A synthetic agent ameliorates polycystic kidney disease by promoting apoptosis of cystic cells through increased oxidative stress.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of chronic kidney disease and the fourth leading cause of end-stage kidney disease, accounting for over 50% of prevalent cases requiring renal replacement therapy. There is a pressing need for improved therapy for ADPKD. Recent insights into the pathophysiology of ADPKD revealed that cyst cells undergo metabolic changes that up-regulate aerobic glycolysis in lieu of mitochondrial respiration for energy production, a process that ostensibly fuels their increased proliferation. The present work leverages this metabolic disruption as a way to selectively target cyst cells for apoptosis. This small-molecule therapeutic strategy utilizes 11beta-dichloro, a repurposed DNA-damaging anti-tumor agent that induces apoptosis by exacerbating mitochondrial oxidative stress. Here, we demonstrate that 11beta-dichloro is effective in delaying cyst growth and its associated inflammatory and fibrotic events, thus preserving kidney function in perinatal and adult mouse models of ADPKD. In both models, the cyst cells with homozygous inactivation of Pkd1 show enhanced oxidative stress following treatment with 11beta-dichloro and undergo apoptosis. Co-administration of the antioxidant vitamin E negated the therapeutic benefit of 11beta-dichloro in vivo, supporting the conclusion that oxidative stress is a key component of the mechanism of action. As a preclinical development primer, we also synthesized and tested an 11beta-dichloro derivative that cannot directly alkylate DNA, while retaining pro-oxidant features. This derivative nonetheless maintains excellent anti-cystic properties in vivo and emerges as the lead candidate for development.

Different effects of Sec61α, Sec62 and Sec63 depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells.

Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co- and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.

Isolated polycystic liver disease genes define effectors of polycystin-1 function.

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, ALG8, GANAB, 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.

Isolated polycystic liver disease genes define effectors of polycystin-1 function.

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, ALG8, GANAB, 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.

Hepatocystin is Essential for TRPM7 Function During Early Embryogenesis.

Mutations in protein kinase C substrate 80K-H (PRKCSH), which encodes for an 80 KDa protein named hepatocystin (80K-H, PRKCSH), gives rise to polycystic liver disease (PCLD). Hepatocystin functions as the noncatalytic beta subunit of Glucosidase II, an endoplasmic reticulum (ER)-resident enzyme involved in processing and quality control of newly synthesized glycoproteins. Patients harboring heterozygous germline mutations in PRKCSH are thought to develop renal cysts as a result of somatic loss of the second allele, which subsequently interferes with expression of the TRP channel polycystin-2 (PKD2). Deletion of both alleles of PRKCSH in mice results in embryonic lethality before embryonic day E11.5. Here, we investigated the function of hepatocystin during Xenopus laevis embryogenesis and identified hepatocystin as a binding partner of the TRPM7 ion channel, whose function is required for vertebrate gastrulation. We find that TRPM7 functions synergistically with hepatocystin. Although other N-glycosylated proteins are critical to early development, overexpression of TRPM7 in Xenopus laevis embryos was sufficient to fully rescue the gastrulation defect caused by loss of hepatocystin. We observed that depletion of hepatocystin in Xenopus laevis embryos decreased TRPM7 expression, indicating that the early embryonic lethality caused by loss of hepatocystin is mainly due to impairment of TRPM7 protein expression.

Sec63 and Xbp1 regulate IRE1α activity and polycystic disease severity.

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.

Polycystin-1: a master regulator of intersecting cystic pathways.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common potentially lethal monogenic disorder, with more than 12 million cases worldwide. The two causative genes for ADPKD, PKD1 and PKD2, encode protein products polycystin-1 (PC1) and polycystin-2 (PC2 or TRPP2), respectively. Recent data have shed light on the role of PC1 in regulating the severity of the cystic phenotypes in ADPKD, autosomal recessive polycystic kidney disease (ARPKD), and isolated autosomal dominant polycystic liver disease (ADPLD). These studies showed that the rate for cyst growth was a regulated trait, a process that can be either sped up or slowed down by alterations in functional PC1. These findings redefine the previous understanding that cyst formation occurs as an 'on-off' process. Here, we review these and other related studies with an emphasis on their translational implications for polycystic diseases.

Steviol retards renal cyst growth through reduction of CFTR expression and inhibition of epithelial cell proliferation in a mouse model of polycystic kidney disease.

Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) is associated with cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel leading to renal failure for which no effective treatment is currently available. We previously reported that steviol retards Madin-Darby canine kidney (MDCK) cyst enlargement by inhibiting CFTR channel activity and promoting proteasomal-mediated CFTR degradation. It is imperative to examine the effect of steviol in animal models of ADPKD. Therefore, we examined the effect of steviol on renal cyst growth in an orthologous mouse model of human ADPKD (Pkd1(flox/flox):Pkhd1-Cre). The results showed that daily treatment with both 200mg/kg BW of steviol and 1000mg/kg BW of stevioside for 14 days markedly decreased kidney weight and cystic index in these mice. However, only steviol markedly reduced blood urea nitrogen and creatinine values. Steviol also reduced cell proliferation but had no effect on cell apoptosis. In addition, steviol suppressed CFTR and mTOR/S6K expression in renal cyst-lining epithelial cells. Interestingly, steviol was found to stimulate AMP-activated protein kinase (AMPK). Our findings indicate that steviol slows cyst progression in ADPKD mouse model, in part, through the activation of AMPK which subsequently inhibits CFTR chloride channel expression and inhibits renal epithelial cell proliferation via mTOR/S6K pathway. Most importantly, steviol could markedly improve kidney function in a mouse model of ADPKD. Steviol thus has potential application for further development as a therapeutic compound for the treatment of polycystic kidney disease.

Altered trafficking and stability of polycystins underlie polycystic kidney disease.

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.

A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation.

Autosomal dominant polycystic liver disease results from mutations in PRKCSH or SEC63. The respective gene products, glucosidase IIß and SEC63p, function in protein translocation and quality control pathways in the endoplasmic reticulum. Here we show that glucosidase IIß and Sec63p are required in mice for adequate expression of a functional complex of the polycystic kidney disease gene products, polycystin-1 and polycystin-2. We find that polycystin-1 is the rate-limiting component of this complex and that there is a dose-response relationship between cystic dilation and levels of functional polycystin-1 following mutation of Prkcsh or Sec63. Reduced expression of polycystin-1 also serves to sensitize the kidney to cyst formation resulting from mutations in Pkhd1, the recessive polycystic kidney disease gene. Finally, we show that proteasome inhibition increases steady-state levels of polycystin-1 in cells lacking glucosidase IIß and that treatment with a proteasome inhibitor reduces cystic disease in orthologous gene models of human autosomal dominant polycystic liver disease.