was not due to a non-specific “anabolic effect” but was specific for cystic kidneys because it was not observed in non-cystic kidneys. When looking for potential metabolic changes we noticed that the urine of control animals led to an increase in the calcium response of LLC-PK1 cells, whereas the urine of doxycycline-treated mice showed the opposite effect and even antagonized the urine of control animals. Further experiments demonstrated that the urine of control animals contained a heat-labile, proteinase K-resistant substance which appears to be responsible for the induction of a calcium response in LLC-PK1 cells. We conclude that doxycycline accelerates cyst growth possibly by the induction of a substance which lowers the intracellular calcium concentration. Our data also add a note of caution when interpreting phenotypes of animal models based upon the tet system. [ABSTRACT FROM AUTHOR]

Background: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in Pkd1 and Pkd2. They encode the polytopic integral membrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively, which are expressed on primary cilia. Formation of kidney cysts in ADPKD starts when a somatic second hit mechanism inactivates the wild-type Pkd allele. Approximately one quarter of families with ADPDK due to Pkd1 have germline nonsynonymous amino acid substitution (missense) mutations. A subset of these mutations is hypomorphic, retaining some residual PC1 function. Previous studies have shown that the highly conserved Ire1 α -XBP1 pathway of the unfolded protein response can modulate levels of functional PC1 in the presence of mutations in genes required for post-translational maturation of integral membrane proteins. We examine how activity of the endoplasmic reticulum chaperone-inducing transcription factor XBP1 affects ADPKD in a murine model with missense Pkd1 ., Methods: We engineered a Pkd1 REJ domain missense murine model, Pkd1 R2216W , on the basis of the orthologous human hypomorphic allele Pkd1 R2220W , and examined the effects of transgenic activation of XBP1 on ADPKD progression., Results: Expression of active XBP1 in cultured cells bearing PC1 R2216W mutations increased levels and ciliary trafficking of PC1 R2216W . Mice homozygous for Pkd1 R2216W or heterozygous for Pkd1 R2216Win trans with a conditional Pkd1 fl allele exhibit severe ADPKD following inactivation in neonates or adults. Transgenic expression of spliced XBP1 in tubule segments destined to form cysts reduced cell proliferation and improved Pkd progression, according to structural and functional parameters., Conclusions: Modulating ER chaperone function through XBP1 activity improved Pkd in a murine model of PC1, suggesting therapeutic targeting of hypomorphic mutations., (Copyright © 2022 by the American Society of Nephrology.)

PKD2 is one of the two genes mutated in ADPKD (autosomal-dominant polycystic kidney disease). The protein product of PKD2, polycystin-2, functions as a non-selective cation channel in the endoplasmic reticulum and possibly at the plasma membrane. Hydrophobicity plots and its assignment to the TRP (transient receptor potential) family of cation channels suggest that polycystin-2 contains six transmembrane domains and that both the N- and C-termini extend into the cytoplasm. However, no experimental evidence for this model has so far been provided. To determine the orientation of the different loops of polycystin-2, we truncated polycystin-2 within the predicted loops 1-5 and tagged the constructs at the C-terminus with an HA (haemagglutinin) epitope. After transient expression and selective membrane permeabilization, immunofluorescence staining for the HA epitope revealed that loops 1, 3 and 5 extend into the lumen of the endoplasmic reticulum or the extracellular space, whereas loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the N- and C-termini of polycystin-2. In accordance with the immunofluorescence data, protease protection assays from microsomal preparations yielded protected fragments when polycystin-2 was truncated in loops 1, 3 and 5, whereas no protected fragments could be detected when polycystin-2 was truncated in loops 2 and 4. The results of the present study therefore provide the first experimental evidence for the topological orientation of polycystin-2.