Characterization of PKD Protein-Positive Exosome-Like Vesicles

Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1^del2/del2 mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease, affecting between 1:400 to 1:1000 individuals.^1,2 There are two genetic loci, PKD1 and PKD2, producing the proteins polycystin-1 (PC1)^3–5 and polycystin-2 (PC2),⁶ respectively. Autosomal recessive polycystic kidney disease (ARPKD), the most common cause of hereditary childhood PKD, is caused by mutations to PKHD1, which encodes fibrocystin/polyductin (FCP).^7,8 These three PKD proteins have been localized to primary cilia,^9,10 where the PC1/PC2 complex acts as a flow sensor on the cilium.¹¹ The role of FCP is less clear, but it complexes with PC2.^12,13Another site of PC1 expression is in urinary exosomes, small vesicles (50 to 100 nm in diameter) present in normal urine, that have been analyzed as a source of biomarkers for various renal diseases.^14,15 Urinary exosomes are thought to be end products of the multivesicular body (MVB)-sorting pathway in which membrane proteins are uniquely packaged into intraluminal vesicles (ILVs) within the MVB, some of which are secreted as exosomes when MVBs fuse with the apical plasma membrane.MVBs and exosomes have been shown to have a role in left/right (L/R) axis determination in the embryonic node. These MVBs, termed nodal vesicular parcels (NVPs), are released from the floor of the node and swept by nodal flow to the left side, where they interact with the “picket-fence” immotile cilia.¹⁶ Symmetry breaking is dependent on a PC2 Ca²⁺-dependent flux.^17,18Transmission electron microscopy studies of dilated bile ducts found in ARPKD mouse model Pkhd1^del2/del2 showed PC1+ exosome-like vesicles surrounding cholangiocyte primary cilia, whereas only occasional single ELVs were found attached to WT cilia.¹⁹ The observations of abundant PC1 in ELVs and of abnormal ELV accumulation in FCP-deficient mice led us to examine whether these may have a functional role in the urinary and biliary systems, analogous to the NVP in the node.     

The Polycystin-1, Lipoxygenase,and α-Toxin Domain Regulates Polycystin-1 Trafficking

Mutations in polycystin-1 (PC1) give rise to autosomal dominant polycystic kidney disease, an important and common cause of kidney failure. Despite its medical importance, the function of PC1 remains poorly understood. Here, we investigated the role of the intracellular polycystin-1, lipoxygenase, and α-toxin (PLAT) signature domain of PC1 using nuclear magnetic resonance, biochemical, cellular, and in vivo functional approaches. We found that the PLAT domain targets PC1 to the plasma membrane in polarized epithelial cells by a mechanism involving the selective binding of the PLAT domain to phosphatidylserine and l-α-phosphatidylinositol-4-phosphate (PI4P) enriched in the plasma membrane. This process is regulated by protein kinase A phosphorylation of the PLAT domain, which reduces PI4P binding and recruits β-arrestins and the clathrin adaptor AP2 to trigger PC1 internalization. Our results reveal a physiological role for the PC1-PLAT domain in renal epithelial cells and suggest that phosphorylation-dependent internalization of PC1 is closely linked to its function in renal development and homeostasis.     

Autosomal dominant polycystic kidney disease: recent advances in pathogenesis and potential therapies

Autosomal dominant polycystic kidney disease (ADPKD) is the most common progressive hereditary kidney disease. In 85–90 % of cases, ADPKD results from a mutation in the PKD1 gene, and the other 10–15 % of the cases are accounted for by mutations in PKD2. PKD1 and PKD2 encode polycystin-1 and polycystin-2. Polycystin-1 may be a receptor that controls the channel activity of polycystin-2 as part of the polycystin signaling complex. ADPKD is characterized by the progressive development of fluid-filled cysts derived from renal tubular epithelial cells that gradually compress the parenchyma and compromise renal function. In recent years, considerable interest has developed in the primary cilia as a site of the proteins that are involved in renal cystogenesis. The pathological processes that facilitate cyst enlargement are hypothesized to result from two specific cellular abnormalities: (1) increased fluid secretion into the cyst lumen and (2) inappropriately increased cell division by the epithelium lining the cyst. Since there is no clinically approved specific or targeted therapy, current practice focuses on blood pressure control and statin therapy to reduce the cardiac mortality associated with chronic kidney disease. However, recent advances in our understanding of the pathways that govern renal cystogenesis have led to a number of intriguing possibilities in regard to therapeutic interventions. The purpose of this article is to review the pathogenesis of renal cyst formation and to review novel targets for the treatment of ADPKD.     

The role of the polycystins in kidney development

 Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease that affects both adults and children. Renal cysts are the cardinal sign of the disease that also causes cysts in liver, pancreas, testis, and ovary, as well as cardiac valvular insufficiency and arterial aneurysms. At least three genes cause ADPKD in humans. PKD1 and PKD2 have been cloned and sequenced, both code for novel proteins. Analyses of their primary structures suggest that polycystin-1, the PKD1 gene product, is a receptor, while similarities between the polycystins and calcium channel subunits suggest that these proteins are subunits of a novel channel. Individuals with mutations in PKD1 or PKD2 have identical phenotypes, which present at a later age in PKD2 patients. Recent evidence suggests that the two polycystins interact, providing a biochemical basis for the similarity of disease caused by mutations in PKD1 and PKD2. Consistent with its protean manifestations, polycystin-1 is widely expressed in both epithelial and non-epithelial tissues during embryological development. Mice with targeted mutations of either the PKD1 or the PKD2 genes die during embryogenesis. Thus, the PKD genes are required for normal fetal development. The observation that loss of polycystin-1 or -2 function causes death during embryogenesis suggests that PKD1 and PKD2 might be part of a morphoregulatory pathway. Received: 16 June 1998 / Revised: 17 September 1998 / Accepted: 18 September 1998     

Comprehensive PKD1 and PKD2 Mutation Analysis in Prenatal Autosomal Dominant Polycystic Kidney Disease

Prenatal forms of autosomal dominant polycystic kidney disease (ADPKD) are rare but can be recurrent in some families, suggesting a common genetic modifying background. Few patients have been reported carrying, in addition to the familial mutation, variation(s) in polycystic kidney disease 1 (PKD1) or HNF1 homeobox B (HNF1B), inherited from the unaffected parent, or biallelic polycystic kidney and hepatic disease 1 (PKHD1) mutations. To assess the frequency of additional variations in PKD1, PKD2, HNF1B, and PKHD1 associated with the familial PKD mutation in early ADPKD, these four genes were screened in 42 patients with early ADPKD in 41 families. Two patients were associated with de novo PKD1 mutations. Forty patients occurred in 39 families with known ADPKD and were associated with PKD1 mutation in 36 families and with PKD2 mutation in two families (no mutation identified in one family). Additional PKD variation(s) (inherited from the unaffected parent when tested) were identified in 15 of 42 patients (37.2%), whereas these variations were observed in 25 of 174 (14.4%, P=0.001) patients with adult ADPKD. No HNF1B variations or PKHD1 biallelic mutations were identified. These results suggest that, at least in some patients, the severity of the cystic disease is inversely correlated with the level of polycystin 1 function.     

Analysis of gene mutations in PKD1/PKD2 by multiplex ligation-dependent probe amplification: some new findings

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a serious genetic disorder that can lead to chronic renal disease. Protein dysfunction caused by mutations in the genes polycystic kidney disease 1 (PKD1) and polycystic kidney disease 2 (PKD2) is an important factor in the pathogenesis of ADPKD. In the present study, 30 Chinese patients with confirmed diagnosis of ADPKD, based on ultrasound or computerized tomography (CT) findings were selected, and the exon copy numbers of PKD1 and PKD2 were determined using multiplex ligation-dependent probe amplification (MLPA). MLPA identified exon deletion in 1 case, suspected exon deletion in 4 cases, and suspected duplications in 3 cases. One case of suspected exon deletion was confirmed using quantitative real-time polymerase chain reaction (q-PCR) and sequencing (PKD2 exon 8). A missense mutation was observed in 1 case of exon deletion using q-PCR and sequencing (PKD1 exon 40, c.11333 C>A). The cases of suspected duplications were verified by q-PCR, and the copy number of exon 6 of PKD1 in 1 case of suspected duplication was 3.8 times greater than that in normal controls. Our findings provide new insights into ADPKD screening and mark a possibly meaningful step toward improved diagnosis and treatment of patients with ADPKD.     

Reduced Ciliary Polycystin-2 in Induced Pluripotent Stem Cells from Polycystic Kidney Disease Patients with PKD1 Mutations

Heterozygous mutations in PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, cause autosomal dominant PKD (ADPKD), whereas mutations in PKHD1, which encodes fibrocystin/polyductin (FPC), cause autosomal recessive PKD (ARPKD). However, the relationship between these proteins and the pathogenesis of PKD remains unclear. To model PKD in human cells, we established induced pluripotent stem (iPS) cell lines from fibroblasts of three ADPKD and two ARPKD patients. Genetic sequencing revealed unique heterozygous mutations in PKD1 of the parental ADPKD fibroblasts but no pathogenic mutations in PKD2. Undifferentiated PKD iPS cells, control iPS cells, and embryonic stem cells elaborated primary cilia and expressed PC1, PC2, and FPC at similar levels, and PKD and control iPS cells exhibited comparable rates of proliferation, apoptosis, and ciliogenesis. However, ADPKD iPS cells as well as somatic epithelial cells and hepatoblasts/biliary precursors differentiated from these cells expressed lower levels of PC2 at the cilium. Additional sequencing confirmed the retention of PKD1 heterozygous mutations in iPS cell lines from two patients but identified possible loss of heterozygosity in iPS cell lines from one patient. Furthermore, ectopic expression of wild-type PC1 in ADPKD iPS-derived hepatoblasts rescued ciliary PC2 protein expression levels, and overexpression of PC1 but not a carboxy-terminal truncation mutant increased ciliary PC2 expression levels in mouse kidney cells. Taken together, these results suggest that PC1 regulates ciliary PC2 protein expression levels and support the use of PKD iPS cells for investigating disease pathophysiology.Polycystic kidney disease (PKD) is associated with defects of primary cilia and replacement of the normal kidney parenchyma with tubular epithelial cysts and fibrosis, leading to progressive deterioration of kidney function. PKD is among the world’s most common life-threatening genetic diseases, affecting approximately 1 in 600 people, and it is a significant contributor to CKD. Autosomal dominant PKD (ADPKD) causes end stage kidney disease by the age of 60 years in approximately 50% of adults with the disease, whereas autosomal recessive PKD (ARPKD) is a more rare form that typically presents earlier in life and causes significant childhood mortality. PKD may be considered a developmental disorder, with renal cysts becoming detectable in utero even in ADPKD.¹ In addition to kidney cysts, hepatic involvement is common, with liver cysts developing in many ADPKD patients and congenital hepatic fibrosis being a hallmark of ARPKD.^1,2ADPKD is inherited as heterozygous mutations in PKD1 or PKD2, whereas ARPKD is caused by biallelic mutations in PKHD1 (polycystic kidney and hepatic disease 1). These three genes encode transmembrane proteins, known as polycystin-1 (PC1), polycystin-2 (PC2), and fibrocystin/polyductin (FPC), respectively. PC1, PC2, and FPC form a receptor channel complex in membrane compartments including the primary cilium,^3,4 a sensory organelle on the apical cell surface, and loss of this localization pattern has been observed in cystic renal epithelia from humans.^5,6 Mutations in more than 50 gene products associated with the cilium cause a spectrum of related diseases known as the ciliopathies, most of which feature cystic kidneys.⁷ Ciliary trafficking signals have recently been identified at the carboxyl terminus of PC1 and the amino terminus of PC2, but the extent to which PC1 is involved in PC2 trafficking is not yet clear.^8–11 The abnormal phenotype in ADPKD has been attributed to loss of epithelial cell heterozygosity as a result of an additional somatic mutation or environmental insult (the two-hit hypothesis), although there is also genetic evidence for a haploinsufficiency model.^12–15There is a need for human disease-specific laboratory models for PKD to better understand disease and develop therapies, because animal models may not fully genocopy or phenocopy the human disease.^16,17 Primary cells taken from nephrectomized ADPKD kidneys have been linked to various epithelial cell phenotypes, but because these cells are derived from kidneys with advanced disease, it remains unclear whether these characteristics represent primary defects central to PKD etiology or secondary consequences of injury or dedifferentiation.^6,18–21 A powerful new technology, induced pluripotent stem (iPS) cells are adult somatic cells which have been reprogrammed into an embryonic pluripotent state.^22,23 The result is a next generation cell culture model that can differentiate into diverse cell types and complex tissues for the purposes of regenerative therapies or investigating disease. As for other hereditary diseases, iPS cells from patients with PKD can be examined for disease-specific abnormalities to better understand the pathophysiology of clinical mutations and screen for potential therapeutics.^7,24 PKD iPS cells derived from unaffected cell types, such as fibroblasts, might be expected to have fewer secondary phenotypes compared with cyst-lining epithelial cells, and they could be used to investigate PKD during development, when PKD disease genes are most highly expressed.^1,16,21,25 Their intrinsic pluripotency, ability to self-renew indefinitely, and immunocompatibility also make PKD iPS cells an attractive potential source for renal replacement tissue. As a first step in this direction, generation of iPS cells from one ADPKD patient was recently reported, although no disease phenotypes were described.²⁶ In our study, we generate iPS cell lines from ADPKD, ARPKD, and healthy control patients and evaluate their ability to ciliate, proliferate, and express PKD disease genes to establish a system in vitro for investigating human PKD. We identify reduced levels of PC2 at the primary cilium in undifferentiated iPS cells, differentiated somatic epithelial cells, and hepatoblasts as a consistent phenotype in three ADPKD patients with PKD1 mutations but not in ARPKD patients. Furthermore, we have found using ADPKD iPS-derived hepatoblasts and cultured kidney cells that wild-type but not mutant PC1 promotes PC2 localization to cilia.     

The PROPKD Score: A New Algorithm to Predict Renal Survival in Autosomal Dominant Polycystic Kidney Disease

The course of autosomal dominant polycystic kidney disease (ADPKD) varies among individuals, with some reaching ESRD before 40 years of age and others never requiring RRT. In this study, we developed a prognostic model to predict renal outcomes in patients with ADPKD on the basis of genetic and clinical data. We conducted a cross-sectional study of 1341 patients from the Genkyst cohort and evaluated the influence of clinical and genetic factors on renal survival. Multivariate survival analysis identified four variables that were significantly associated with age at ESRD onset, and a scoring system from 0 to 9 was developed as follows: being male: 1 point; hypertension before 35 years of age: 2 points; first urologic event before 35 years of age: 2 points; PKD2 mutation: 0 points; nontruncating PKD1 mutation: 2 points; and truncating PKD1 mutation: 4 points. Three risk categories were subsequently defined as low risk (0–3 points), intermediate risk (4–6 points), and high risk (7–9 points) of progression to ESRD, with corresponding median ages for ESRD onset of 70.6, 56.9, and 49 years, respectively. Whereas a score ≤3 eliminates evolution to ESRD before 60 years of age with a negative predictive value of 81.4%, a score >6 forecasts ESRD onset before 60 years of age with a positive predictive value of 90.9%. This new prognostic score accurately predicts renal outcomes in patients with ADPKD and may enable the personalization of therapeutic management of ADPKD.     

Current advances in molecular genetics of autosomal-dominant polycystic kidney disease

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.     

Hyperproliferation of PKD1 cystic cells is induced by insulin-like growth factor-1 activation of the Ras/Raf signalling system

Autosomal dominant polycystic kidney disease (ADPKD) largely results from mutations in the PKD1 gene leading to hyperproliferation of renal tubular epithelial cells and consequent cyst formation. Rodent models of PKD suggest that the multifunctional hormone insulin-like growth factor-1 (IGF-1) could play a pathogenic role in renal cyst formation. In order to test this possibility, conditionally immortalized renal epithelial cells were prepared from normal individuals and from ADPKD patients with known germline mutations in PKD1. All patient cell lines had a decreased or absence of polycystin-1 but not polycystin-2. These cells had an increased sensitivity to IGF-1 and to cyclic AMP, which required phosphatidylinositol-3 (PI3)-kinase and the mitogen-activated protein kinase, extracellular signal-regulated protein kinase (ERK) for enhanced growth. Inhibition of Ras or Raf abolished the stimulated cell proliferation. Our results suggest that haploinsufficiency of polycystin-1 lowers the activation threshold of the Ras/Raf signalling system leading to growth factor-induced hyperproliferation. Inhibition of Ras or Raf activity may be a therapeutic option for decreasing tubular cell proliferation in ADPKD.     

Soluble TNF Receptors and Kidney Dysfunction in the Elderly

The importance of TNF-α and its soluble receptors (sTNFR1 and sTNFR2) in the development of kidney disease is being unraveled. Yet, community-based data regarding the role of sTNFRs are lacking. We assessed serum sTNFRs and aspects of kidney damage cross-sectionally in two independent community-based cohorts of elderly participants: Prospective Investigation of the Vasculature in Uppsala Seniors (n=815; mean age, 75 years; 51% women) and Uppsala Longitudinal Study of Adult Men (n=778; mean age, 78 years). Serum sTNFR1 correlated substantially with different aspects of kidney pathology in the Uppsala Longitudinal Study of Adult Men cohort (R=−0.52 for estimated GFR, R=0.22 for urinary albumin-to-creatinine ratio, and R=0.17 for urinary kidney injury molecule-1; P<0.001 for all), with similar correlations in the Prospective Investigation of the Vasculature in Uppsala Seniors cohort. These associations remained significant after adjustment for age, sex, inflammatory markers, and cardiovascular risk factors and were also evident in participants without diabetes. Serum sTNFR2 was associated with all three markers in the Prospective Investigation of the Vasculature in Uppsala Seniors cohort (P<0.001 for all). Our findings from two independent community-based cohorts confirm and extend results of previous studies supporting circulating sTNFRs as relevant biomarkers for kidney damage and dysfunction in elderly individuals, even in the absence of diabetes.     

Competitive Interaction Between Fibroblast Growth Factor 23 And Asymmetric Dimethylarginine in Patients With CKD

Both fibroblast growth factor 23 (FGF-23) and asymmetric dimethylarginine (ADMA) are associated with progression of CKD. We tested the hypothesis that ADMA and FGF23 are interactive factors for CKD progression in a cohort of 758 patients with CKD in Southern Europe (mean eGFR±SD, 36±13 ml/min per 1.73 m²) and in a central European cohort of 173 patients with CKD (MMKD study, mean eGFR, 64±39 ml/min per 1.73 m²). In the first cohort, 214 patients had renal events (decrease in eGFR of >30%, dialysis, or kidney transplantation) during a 3-year follow-up. Both intact FGF-23 and ADMA predicted the incidence rate of renal events in unadjusted and adjusted analyses (P<0.001). There was a strong competitive interaction between FGF-23 and ADMA in the risk of renal events (P<0.01 in adjusted analyses); the risk associated with raised ADMA levels was highest in patients with low FGF-23 levels. These results were confirmed in the MMKD cohort, in which FGF-23 level was again an effect modifier of the relationship between plasma ADMA level and renal events (doubling of baseline serum creatinine, dialysis, or kidney transplantation) in the adjusted analyses (P<0.01). Furthermore, in the MMKD cohort there was a parallel, independent competitive interaction between symmetric dimethylarginine level and c-terminal FGF-23 level for the risk for renal events (P=0.001). These findings indicate that the association of ADMA level with the risk of CKD progression is modified by FGF-23 level and provide further evidence that dysregulation of the nitric oxide system is involved in CKD progression.     

ADPKD: molecular characterization and quest for treatment

Autosomal-dominant polycystic kidney disease (ADPKD) is a common hereditary disease that features multiple cystogenesis in various organs and vascular defects. The genes responsible for ADPKD, PKD1, and PKD2 have been identified, and the pathological processes of the disease are becoming clearer. This review focuses on recent findings about the molecular and cellular biology of ADPKD, and especially on PKD1. PKD1 and its product, polycystin-1, play pivotal roles in cellular differentiation because they regulate the cell cycle and because polycystin-1 is a component of adherens junctions. A possible link between polycystin-1 and PPARγ is discussed. The extraordinarily fast research progress in this area in the last decade has now reached a stage where the development of a remedy for ADPKD might become possible in the near future.     

Polycystin-1 expression in PKD1, early-onset PKD1, and TSC2/PKD1 cystic tissue.

BACKGROUND: The mutational mechanism responsible for cyst formation in polycystic kidney disease 1 gene (PKD1) remains controversial, with data indicating a two-hit mechanism, but also evidence of polycystin-1 expression in cystic tissue. METHODS: To investigate this apparent paradox, we analyzed polycystin-1 expression in cystic renal or liver tissue from 10 patients with truncating PKD1 mutations (including one early-onset case) and 2 patients with severe disease associated with contiguous deletions of TSC2 and PKD1, using monoclonal antibodies (mAbs) to both extreme N-(7e12) and C-terminal (PKS-A) regions of the protein. Truncation of the C-terminal epitope from the putative mutant proteins in each case allowed exclusive assessment of the nontruncated protein with PKS-A. RESULTS: In adult PKD1 tissue, the majority of cysts (approximately 80%) showed polycystin-1 expression, although staining was absent in a variable but significant minority (approximately 20%), in spite of the normal expression of marker proteins. Unlike adult PKD1, however, negative cysts were rarely found in infantile PKD1 or TSC2/PKD1 deletion cases. CONCLUSIONS: If a two-hit mutational mechanism is operational, these results suggest that the majority of somatic mutations in adult PKD1 are likely to be missense changes. The low level of polycystin-1-negative cysts in the three "early-onset" cases, however, suggests that a somatic PKD1 mutation may not always be required for cyst formation.     

The polycystic kidney disease proteins,polycystin-1, polycystin-2, polaris,and cystin,are co-localized in renal cilia

Recent evidence has suggested an association between structural and/or functional defects in the primary apical cilium of vertebrate epithelia and polycystic kidney disease (PKD). In Caenorhabditis elegans, the protein orthologues of the PKD-related proteins, polycystin-1 (LOV-1), polycystin-2 (PKD2), and polaris (OSM-5), co-localize in the cilia of male-specific sensory neurons, and defects in these proteins cause abnormalities of cilia structure and/or function. This study sought to determine whether the mammalian polycystins are expressed in primary cilia of renal epithelia and whether these proteins co-localize with polaris and cystin, the newly described, cilia-associated protein that is disrupted in the cpk mouse. To begin to address this issue, the expression of the protein products encoded by the PKD1, PKD2, Tg737, and cpk genes were examined in mouse cortical collecting duct (mCCD) cells using an immunofluorescence-based approach with a series of previously well-characterized antibodies. The mCCD cells were grown on cell culture inserts to optimize cell polarization and cilia formation. The data demonstrate co-localization in cilia of polycystin-1 and polycystin-2, which are the principal proteins involved in autosomal dominant polycystic kidney disease, with polaris and cystin, which are proteins that are disrupted in the Tg737(orpk)and cpk mouse models of autosomal recessive polycystic kidney disease, respectively. These data add to a growing body of evidence that suggests that primary cilium plays a key role in normal physiologic functions of renal epithelia and that defects in ciliary function contribute to the pathogenesis of PKD.     

Copeptin Is Associated with Kidney Length,Renal Function,and Prevalence of Simple Cysts in a Population-Based Study

Arginine vasopressin (AVP) has a key role in osmoregulation by facilitating water transport in the collecting duct. Recent evidence suggests that AVP may have additional effects on renal function and favor cyst growth in polycystic kidney disease. Whether AVP also affects kidney structure in the general population is unknown. We analyzed the association of copeptin, an established surrogate for AVP, with parameters of renal function and morphology in a multicentric population-based cohort. Participants from families of European ancestry were randomly selected in three Swiss cities. We used linear multilevel regression analysis to explore the association of copeptin with renal function parameters as well as kidney length and the presence of simple renal cysts assessed by ultrasound examination. Copeptin levels were log-transformed. The 529 women and 481 men had median copeptin levels of 3.0 and 5.2 pmol/L, respectively (P<0.001). In multivariable analyses, the copeptin level was associated inversely with eGFR (β=−2.1; 95% confidence interval [95% CI], −3.3 to −0.8; P=0.002) and kidney length (β=−1.2; 95% CI, −1.9 to −0.4; P=0.003) but positively with 24-hour urinary albumin excretion (β=0.11; 95% CI, 0.01 to 0.20; P=0.03) and urine osmolality (β=0.08; 95% CI, 0.05 to 0.10; P<0.001). A positive association was found between the copeptin level and the presence of renal cysts (odds ratio, 1.6; 95% CI, 1.1 to 2.4; P=0.02). These results suggest that AVP has a pleiotropic role in renal function and may favor the development of simple renal cysts.     

Inactivation of Integrin-β1 Prevents the Development of Polycystic Kidney Disease after the Loss of Polycystin-1

Dysregulation of polycystin-1 (PC1) leads to autosomal dominant polycystic kidney disease (ADPKD), a disorder characterized by the formation of multiple bilateral renal cysts, the progressive accumulation of extracellular matrix (ECM), and the development of tubulointerstitial fibrosis. Correspondingly, cystic epithelia express higher levels of integrins (ECM receptors that control various cellular responses, such as cell proliferation, migration, and survival) that are characteristically altered in cystic cells. To determine whether the altered expression of ECM and integrins could establish a pathologic autostimulatory loop, we tested the role of integrin-β1 in vitro and on the cystic development of ADPKD in vivo. Compared with wild-type cells, PC1-depleted immortalized renal collecting duct cells had higher levels of integrin-β1 and fibronectin and displayed increased integrin-mediated signaling in the presence of Mn²⁺. In mice, conditional inactivation of integrin-β1 in collecting ducts resulted in a dramatic inhibition of Pkd1-dependent cystogenesis with a concomitant suppression of fibrosis and preservation of normal renal function. Our data provide genetic evidence that a functional integrin-β1 is required for the early events leading to renal cystogenesis in ADPKD and suggest that the integrin signaling pathway may be an effective therapeutic target for slowing disease progression.     

Vascular expression of polycystin-2

The expression of polycystin-1 in the vascular smooth muscle cells (VSMC) of elastic and large distributive arteries suggests that some vascular manifestations of autosomal-dominant polycystic kidney disease (ADPKD) result directly from the genetic defect. Intracranial aneurysms have been reported in PKD2, as well as in PKD1 families. To determine whether the vascular expression of polycystin-2 is similar to that of polycystin-1, the expression of PKD2 mRNA and protein in cultured pig aortic VSMC was studied and immunofluorescence and immunohistochemistry were used to study the localization of polycystin-2 in cultured pig aortic VSMC, pig ascending thoracic aorta, and normal elastic and intracranial arteries and intracranial aneurysms obtained at autopsy from patients without or with ADPKD. Tissues derived from Pkd2 wild-type and Pkd2 null mice were used to confirm the specificity of the immunostaining for polycystin-2. Northern blots of VSMC revealed the expected 5.3-kb band. Western blotting detected a 110-kb band in a 100,000 x g fraction of VSMC homogenates. Cultured VSMC as well as VSMC between the elastic lamellae of pig thoracic aorta were positive for polycystin-2 by immunofluorescence. The staining pattern was cytoplasmic. Treatment of the cells before fixation with Taxol, colchicine, or cytochalasin-D altered the pattern of staining in a way suggesting alignment with the cytoskeleton. The immunohistochemical staining for polycystin-2 was abolished by extraction with 0.5% Triton X-100, indicating that polycystin-2 is not associated with the cytoskeleton. Weak immunoreactivity for polycystin-2, which was markedly enhanced by protease digestion, was detected in formaldehyde-fixed normal human elastic and intracranial arteries. Immunostaining of variable intensity for polycystin-2, which was not consistently enhanced by protease digestion, was seen in the spindle-shaped cells of the wall of the intracranial aneurysms. The similar expression of polycystin-1 and polycystin-2 in the vascular smooth muscle is consistent with the proposed interaction of these proteins in a single pathway. These observations suggest a direct pathogenic role for PKD1 and PKD2 mutations in the vascular complications of ADPKD.     

Double inhibition of cAMP and mTOR signalling may potentiate the reduction of cell growth in ADPKD cells

Background

ADPKD is a renal pathology caused by mutations of PKD1 and PKD2 genes, which encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC1 plays an important role regulating several signal transducers, including cAMP and mTOR, which are involved in abnormal cell proliferation of ADPKD cells leading to the development and expansion of kidney cysts that are a typical hallmark of this disease. Therefore, the inhibition of both pathways could potentiate the reduction of cell proliferation enhancing benefits for ADPKD patients.

Methods

The inhibition of cAMP- and mTOR-related signalling was performed by Cl-IB-MECA, an agonist of A3 receptors, and rapamycin, respectively. Protein kinase activity was evaluated by immunoblot and cell growth was analyzed by direct cell counting.

Results

The activation of A₃AR by the specific agonist Cl-IB-MECA causes a marked reduction of CREB, mTOR, and ERK phosphorylation in kidney tissues of Pkd1 ^flox/?: Ksp-Cre polycystic mice and reduces cell growth in ADPKD cell lines, but not affects the kidney weight. The combined sequential treatment with rapamycin and Cl-IB-MECA in ADPKD cells potentiates the reduction of cell proliferation compared with the individual compound by the inhibition of CREB, mTOR, and ERK kinase activity. Conversely, the simultaneous application of these drugs counteracts their effect on cell growth, because the inhibition of ERK kinase activity is lost.

Conclusion

The double treatment with rapamycin and Cl-IB-MECA may have synergistic effects on the inhibition of cell proliferation in ADPKD cells suggesting that combined therapies could improve renal function in ADPKD patients.