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Although translational research into autosomal dominant polycystic kidney disease (ADPKD) and its pathogenesis has made considerable progress, there is presently lack of standardized animal model for preclinical trials. In this study, we developed an orthologous mouse model of human ADPKD by cross‐mating Pkd2 conditional‐knockout mice (Pkd2 f3) to Cre transgenic mice in which Cre is driven by a spectrum of kidney‐related promoters. By systematically characterizing the mouse model, we found that Pkd2 f3/f3 mice with a Cre transgene driven by the mouse villin‐1 promoter (Vil‐Cre;Pkd2 f3/f3) develop overt cysts in the kidney, liver and pancreas and die of end‐stage renal disease (ESRD) at 4–6 months of age. To determine whether these Vil‐Cre;Pkd2 f3/f3 mice were suitable for preclinical trials, we treated the mice with the high‐dose mammalian target of rapamycin (mTOR) inhibitor rapamycin. High‐dose rapamycin significantly increased the lifespan, lowered the cystic index and kidney/body weight ratio and improved renal function in Vil‐Cre;Pkd2 f3/f3 mice in a time‐ and dose‐dependent manner. In addition, we further found that rapamycin arrested aberrant epithelial‐cell proliferation in the ADPKD kidney by down‐regulating the cell‐cycle‐associated cyclin‐dependent kinase 1 (CDK1) and cyclins, namely cyclin A, cyclin B, cyclin D1 and cyclin E, demonstrating a direct link between mTOR signalling changes and the polycystin‐2 dysfunction in cystogenesis. Our newly developed ADPKD model provides a practical platform for translating in vivo preclinical results into ADPKD therapies. The newly defined molecular mechanism by which rapamycin suppresses proliferation via inhibiting abnormally elevated CDK1 and cyclins offers clues to new molecular targets for ADPKD treatment.

// Xueyou Ma 1, * , Kun Tang 1, * , Chunguang Yang 1 , Guanqing Wu 1 , Nan Xu 1 , Meng Wang 1 , Xing Zeng 1 , Zhiquan Hu 1 , Ranran Song 2 , Bertram Yuh 3 , Zhihua Wang 1 , Zhangqun Ye 1 1 Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 2 Department of Maternal and Child Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 3 Division of Urologic Oncology, City of Hope National Cancer Center, Duarte, CA, USA * These authors have contributed equally to this work Correspondence to: Zhihua Wang, email: zhwang_hust@hotmail.com Keywords: bladder neck preservation, radical prostatectomy, prostate cancer, urinary incontinence, meta-analysis Received: June 13, 2016 Accepted: August 26, 2016 Published: September 13, 2016 ABSTRACT Bladder neck preservation (BNP) during radical prostatectomy (RP) may improve postoperative urinary continence, although its overall effectiveness remains controversial. We systematically searched PubMed, Ovid Medline, Embase, CBM and the Cochrane Library to identify studies published before February 2016 that assessed associations between BNP and post-RP urinary continence. Thirteen trials (1130 cases and 1154 controls) assessing BNP versus noBNP (or with bladder neck reconstruction, BNR) were considered suitable for meta-analysis, including two randomized controlled trials (RCT), six prospective and five retrospective studies. Meta-analysis demonstrated that BNP improved early urinary continence rates (6 mo, OR = 1.66; 95% CI, 1.21–2.27; P = 0.001) and long-term urinary continence outcomes (>12 mo, OR = 3.99; 95% CI, 1.94–8.21; P = 0.0002). Patients with BNP also had lower bladder neck stricture frequencies (OR = 0.49; 95% CI, 0.29–0.81; P = 0.006). Anastomotic leak rates, positive surgical margins and biochemical failure rates were comparable between the two groups (P>0.05). There were no differences in baseline characteristics except for a smaller average prostate volume (WMD = -2.24 ml; 95% CI, -4.27 to -0.22; P = 0.03) in BNP patients. Our analyses indicated that BNP during RP improved early recovery and overall long-term (1 year) urinary continence and decreased bladder neck stricture rates without compromising oncologic control.

Aim: Autosomal dominant polycystic kidney disease is one of the most common genetic renal diseases. Cyclooxygenase plays an important role in epithelial cell proliferation and may contribute to the mechanisms underlying cyst formation. The aim of the present study was to evaluate the role of cyclooxygenase inhibition in the cyst progression in polycystic kidney disease. Method: Pkd2WS25/- mice, a murine model which harbors a compound cis-heterozygous mutation of the Pkd2 gene were used. Cyclooxygenase expression was assessed in both human and murine kidney specimens. Pkd2WS25/- mice were treated with Sulindac (a nonselective cyclooxygenase inhibitor) or vehicle for 8 months starting at three weeks age, and then renal cyst burden was assessed by kidney weight and volume. Results: Cyclooxygenase-2 expression was up-regulated compared to control kidneys as shown by RNase protection in human polycystic kidneys and immunoblot in mouse Pkd2WS25/- kidneys. Cyclooxygenase-2 expression was up-regulated in the renal interstitium as well as focal areas of the cystic epithelium (p

Background Dysregulation of transforming growth factor β (TGF-β) signaling and hypoxic microenvironment have respectively been reported to be involved in disease progression in malignancies of prostate. Emerging evidence indicates that downregulation of TGFBR2, a pivotal regulator of TGF-β signaling, may contribute to carcinogenesis and progression of prostate cancer (PCa). However, the biological function and regulatory mechanism of TGFBR2 in PCa remain poorly understood. In this study, we propose to investigate the crosstalk of hypoxia and TGF-β signaling and provide insight into the molecular mechanism underlying the regulatory pathways in PCa. Methods Prostate cancer cell lines were cultured in hypoxia or normoxia to evaluate the effect of hypoxia on TGFBR2 expression. Methylation specific polymerase chain reaction (MSP) and demethylation agents was used to evaluate the methylation regulation of TGFBR2 promoter. Besides, silencing of EZH2 via specific siRNAs or chemical inhibitor was used to validate the regulatory effect of EZH2 on TGFBR2. Moreover, we conducted PCR, western blot, and luciferase assays which studied the relationship of miR-93 and TGFBR2 in PCa cell lines and specimens. We also detected the impacts of hypoxia on EZH2 and miR-93, and further examined the tumorigenic functions of miR-93 on proliferation and epithelial-mesenchymal transition via a series of experiments. Results TGFBR2 expression was attenuated under hypoxia. Hypoxia-induced EZH2 promoted H3K27me3 which caused TGFBR2 promoter hypermethylation and contributed to its epigenetic silencing in PCa. Besides, miR-93 was significantly upregulated in PCa tissues and cell lines, and negatively correlated with the expression of TGFBR2. Ectopic expression of miR-93 promoted cell proliferation, migration and invasion in PCa, and its expression could also be induced by hypoxia. In addition, TGFBR2 was identified as a bona fide target of miR-93. Conclusions Our findings elucidate diverse hypoxia-regulated pathways including EZH2-mediated hypermethylation and miR-93-induced silencing contribute to attenuation of TGFBR2 expression and promote cancer progression in prostate cancer. Electronic supplementary material The online version of this article (10.1186/s13046-018-0764-9) contains supplementary material, which is available to authorized users.

Although much is known about the molecular genetic mechanisms of autosomal-dominant polycystic kidney disease (ADPKD), few effective treatment is currently available. Here, we explore the in vivo effects of causal gene replacement in orthologous gene models of ADPKD in mice. Wild-type mice with human PKD2 transgene ( PKD2 tg ) overexpressed polycystin (PC)-2 in several tissues, including the kidney and liver, and showed no significant cyst formation in either organ. We cross-mated PKD2 tg with a Pkd2 -null mouse model, which is embryonically lethal and forms renal and pancreatic cysts. Pkd2 −/− mice with human PKD2 transgene ( Pkd2 −/− ; PKD2 tg ) were born in expected Mendelian ratios, indicating that the embryonic lethality of the Pkd2 −/− mice was rescued. Pkd2 −/− ; PKD2 tg mice survived up to 12 months and exhibited moderate to severe cystic phenotypes of the kidney, liver, and pancreas. Moreover, Pkd2 −/− mice with homozygous PKD2 tg -transgene alleles ( Pkd2 −/− ; PKD2 tg/tg ) showed significant further amelioration of the cystic severity compared to that in Pkd2 −/− mice with a hemizygous PKD2 tg allele ( Pkd2 −/− ; PKD2 tg ), suggesting that the ADPKD phenotype was improved by increased transgene dosage. On further analysis, cystic improvement mainly resulted from reduced proliferation, rather apoptosis, of cyst-prone epithelial cells in the mouse model. The finding that the functional restoration of human PC2 significantly rescued ADPKD phenotypes in a dose-dependent manner suggests that increasing PC2 activity may be beneficial in some forms of ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) can be caused by mutations in the PKD1 or PKD2 genes. The PKD1 gene product is a Wnt cell-surface receptor. We previously showed that a lack of the PKD2 gene product, PC2, increases β-catenin signaling in mouse embryonic fibroblasts, kidney renal epithelia, and isolated renal collecting duct cells. However, it remains unclear whether β-catenin signaling plays a role in polycystic kidney disease phenotypes or if a Wnt inhibitor can halt cyst formation in ADPKD disease models. Here, using genetic and pharmacologic approaches, we demonstrated that the elevated β-catenin signaling caused by PC2 deficiency contributes significantly to disease phenotypes in a mouse ortholog of human ADPKD. Pharmacologically inhibiting β-catenin stability or the production of mature Wnt protein, or genetically reducing the expression of Ctnnb1 (which encodes β-catenin), suppressed the formation of renal cysts, improved renal function, and extended survival in ADPKD mice. Our study clearly demonstrates the importance of β-catenin signaling in disease phenotypes associated with Pkd2 mutation. It also describes the effects of two Wnt inhibitors, XAV939 and LGK974, on various Wnt signaling targets as a potential therapeutic modality for ADPKD, for which there is currently no effective therapy.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2 which encodes polycystin-1 (PC1) and polycystin-2, respectively. PC1 was previously shown to slow cell proliferation and inhibit apoptosis but the underlying mechanisms remain elusive or controversial. Here we showed in cultured mammalian cells and Pkd1 knockout mouse kidney epithelial cells that PC1 and its truncation mutant comprising the last five transmembrane segments and the intracellular C-terminus (PC1-5TMC) down-regulate the phosphorylation of protein kinase R (PKR) and its substrate eukaryotic translation initiation factor 2 alpha (eIF2α). PKR is known to be activated by interferons and dsRNAs, inhibits protein synthesis and induces apoptosis. By co-immunoprecipitation experiments we found that PC1 truncation mutants associate with PKR, or with PKR and its activator PACT. Further experiments showed that PC1 and PC1-5TMC reduce phosphorylation of eIF2α through inhibiting PKR phosphorylation. Our TUNEL experiments using tunicamycin, an apoptosis inducer, and GADD34, an inhibitor of eIF2α phosphorylation, demonstrated that PC1-5TMC inhibits apoptosis of HEK293T cells in a PKR-eIF2α-dependent manner, with concurrent up- and down-regulation of Bcl-2 and Bax, respectively, revealed by Western blotting. Involvement of PC1-regulated eIF2α phosphorylation and a PKR-eIF2α pathway in cell apoptosis may be an important part of the mechanism underlying ADPKD pathogenesis.

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-β 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-β 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.

Background Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease in the clinic. The predominant clinical manifestation is bilateral and progressive cysts formation in the kidneys, impairs normal renal parenchyma, and ultimately leads to endstage renal disease (ESRD). ADPKD is a heterogenic disease which is resulted from the mutations of PKD1 or PKD2 genes which encode polycystin-1 (PC1) and -2 (PC2), thereby multiple cell signaling pathways are involved. Method Although causative genes and aberrant signaling pathways have been investigated for decades, lack of effective and less side-effect treatment for the disease still perplex vast clinicians. Therefore, development of new therapeutic approaches for ADPKD is currently very much desired. Conclusion This review will center on pathogenesis of ADPKD, and thereafter gene transfer will be discussed as potential treatment for the disease. New therapeutic interventions will bring further hope to improve prognosis of this incurable disease.

Mutations in the PKHD1 gene result in autosomal recessive polycystic kidney disease (ARPKD) in humans. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted mutation in the mouse orthologue of human PKHD1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. By littermates of this mouse, we developed two immortalized renal collecting duct cell lines with Pkhd1 and two without. Under nonpermissive culture conditions, the Pkhd1(-/-) renal cells displayed aberrant cell-cell contacts and tubulomorphogenesis. The Pkhd1(-/-) cells also showed significantly reduced cell proliferation and elevated apoptosis. To validate this finding in vivo, we examined proliferation and apoptosis in the kidneys of Pkhd1(-/-) mice and their wildtype littermates. Using proliferation (PCNA and Histone-3) and apoptosis (TUNEL and caspase-3) markers, similar results were obtained in the Pkhd1(-/-) kidney tissues as in the cells. To identify the molecular basis of these findings, we analyzed the effect of Pkhd1 loss on multiple putative signaling regulators. We demonstrated that the loss of Pkhd1 disrupts multiple major phosphorylations of focal adhesion kinase (FAK), and these disruptions either inhibit the Ras/C-Raf pathways to suppress MEK/ERK activity and ultimately reduce cell proliferation, or suppress PDK1/AKT to upregulate Bax/caspase-9/caspase-3 and promote apoptosis. Our findings indicate that apoptosis may be a major player in the cyst formation in ARPKD, which may lead to new therapeutic strategies for human ARPKD.

Autosomal recessive polycystic kidney disease is caused by mutations in PKHD1, which encodes the membrane-associated receptor-like protein fibrocystin/polyductin (FPC). FPC associates with the primary cilia of epithelial cells and co-localizes with the Pkd2 gene product polycystin-2 (PC2), suggesting that these two proteins may function in a common molecular pathway. For investigation of this, a mouse model with a gene-targeted mutation in Pkhd1 that recapitulates phenotypic characteristics of human autosomal recessive polycystic kidney disease was produced. The absence of FPC is associated with aberrant ciliogenesis in the kidneys of Pkhd1-deficient mice. It was found that the COOH-terminus of FPC and the NH2-terminus of PC2 interact and that lack of FPC reduced PC2 expression but not vice versa, suggesting that PC2 may function immediately downstream of FPC in vivo. PC2-channel activities were dysregulated in cultured renal epithelial cells derived from Pkhd1 mutant mice, further supporting that both cystoproteins function in a common pathway. In addition, mice with mutations in both Pkhd1 and Pkd2 had a more severe renal cystic phenotype than mice with single mutations, suggesting that FPC acts as a genetic modifier for disease severity in autosomal dominant polycystic kidney disease that results from Pkd2 mutations. It is concluded that a functional and molecular interaction exists between FPC and PC2 in vivo.

Polycystin-2 (PC2) is the product of the PKD2 gene, which is mutated in 10-15% patients of autosomal dominant polycystic kidney disease (ADPKD). PC2 is an integral transmembrane protein and acts as a calcium-permeable cation channel. The functional modulation of this channel by other protein partners remains largely unknown. In the present study, using a yeast two-hybrid approach, we discovered that both intracellular N- and C-termini of PC2 associate with alpha-actinins, actin-binding and actin-bundling proteins important in cytoskeleton organization, cell adhesion, proliferation and migration. The PC2-alpha-actinin association was confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. In addition, the in vivo interaction between endogenous PC2 and alpha-actinins was demonstrated by co-immunoprecipitation in human embryonic kidney 293 and Madin-Darby canine kidney (MDCK) cells, rat kidney and heart tissues and human syncytiotrophoblast (hST) apical membrane vesicles. Immunofluorescence experiments showed that PC2 and alpha-actinin were partially co-localized in epithelial MDCK and inner medullary collecting duct cells, NIH 3T3 fibroblasts and hST vesicles. We studied the functional modulation of PC2 by alpha-actinin in a lipid bilayer electrophysiology system using in vitro translated PC2 and found that alpha-actinin substantially stimulated the channel activity of reconstituted PC2. A similar stimulatory effect of alpha-actinin on PC2 was also observed when hST vesicles were reconstituted in lipid bilayer. Thus, physical and functional interactions between PC2 and alpha-actinin may play an important role in abnormal cell adhesion, proliferation and migration observed in ADPKD.

Mutations of the polycystic kidney and hepatic disease 1 ( PKHD1 ) gene have been shown to cause autosomal recessive polycystic kidney disease (ARPKD), but the cellular functions of the gene product (PKHD1) remain uncharacterized. To illuminate its properties, the spatial and temporal expression patterns of PKHD1 were determined in mouse, rat, and human tissues by using polyclonal Abs and mAbs recognizing various specific regions of the gene product. During embryogenesis, PKHD1 is widely expressed in epithelial derivatives, including neural tubules, gut, pulmonary bronchi, and hepatic cells. In the kidneys of the pck rats, the rat model of which is genetically homologous to human ARPKD, the level of PKHD1 was significantly reduced but not completely absent. In cultured renal cells, the PKHD1 gene product colocalized with polycystin-2, the gene product of autosomal dominant polycystic disease type 2, at the basal bodies of primary cilia. Immunoreactive PKHD1 localized predominantly at the apical domain of polarized epithelial cells, suggesting it may be involved in the tubulogenesis and/or maintenance of duct–lumen architecture. Reduced PKHD1 levels in pck rat kidneys and its colocalization with polycystins may underlie the pathogenic basis for cystogenesis in polycystic kidney diseases.

Polycystin-2 (PC2) is the product of the second cloned gene (PKD2) responsible for autosomal dominant polycystic kidney disease and has recently been shown to be a calcium-permeable cation channel. PC2 has been shown to connect indirectly with the actin microfilament. Here, we report a direct association between PC2 and the actin microfilament. Using a yeast two-hybrid screen, we identified a specific interaction between the PC2 cytoplasmic C-terminal domain and tropomyosin-1 (TM-1), a component of the actin microfilament complex. Tropomyosins constitute a protein family of more than 20 isoforms arising mainly from alternative splicing and are present in muscle as well as non-muscle cells. We identified a new TM-1 splicing isoform in kidney and heart (TM-1a) that differs from TM-1 in the C terminus and interacted with PC2. In vitro biochemical methods, including GST pull-down, blot overlay and microtiter binding assays, confirmed the interaction between PC2 and the two TM-1 isoforms. Further experiments targeted the interacting domains to G821-R878 of PC2 and A152-E196, a common segment of TM-1 and TM-1a. Indirect double immunofluorescence experiments showed partial co-localization of PC2 and TM-1 in transfected mouse fibroblast NIH 3T3 cells. Co-immunoprecipitation (co-IP) studies using 3T3 cells and Xenopus oocytes co-expressing PC2 and TM-1 (or TM-1a) revealed in vivo association between the protein pairs. Furthermore, the in vivo interaction between the endogenous PC2 and TM-1 was demonstrated also by reciprocal co-IP using native human embryonic kidney cells and human adult kidney. Considering previous reports that TM-1 acts as a suppressor of neoplastic growth of transformed cells, it is possible that TM-1 contributes to cyst formation/growth when the anchorage of PC2 to the actin microfilament via TM-1 is altered.

Autosomal recessive polycystic kidney disease (ARPKD) is a common hereditary renal cystic disease in infants and children. By genetic linkage analyses, the gene responsible for this disease, termed polycystic kidney and hepatic disease 1 (PKHD1), was mapped on human chromosome 6p21.1-p12, and has been further localized to a 1-cM genetic interval flanked by the D6S1714/D6S243 (telomeric) and D6S1024 (centromeric) markers. We recently identified a novel gene in this genetic interval from kidney cDNA, using cloning strategies. The gene PKHD1 (PKHD1-tentative) encodes a novel 3396-amino-acid protein with no apparent homology with any known proteins. We named its gene product "tigmin" because it contains multiple TIG domains, which usually are seen in proteins containing immunoglobulin-like folds. PKHD1 encodes an 11.6-kb transcript and is composed of 61 exons spanning an approximately 365-kb genomic region on chromosome 6p12-p11.2 adjacent to the marker D6S1714. Northern blot analyses demonstrated that the gene has discrete bands with one peak signal at approximately 11 kb, indicating that PKHD1 is likely to have multiple alternative transcripts. PKHD1 is highly expressed in adult and infant kidneys and weakly expressed in liver in northern blot analysis. This expression pattern parallels the tissue involvement observed in ARPKD. In situ hybridization analysis further revealed that the expression of PKHD1 in the kidney is mainly localized to the epithelial cells of the collecting duct, the specific tubular segment involved in cyst formation in ARPKD. These features of PKHD1 make it a strong positional candidate gene for ARPKD.

Considerable progress toward understanding pathogenesis of autosomal dominant polycystic disease (ADPKD) has been made during the past 15 years. ADPKD is a heterogeneous human disease resulting from mutations in either of two genes, PKD1 and PKD2. The similarity in the clinical presentation and evidence of direct interaction between the COOH termini of polycystin-1 and polycystin-2, the respective gene products, suggest that both proteins act in the same molecular pathway. The fact that most mutations from ADPKD patients result in truncated polycystins as well as evidence of a loss of heterozygosity mechanism in individual PKD cysts indicate that the loss of the function of either PKD1 or PKD2 is the most likely pathogenic mechanism for ADPKD. A novel mouse model, WS25, has been generated with a targeted mutation at Pkd2 locus in which a mutant exon 1 created by inserting a neo r cassette exists in tandem with the wild-type exon 1. This causes an unstable allele that undergoes secondary recombination to produce a true null allele at Pkd2 locus. Therefore, the model Pkd2 WS25/− , which carries the WS25 unstable allele and a true null allele, produces somatic second hits during mouse development or adult life and establishes an extremely faithful model of human ADPKD.

PKD2, the second gene for the autosomal dominant polycystic kidney disease (ADPKD), encodes a protein, polycystin-2, with predicted structural similarity to cation channel subunits. However, the function of polycystin-2 remains unknown. We used polyclonal antisera specific for the intracellular NH(2) and COOH termini to identify polycystin-2 as an approximately 110-kDa integral membrane glycoprotein. Polycystin-2 from both native tissues and cells in culture is sensitive to Endo H suggesting the continued presence of high-mannose oligosaccharides typical of pre-middle Golgi proteins. Immunofluorescent cell staining of polycystin-2 shows a pattern consistent with localization in the endoplasmic reticulum. This finding is confirmed by co-localization with protein-disulfide isomerase as determined by double indirect immunofluorescence and co-distribution with calnexin in subcellular fractionation studies. Polycystin-2 translation products truncated at or after Gly(821) retain their exclusive endoplasmic reticulum localization while products truncated at or before Glu(787) additionally traffic to the plasma membrane. Truncation mutants that traffic to the plasma membrane acquire Endo H resistance and can be biotinylated on the cell surface in intact cells. The 34-amino acid region Glu(787)-Ser(820), containing two putative phosphorylation sites, is responsible for the exclusive endoplasmic reticulum localization of polycystin-2 and is the site of specific interaction with an as yet unidentified protein binding partner for polycystin-2. The localization of full-length polycystin-2 to intracellular membranes raises the possibility that the PKD2 gene product is a subunit of intracellular channel complexes.

Fractionated human bone marrow cells, isolated by density centrifugation, could be well cryopreserved in both a DMSO/HES/albumin mixture and the conventional cryoprotectant (DMSO/serum) with either a programmed freezer or a -80 degrees C freezer. A lower initial temperature rise and a delayed plateau phase in the mixture in comparison with those in the DMSO/serum were observed using a programmed procedure without heat fusion compensation. Plateau phase in the mixture could also be shortened with increased liquid nitrogen to minimize the transition phase duration. However, the method of mononuclear cells immersed in a -80 degrees C freezer and stored in liquid nitrogen is simple and easily standardized. Additionally, widely used lactated Ringer's solution instead of Normosol-R can be employed to prepare the mixture for the preservation of fractionated cells.

A 2184-nucleotide (nt) sequence of the p53 gene in Rhesus monkey (Macaca mulatta) was determined in order to facilitate the use of the Rhesus as an animal model in the testing of novel antisense oligodeoxyribonucleotides for a variety of human cancers, including acute myelogenous leukemia (AML). Within overlapping regions, we found greater than 95% identity between the Rhesus and human p53 sequences, and greater than 98% identity between Rhesus and African green monkey p53 sequences. The deduced amino acid (aa) sequence of the p53 protein is highly conserved between human and Rhesus monkey, with only 18 minor differences in 393 aa.

Autosomal dominant (ADPKD) and autosomal recessive (ARPKD) polycystic kidney disease are caused by mutations in Pkd1/Pkd2 and Pkhd1, which encode polycystins (PCs) and fibrocystin/polyductin (FPC). Our recent study reported that a deficiency in FPC increases the severity of cystic disease in Pkd2 mutants and down-regulates PC2 in vivo, but the precise molecular mechanism of these effects is unknown (Kim, I., Fu, Y., Hui, K., Moeckel, G., Mai, W., Li, C., Liang, D., Zhao, P., Ma, J., Chen, X.-Z., George, A. L., Jr., Coffey, R. J., Feng, Z. P., and Wu, G. (2008) J. Am. Soc. Nephrol. 19, 455–468). In this study, through the use of deletion and mutagenesis strategies, we identified a PC2-binding domain in the intracellular C terminus of FPC and an FPC-binding domain in the intracellular N terminus of PC2. These binding domains provide a molecular basis for the physical interaction between PC2 and FPC. In addition, we also found that physical interaction between the binding domains of PC2 and FPC is able to prevent down-regulation of PC2 induced by loss of FPC. In vivo, we generated a mouse model of ADPKD with hypomorphic Pkd2 alleles (Pkd2nf3/nf3) and show that PC2 down-regulation is accompanied by a phenotype similar to that of Pkhd1–/– mice. These findings demonstrate a common mechanism underlying cystogenesis in ADPKD and ARPKD and provide insight into the molecular relationship between PC2 and FPC.

Endoplasmic reticulum(ER)-associated degradation (ERAD) is an essential process for cell homeostasis and remains not well understood. During ERAD, misfolded proteins are recognized, ubiquitinated on ER and subsequently retro-translocated/dislocated from ER to the 26S proteasome in the cytosol for proteolytic elimination. Polycystin-2 (PC2), a member of the transient receptor potential superfamily of cation channels, is a Ca channel mainly located on ER and primary cilium membranes of cells. Mutations in PC2 are associated with autosomal dominant polycystic kidney disease (ADPKD). The cellular and molecular mechanisms underlying the PC2-associated pathogenesis remain unclear. Here we show that PC2 degradation is regulated by the ERAD pathway through the ubiquitin-proteasome system. PC2 interacted with ATPase p97, a well-known ERAD component extracting substrates from ER, and immobilized it in perinuclear regions. PC2 also interacted with Herp, an ubiquitin-like protein implicated in regulation of ERAD. We found that Herp is required for and promotes PC2 degradation. ER stress accelerates the retro-translocation of PC2 for cytosolic degradation, at least in part through increasing the Herp expression. Thus, PC2 is a novel ERAD substrate. Herp also promoted, to varied degrees, the degradation of PC2 truncation mutants, including two pathogenic mutants R872X and E837X, as long as they interact with Herp. In contrast, Herp did not interact with, and has no effect on the degradation of, PC2 mutant missing both the N- and C-termini. The ERAD machinery may thus be important for ADPKD pathogenesis because the regulation of PC2 expression by the ERAD pathway is altered by mutations in PC2.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1, encoding polycystin-1 (PC1), or PKD2 (polycystin-2, PC2). Autosomal recessive PKD (ARPKD) is caused by mutations in PKHD1, encoding fibrocystin/polyductin (FPC). No molecular link between ADPKD and ARPKD has been determined. Here, we demonstrated, by yeast two-hybrid and biochemical assays, that KIF3B, a motor subunit of kinesin-2, associates with PC2 and FPC. Co-immunoprecipitation experiments using Madin-Darby canine kidney (MDCK) and inner medullary collecting duct (IMCD) cells and human kidney revealed that PC2 and KIF3B, FPC and KIF3B and, furthermore, PC2 and FPC are endogenously in the same complex(es), though no direct association between the PC2 and FPC intracellular termini was detected. In vitro binding and Far Western blot experiments demonstrated that PC2 and FPC are in the same complex only if KIF3B is present, presumably by forming a PC2-KIF3B-FPC complex. This was supported by our observation that altering KIF3B level in IMCD cells by over-expression or siRNA significantly affected complexing between PC2 and FPC. Immunofluorescence experiments showed that PC2, FPC and KIF3B partially co-localized in primary cilia of over-confluent and perinuclear regions of sub-confluent cells. Furthermore, KIF3B mediated functional modulation of purified PC2 channels by FPC in a planer lipid bilayer electrophysiology system. The FPC C-terminus substantially stimulated PC2 channel activity in the presence of KIF3B, whereas FPC or KIF3B alone had no effect. Taken together, we discovered that kinesin-2 is a linker between PC2 and FPC and mediates the regulation of PC2 channel function by FPC. Our study may be important for elucidating common molecular pathways for PKD of different genotypes.

Polycystin-2 (PC2), encoded by the PKD2 gene, is mutated in 10-15% of autosomal dominant polycystic kidney disease (ADPKD) patients. PC2 is a Ca(2+)-permeable nonselective cation channel and is present in kidney and many other organs. Likewise, PKD2-mutated patients and mice exhibit extrarenal abnormalities. In comparison with cysts in the kidney, liver, and pancreas, abnormalities in the heart, brain, and vascular vessels are less understood. In particular, roles of PC2 in muscle and endothelia remain largely unknown. In the present study, using a yeast two-hybrid screening, we discovered that the PC2 carboxyl terminal domain (D682-V968) interacts with the cardiac troponin I, an important regulatory component of the actin microfilament in cardiac muscle cells. This interaction was demonstrated by GST pull-down and microtiter binding assays. Dose-dependent binding between PC2 and troponin I followed a Michaelis-Menten relationship, indicating a 1:1 binding stoichiometry. The interacting domains were located to the R872-H927 segment of PC2 and the M1-V107 and K106-L158 segments of troponin I. Co-immunoprecipitation experiments demonstrated that the cardiac and two skeletal isoforms of troponin I were all associated with PC2, when coexpressed in mouse fibroblast NIH 3T3 cells and Xenopus oocytes. Furthermore, reciprocal co-immunoprecipitation verified the interaction between the native polycystin-2 and troponin I in human adult heart tissues. This study thus provides new evidence for a direct attachment of PC2 to the actin microfilament network, in addition to the recently identified association between PC2 and trypomyosin-1. Troponin I functions as an inhibitory subunit of the troponin complex for calcium-dependent regulation of muscle contraction and as an inhibitor of angiogenesis seen in ADPKD. It is possible that altered interaction due to pathogenic polycystin-1 or -2 mutations can account for angiogenesis in ADPKD and may be corrected to some extent by exogenous troponin I.

Autosomal dominant polycystic kidney disease (ADPKD) occurs by germline mutation in PKD1 or PKD2. Evidence of homozygous inactivation of either gene in human cyst lining cells as well as in mouse knockout models strongly supports a two-hit mechanism for cyst formation. Discovery of trans-heterozygous mutations in PKD1 and PKD2 in a minority of human renal cysts has led to the proposal that such mutations also can play a role in cyst formation. In the current study, we investigated the role of trans-heterozygous mutations in mouse models of polycystic kidney disease. In Pkd1(+/-), Pkd2 (+/-) and Pkd1(+/-) : Pkd2 (+/-) mice, the renal cystic lesion was mild and variable with no adverse effect on survival at 1 year. In keeping with the two-hit mechanism of cyst formation, approximately 70% of kidney cysts in Pkd2 (+/-) mice exhibited uniform loss of polycystin-2 expression. Cystic disease in trans-heterozygous Pkd1(+/-) : Pkd2 (+/-) mice, however, was notable for severity in excess of that predicted by a simple additive effect based on cyst formation in singly heterozygous mice. The data suggest a modifier role for the 'trans' polycystin gene in cystic kidney disease, and support a contribution from threshold effects to cyst formation and growth.

Autosomal-dominant polycystic kidney disease results from at least two causal genes, PKD1 and PKD2. The identical clinical phenotype in human patients and targeted Pkd1 and Pkd2 mutant mouse models provides evidence that both gene products act in the same pathogenic pathway. The discovery of direct PKD1 and PKD2 interactions implies that both gene products, polycystin-1 and polycystin-2, play a functional role in the same molecular complex. The spectrum of germ-line mutations in both genes and the somatic mutations identified from individual PKD1 or PKD2 cysts indicate that loss of function of either PKD1 or PKD2 is the mechanism of cystogenesis in autosomal-dominant polycystic kidney disease. A novel mouse model, Pkd2WS25/-, has proved that loss of heterozygosity is the molecular mechanism of autosomal-dominant polycystic kidney disease. Recently, studies on the expression patterns of PKD1 and PKD2 in humans or mice indicate that polycystin 1 and polycystin 2 seem to have their own respective functional roles, even though most of the functions of these polycystins are parallel during human and mouse development. Pkd2-deficient mice have cardiac septum defects, but Pkd1 knockout mice do not have this phenotype. On the other hand, Pkd2 has a very low level of expression in the central nervous system when compared with Pkd1. In addition, the level of expression of Pkd1 is increased during mesenchymal condensation, whereas Pkd2 expression is unchanged. Preliminary data have shown that the PKD1/PKD2 compound trans-heterozygous has a more severe cystic phenotype in the kidney than that of an age-matched heterozygous type 1 or type 2 of autosomal-dominant polycystic kidney disease alone. This finding suggests that PKD1 may be a modifier of disease severity for PKD2, and vice versa. The characteristics of the contiguous PKD1/TSC2 syndrome phenotypes and the data from Krd mice imply that TSC2 and PAX2 may also serve as potential modifiers for the disease severity of autosomal-dominant polycystic kidney disease.

Pkd2, the mouse homologue of PKD2, the gene responsible for the second form of autosomal dominant polycystic kidney disease, is highly expressed in fetal and adult mouse tissues. The expression of Pkd2 is developmentally regulated. To begin to dissect out the regulatory mechanism of Pkd2 expression, we characterized the basic features of the gene structure and identified potential cis-regulatory elements of Pkd2 transcription. Pkd2 spans 42 kb with a transcription start site 165 bp upstream of the translation start codon. Exon 1 of Pkd2 is 755 bp long, and the full-length transcript is 5215 bp. The Pkd2 promoter region is GC-rich and lacks a consensus TATA or CCAAT box. Consensus binding sites for the transcription factors Sp-1, NF-1, and Ap-2 lie in the 5' upstream region of Pkd2. The Sp-1 binding site is conserved in 5' upstream sequences of both the mouse and the human genes. The CAT activity of a series of upstream segments from +178 to -2749 was assessed in MDCK, LLCPK1, COS-7, and HEK293 cells. Deletion analysis identified a 409-bp fragment from position -221 to +178 responsible for basal promoter activity. A 922-bp fragment from -744 to +178 showed the highest level of CAT activity in the cell lines tested. These data define a functional promoter candidate region for Pkd2.

It is estimated that approximately 15% of families with autosomal dominant polycystic kidney disease (ADPKD) have mutations in PKD2. Identification of these mutations is central to identifying functionally important regions of gene and to understanding the mechanisms underlying the pathogenesis of the disorder. The current study describes mutations in six type 2 ADPKD families. Two single base substitution mutations discovered in the ORF in exon 14 constitute the most COOH-terminal pathogenic variants described to date. One of these mutations is a nonsense change and the other encodes an apparent missense variant. Reverse transcription-PCR from patient lymphoblast RNA showed that, in addition, both mutations resulted in out-of-frame splice variants by activating cryptic splice sites via different mechanisms. The apparent missense variant produced such a strong splicing signal that the processed transcript from the mutant chromosome did not contain any of the normally spliced, missense product. A third mutation, a nonconservative missense change effecting a negatively charged residue in the third transmembrane span, is likely pathogenic and defines a highly conserved residue consistent with a potential channel subunit function for polycystin-2. The remaining three mutations included two frame shifts resulting from deletion of one or two bases in exons 6 and 10, respectively, and a nonsense mutation due to a single base substitution in exon 4. The study also defined a novel intragenic polymorphism in exon 1 that will be useful in analyzing “second hits” in PKD2. Finally, the study demonstrates that there are reduced levels of normal polycystin-2 protein in lymphoblast lines from PKD2-affected individuals and that truncated mutant polycystin-2 cannot be detected in patient lymphoblasts, suggesting that the latter may be unstable in at least some tissues. The mutations described will serve as critical reagents for future functional studies in PKD2.

The PKHD1 (polycystic kidney and hepatic disease 1) gene responsible for autosomal recessive polycystic kidney disease has been mapped to 6p21.1-p12 to an approximately 1-cM interval flanked by the markers D6S1714/D6S243 and D6S1024. We have developed a sequence-ready BAC/PAC-based contig map of this region as the next step for the positional cloning of PKHD1. This contig comprising 52 clones spanning approximately 1 Mb was established by content mapping of 44 BAC/PAC-end-derived STSs, 3 known genetic markers, 5 YAC-end-derived STSs, 3 random STSs, 1 previously mapped gene, and 1 EST. The average depth per marker is 6.3 clones, and the average STS density is 20 kb. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. A high-resolution BAC/PAC-based contig map is essential to the ultimate goal of identifying the PKHD1 gene.

Mutations in PKD2 cause autosomal dominant kidney disease (ADPKD). Polycystin-2, the PKD2 gene product, is an integral membrane glycoprotein of unknown function. We have identified PKD2L, another member of the PKD2 gene family. PKD2L is expressed in adult heart and skeletal muscle, brain, spleen, testis, and retina, and alternative transcripts of 2.4, 2.7, and 3.0 kb are seen. PKD2L shows 56% identity and 76% similarity with polycystin-2 over a 581-amino-acid span; however, the COOH-terminal 65 residues of PKD2L are unrelated to PKD2. PKD2L is localized to chromosome 10q25 and is excluded as a candidate gene for autosomal recessive polycystic kidney disease, autosomal dominant polycystic liver disease, and the third form of ADPKD. Given the high degree of homology between PKD2L and PKD2, it is likely that the respective functions of these proteins are also closely related.

The gene responsible for the second form of autosomal dominant polycystic kidney disease, PKD2, has recently been identified. We now describe the cloning, genomic localization, cDNA sequence, and expression analysis of its murine homologue, Pkd2. The cloned cDNA sequence is 5134 bp long and is predicted to encode a 966-amino-acid integral membrane protein with six membrane-spanning domains and intracellular NH 2 and COOH termini. Pkd2 is highly conserved with 91% identity and 98% similarity to polycystin-2 at the amino acid level. Pkd2 mRNA is widely expressed in mouse tissues. Pkd2 maps to mouse Chromosome 5 and is excluded as a candidate gene for previously mapped mouse mutations resulting in a polycystic kidney phenotype.

PKD2, the gene defective in the second form of autosomal dominant polycystic kidney disease (ADPKD), has been identified by positional cloning and found to encode an integral membrane protein with similarity to the gene for the more common form of ADPKD and to calcium channels. We have determined the exon-intron structure of the PKD2 gene. PKD2 is encoded in at least 15 exons with the translation start site in exon 1. All the splice acceptor and donor sites conform to the AG/GT rule. We have designed a series of intronic oligonucleotide primers for amplifying the entire coding sequence from genomic DNA in segments well suited to mutation analysis using conventional screening strategies such as SSCA or heteroduplex analysis.

Autosomal recessive polycystic kidney disease is one of the most common hereditary renal cystic diseases in children. Genetic studies have recently assigned the only known locus for this disorder, PKHD1, to chromosome 6p21-p12. We have generated a YAC contig that spans approximately 5 cM of this region, defined by the markers D6S1253-D6S295, and have mapped 43 sequence-tagged sites (STS) within this interval. This set includes 20 novel STSs, which define 12 unique positions in the region, and three ESTs. A minimal set of two YACs spans the segment D6S465-D6S466, which contains PKHD1, and estimates of their sizes based on information in public databases suggest that the size of the critical region is3.1 Mb. Twenty-eight STSs map to this interval, giving an average STS density of1/150 kb. These resources will be useful for establishing a complete transcription map of the PKHD1 region.

A second gene for autosomal dominant polycystic kidney disease was identified by positional cloning. Nonsense mutations in this gene (PKD2) segregated with the disease in three PKD2 families. The predicted 968-amino acid sequence of the PKD2 gene product has six transmembrane spans with intracellular amino- and carboxyl-termini. The PKD2 protein has amino acid similarity with PKD1, the Caenorhabditis elegans homolog of PKD1, and the family of voltage-activated calcium (and sodium) channels, and it contains a potential calcium-binding domain.

Lymphomatous polyposis (LP) is generally thought to be an expression of non-Hodgkin's lymphoma (NHL) of follicular mantle cell (MC) origin. We report nine patients with LP from more than 3,500 cases of NHL studied by the Nebraska Lymphoma Study Group. Our patients differed from those reported previously in that LP represented a follicular center cell (FCC) NHL in two of the nine cases, with the remainder consisting of MC NHL. Three patients developed LP during a relapse of previously diagnosed and treated extraintestinal MC NHL (parotid gland, tonsil, and inguinal lymph node, respectively), whereas the other six patients presented with primary LP. In seven of the nine LP cases, a large mass predominated among a myriad of small polyps. The FCC cases were confined to the small intestine, whereas the MC cases were either pan-intestinal or colonic on their localization. Two MC cases studied by Southern blotting exhibited rearrangement of the bcl-1 locus. Bcl-2 rearrangement was not detected in any of the nine cases when studied by either a polymerase chain reaction-based assay (seven cases) or by Southern blotting (two cases). To date, four patients (three MC, one FCC) have experienced recurrent NHL in gastrointestinal sites. With follow-up ranging from 13 to 147 months, the entire group had a median survival of 41 months (primary MC LP:13, 13, 41, and 77 months; primary FCC LP:45 and 147 months; secondary MC LP:17, 41 and 76 months), and only one patient has died. We conclude that LP is a rare manifestation of NHL of either follicular MC or germinal center cell origin.

A simple and practical method of unfractionated bone marrow processing and cryopreservation was studied. The date shows that RBCs can be rapidly sedimented by methylcellulose or sodium carboxymethyl starch within 15-45 min. The cells can be cryopreserved in a mixture consisting of 5% DMSO, 6% HES and 4% Albumin prepared in a Lactated Ringer solution which is widely used, and can be simply immersed into a -80 degrees C freezer and stored in liquid nitrogen until infusion. Recovery percentages of nucleated cell, cell viability and CFU-G were similar to those cryopreserved with the conventional method. Clinical toxicity was mild in 12 infused patients. Of them 8 patients had received high dose chemotherapy +/- TBI and their peripheral WBC recovery was rapid. The recovery of WBC or Platelet (PLT) in the study group was similar to that of the control group whose marrow cells were cryopreserved in 10% DMSO. Therefore, cells cryopreserved with this method can also accelerate the hematopoietic recovery in myeloablatively treated patients.