Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of ESRD. Affected individuals inherit a defective copy of either PKD1 or PKD2, which encode polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 and PC2 are secreted on urinary exosome-like vesicles (ELVs) (100-nm diameter vesicles), in which PC1 is present in a cleaved form and may be complexed with PC2. Here, label-free quantitative proteomic studies of urine ELVs in an initial discovery cohort (13 individuals with PKD1 mutations and 18 normal controls) revealed that of 2008 ELV proteins, 9 (0.32%) were expressed at significantly different levels in samples from individuals with PKD1 mutations compared to controls (P,0.03). In samples from individuals with PKD1 mutations, levels of PC1 and PC2 were reduced to 54% (P,0.02) and 53% (P,0.001), respectively. Transmembrane protein 2 (TMEM2), a protein with homology to fibrocystin, was 2.1-fold higher in individuals with PKD1 mutations (P,0.03). The PC1/TMEM2 ratio correlated inversely with height-adjusted total kidney volume in the discovery cohort, and the ratio of PC1/TMEM2 or PC2/TMEM2 could be used to distinguish individuals with PKD1 mutations from controls in a confirmation cohort. In summary, results of this study suggest that a test measuring the urine exosomal PC1/TMEM2 or PC2/TMEM2 ratio may have utility in diagnosis and monitoring of polycystic kidney disease. Future studies will focus on increasing sample size and confirming these studies. The data were deposited in the ProteomeXchange (identifier PXD001075).

Mutations in the PKHD1 gene, which encodes fibrocystin, cause autosomal recessive polycystic kidney disease (ARPKD). Unfortunately, the lack of specific antibodies to the mouse protein impairs the study of splicing, post-translational processing, shedding, and temporal and spatial expression of endogenous fibrocystin at the cellular and subcellular level. Here, we report using a knock-in strategy to generate a null Pkhd1 strain and a strain that expresses fibrocystin along with two SV5-Pk epitope tags engineered in-frame into the third exon, immediately C-terminal to the signal-peptide cleavage site in a poorly conserved region. By 6 mo of age, the Pkhd1-null mouse develops massive cystic hepatomegaly and proximal tubule dilation, whereas the mouse with epitope-tagged fibrocystin has histologically normal liver and kidneys at 14 mo. Although Pkhd1 was believed to generate many splice forms, our western analysis resolved fibrocystin as a 500 kD product without other forms in the 15-550 kD range. Western analysis also revealed that exosome-like vesicles (ELVs) secrete the bulk of fibrocystin in its mature cleaved form, and scanning electron microscopy identified that fibrocystin on ELVs attached to cilia. Furthermore, the addition of ELVs with epitope-tagged fibrocystin to wild-type cells showed that label transferred to primary cilia within 5 min. In summary, tagging of the endogenous Pkhd1 gene facilitates the study of the glycosylation, proteolytic cleavage, and shedding of fibrocystin.

Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40–200nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV) enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin positive irregularly shaped membranous vesicles and podocin/podocalyxin negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton and RhoGDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases and complement proteins involved in glomerular disease are in GMVs and some were shed in the disease state (nephrin, TRPC6 and INF2 and PLA2R). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease.

Background & Aims In polycystic kidney (PKD) and liver (PLD) diseases, the normally non-proliferative hepato-renal epithelia acquire a proliferative, cystic phenotype, which is linked to overexpression of Cdc25A and cell cycle deregulation. We investigated the effects of Cdc25A inhibition in mice and rats, via genetic and pharmacological approaches. Methods Cdc25A+/− mice (which have reduced levels of Cdc25A) were cross-bred with Pkhd1del2/del2 mice (which have increased levels of Cdc25A and develop hepatic cysts). Cdc25A expression was analyzed in livers of control and PCK rats, control and Pkd2ws25/− mice, healthy individuals, and patients with PLD. We examined effects of pharmacologic inhibition of Cdc25A with Vitamin K3 (VK3) on the cell cycle, proliferation, and cyst expansion in vitro; hepato-renal cystogenesis in PCK rats and Pkd2ws25/− mice; and expression of Cdc25A and the cell cycle proteins regulated by Cdc25A. We also examined effects of the Cdc25A inhibitor PM-20 on hepato-renal cystogenesis in Pkd2ws25/− mice. Results Liver weights and hepatic and fibrotic areas were decreased by 32%–52% in Cdc25A+/−:Pkhd1del2/del2 mice, compared to Pkhd1del2/del2 mice.VK3 altered the cell cycle and reduced proliferation of cultured cholangiocytes by 32%–83% and decreased growth of cultured cysts by 23%–67%. In PCK rats and Pkd2ws25/− mice, VK3 reduced liver and kidney weights and hepato-renal cystic and fibrotic areas by 18%–34%. PM-20 decreased hepato-renal cystogenesis in Pkd2ws25/− mice by 15%. Conclusions Cdc25A inhibitors block cell cycle progression and proliferation, reduce liver and kidney weights and cyst growth in animal models of PKD and PLD, and might be developed as therapeutics for these diseases.

Aberrant intracellular calcium levels and increased cAMP signaling contribute to the development of polycystic kidney disease (PKD). cAMP can be hydrolyzed by various phosphodiesterases (PDEs). To examine the role of cAMP hydrolysis and the most relevant PDEs in the pathogenesis of PKD, we examined cyst development in Pde1-or Pde3-knockout mice on the Pkd2 2/WS25 background (WS25 is an unstable Pkd2 allele). These PDEs were selected because of their importance in cross-talk between calcium and cyclic nucleotide signaling (PDE1), control of cell proliferation and cystic fibrosis transmembrane conductance regulator (CFTR) -driven fluid secretion (PDE3), and response to vasopressin V2 receptor activation (both). In Pkd2 2/WS25mice, knockout of Pde1a, Pde1c, or Pde3a but not of Pde1b or Pde3b aggravated the development of PKD and was associated with higher levels of protein kinase Aphosphorylated (Ser133) cAMP-responsive binding protein (P-CREB), activating transcription factor-1, and CREB-induced CRE modulator proteins in kidney nuclear preparations. Immunostaining also revealed higher expression of P-CREB in Pkd2 2/WS25 ;Pde1a 2/2 , Pkd2 2/WS25 ;Pde1c 2/2 , and Pkd2 2/WS25 ;Pde3a 2/2 kidneys.The cystogenic effect of desmopressin administration was markedly enhanced in Pkd2 2/WS25 ;Pde3a 2/2 mice, despite PDE3 accounting for only a small fraction of renal cAMP PDE activity. These observations show that calcium-and calmodulindependent PDEs (PDE1A and PDE1C) and PDE3A modulate the development of PKD, possibly through the regulation of compartmentalized cAMP pools that control cell proliferation and CFTR-driven fluid secretion. Treatments capable of increasing the expression or activity of these PDEs may, therefore, retard the development of PKD.