Linkage confirms canine pkd1 orthologue as a candidate for bull terrier polycystic kidney disease

Bull terrier polycystic kidney disease (BTPKD) is a Mendelian disorder with many features reminiscent of human autosomal dominant polycystic disease, the latter disease being due to mutations at PKD1 and PKD2 loci. We investigated the role of the canine pkd1 orthologue in BTPKD via linkage analysis of a large kindred in which the disorder is segregating. Twelve microsatellite markers around the canine pkd1 locus (CFA6) were amplified from the genomic DNA of 20 affected and 16 unaffected bull terriers. An additional 28 affected dogs were genotyped at five key microsatellites. A highly significant multi-point LOD score that peaked over the canine pkd1 locus was observed (LOD = 6.59, best two-point LOD score LOD = 6.02), implicating this as the BTPKD locus.     

PKHD1基因缺陷与常染色体隐性遗传性多囊肾病的研究进展

PKHD1是目前所知人类常染色体隐性遗传多囊肾病(autosomal recessive polycystic kidney disease,ARPKD)的惟一致病基因。ARPKD临床病变以双肾多发性进行性充液囊泡为主要特征。目前对PKHDl基因在ARPKD发病中的作用了解并不多。该文对ARPKD的发病机制和PKHD1基因功能最新研究进展进行综述。     

A non-synonymous mutation in the canine Pkd1 gene is associated with autosomal dominant polycystic kidney disease in Bull Terriers

Polycystic Kidney Disease is an autosomal dominant disease common in some lines of Bull Terriers (BTPKD). The disease is linked to the canine orthologue of human PKD1 gene, Pkd1, located on CFA06, but no disease-associated mutation has been reported. This study sequenced genomic DNA from two Bull Terriers with BTPKD and two without the disease. A non-synonymous G>A transition mutation in exon 29 of Pkd1 was identified. A TaqMan® SNP Genotyping Assay was designed and demonstrated the heterozygous detection of the mutation in 47 Bull Terriers with BTPKD, but not in 102 Bull Terriers over one year of age and without BTPKD. This missense mutation replaces a glutamic acid residue with a lysine residue in the predicted protein, Polycystin 1. This region of Polycystin 1 is highly conserved between species, and is located in the first cytoplasmic loop of the predicted protein structure, close to the PLAT domain and the second transmembrane region. Thus, this change could alter Polycystin 1 binding or localization. Analytic programs PolyPhen 2, Align GVGD and SIFT predict this mutation to be pathogenic. Thus, BTPKD is associated with a missense mutation in Pkd1, and the application of this mutation specific assay could reduce disease transmission by allowing diagnosis of disease in young animals prior to breeding.     

Molecular and cellular pathophysiology of autosomal recessive polycystic kidney disease (ARPKD)

Autosomal recessive polycystic kidney disease (ARPKD) belongs to a group of congenital hepatorenal fibrocystic syndromes characterized by dual renal and hepatic involvement of variable severity. Despite the wide clinical spectrum of ARPKD (MIM 263200), genetic linkage studies indicate that mutations at a single locus, PKHD1 (polycystic kidney and hepatic disease 1), located on human chromosome region 6p21.1–p12, are responsible for all phenotypes of ARPKD. Identification of cystic disease genes and their encoded proteins has provided investigators with critical tools to begin to unravel the molecular and cellular mechanisms of PKD. PKD cystic epithelia share common phenotypic abnormalities despite the different genetic mutations that underlie the disease. Recent studies have shown that many cyst-causing proteins are expressed in multimeric complexes at distinct subcellular locations within epithelia. This co-expression of cystoproteins suggests that cyst formation, regardless of the underlying disease gene, results from perturbations in convergent and/or integrated signal transduction pathways. To date, no specific therapies are in clinical use for ameliorating cyst growth in ARPKD. However, studies noted in this review suggest that therapeutic targeting of the cAMP and epidermal growth factor receptor (EGFR)-axis abnormalities in cystic epithelia may translate into effective therapies for ARPKD and, by analogy, autosomal dominant polycystic kidney disease (ADPKD). A particularly promising approach appears to be the targeting of downstream intermediates of both the cAMP and EGFR axis. This review focuses on ARPKD and presents a concise summary of the current understanding of the molecular genetics and cellular pathophysiology of this disease. It also highlights phenotypic and mechanistic similarities between ARPKD and ADPKD.The authors are supported by the National Institutes of Health (grant no. 1-P50-DK57306), the PKD Foundation (grant no. 76a2r), and the Children’s Research Institute, Children’s Hospital of Wisconsin.     

Autosomal dominant polycystic kidney disease: Genetics, mutations and microRNAs

Autosomal dominant polycystic kidney disease (ADPKD) is a common, monogenic multi-systemic disorder characterized by the development of renal cysts and various extrarenal manifestations. Worldwide, it is a common cause of end-stage renal disease. ADPKD is caused by mutation in either one of two principal genes, PKD1 and PKD2, but has large phenotypic variability among affected individuals, attributable to PKD genic and allelic variability and, possibly, modifier gene effects. Recent studies have generated considerable information regarding the genetic basis and molecular diagnosis of this disease, its pathogenesis, and potential strategies for targeted treatment. The purpose of this article is to provide a comprehensive review of the genetics of ADPKD, including mechanisms responsible for disease development, the role of gene variations and mutations in disease presentation, and the putative role of microRNAs in ADPKD etiology. The emerging and important role of genetic testing and the advent of novel molecular diagnostic applications also are reviewed. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Epigenetics and autosomal dominant polycystic kidney disease

The roles of epigenetic modulation of gene expression and protein functions in autosomal dominant polycystic kidney disease (ADPKD) have recently become the focus of scientific investigation. Evidence generated to date indicates that one of the epigenetic modifiers, histone deacetylases (HDACs), are important regulators of ADPKD. HDACs are involved in regulating the expression of the Pkd1 gene and are the target of fluid flow-induced calcium signal in kidney epithelial cells. Pharmacological inhibition of HDAC activity has been found to reduce the progression of cyst formation and slow the decline of kidney function in Pkd1 conditional knockout mice and Pkd2 knockout mice, respectively, implicating the potential clinical application of HDAC inhibitors on ADPKD. Since the expression of HDAC6 is upregulated in cystic epithelial cells, the potential roles of HDAC6 in regulating cilia resorption and epidermal growth factor receptor (EGFR) trafficking through deacetylating α-tubulin and regulating Wnt signaling through deacetylating β-catenin are also discussed. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Growth characteristics of cells cultured from two murine models of polycystic kidney disease

Summary Polycystic kidney disease (PKD) is characterized by multiple renal cysts that are lined by epithelium and filled with fluid. PKD may result from one of a number of factors, either inherited or environmental. In this study, we have compared two mouse models in which PKD results from a genetic cause. In the C57BL/6J-cpk model, the mutated gene is unknown. In the other model, an SV40 large T antigen transgene causes renal cysts. We examined cultured cells from the kidneys of these mouse models, comparing growth characteristics. Although several features of PKD lead one to expect that the epithelial cells lining the cysts would have an increased rate of proliferation in culture, we found that they did not. The implications of these findings are discussed.     

Apoptosis in polycystic kidney disease

Apoptosis is the process of programmed cell death. It is a ubiquitous, controlled process consuming cellular energy and designed to avoid cytokine release despite activation of local immune cells, which clear the cell fragments. The process occurs during organ development and in maintenance of homeostasis. Abnormalities in any step of the apoptotic process are associated with autoimmune diseases and malignancies. Polycystic kidney disease (PKD) is the most common inherited kidney disease leading to end-stage renal disease (ESRD). Cyst formation requires multiple mechanisms and apoptosis is considered one of them. Abnormalities in apoptotic processes have been described in various murine and rodent models of PKD as well as in human PKD kidneys. The purpose of this review is to outline the role of apoptosis in progression of PKD as well as to describe the mechanisms involved. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Compromised cytoarchitecture and polarized trafficking in autosomal dominant polycystic kidney disease cells

Cystogenesis associated with autosomal dominant polycystic kidney disease (ADPKD) is characterized by perturbations in the polarized phenotype and function of cyst-lining epithelial cells. The polycystins, the protein products of the genes mutated in the majority of ADPKD cases, have been described recently, but the pathological mechanism by which causal mutations result in the mislocalization of cell membrane proteins has remained unclear. This report documents the dissociation from the ADPKD cell basolateral membrane of three molecules essential for spatial organization and exocytosis. The adherens junction protein E-cadherin, the subcellular disposition of which governs intercellular and intracellular architecture, was discovered sequestered in an internal ADPKD cell compartment. At the same time, sec6 and sec8, components of a complex critical for basolateral cargo delivery normally arrayed at the apico-lateral apex, were depleted from the ADPKD cell plasma membrane. An analysis of membrane transport revealed that basolateral trafficking of proteins and lipids was impaired as a result of delayed cargo exit from the ADPKD cell Golgi apparatus. Apical transport proceeded normally. Taken together with recent documentation of an association between polycystin-1 and E-cadherin (Huan and van Adelsberg 1999), the data suggest that causal mutations disrupt E-cadherin-dependent cytoarchitecture, adversely affecting protein assemblies crucial for basolateral trafficking.     

Epithelin mRNA expression in polycystic kidney disease

Epithelins are polypeptides that are preferentially expressed in epithelial cells and modulate growth. Epithelin expression is predominant in tissues of epithelial origin such as the kidney, spleen, lung, placenta, and colon. Because polycystic kidney disease involves abnormal proliferation of the proximal and/or distal tubule epithelial cells, we investigated epithelin mRNA expression in polycystic kidneys of mice homozygous for the mutation. Epithelin mRNA was highly expressed in the polycystic kidneys of homozygous mice when compared with the heterozygotes or wild type controls. A study on the time course of epithelin expression indicated that epithelin mRNA expression paralleled cyst formation and progression of the disease. A 2-fold increase in expression was observed at Day 15, a stage when cystic changes were first visible. This increase in expression was also observed at Day 21, a stage of maximum disease pathology, which ultimately results in the death of the animal. In situ hybridization localized epithelin mRNA predominantly to the epithelial cell layer surrounding the cysts. The high levels of epithelin in epithelial cells suggest a role in renal epithelial cell proliferation and cyst formation in polycystic kidney disease.     

Two polymorphic microsatellites in a coding segment of the canine androgen receptor gene

A 0.6-kb segment of exon 1 of the canine androgen receptor gene contains two polymorphic CAG tandem repeats which encode strings of glutamine homopolymers. The number of CAGs in each tandem repeat was determined by (1) polymerase chain reaction (PCR) amplification of a gene segment containing both repeats, (2) cleavage between repeats with restriction enzyme EcoO109I and (3) fractionation of the restriction fragments containing individual CAG repeats by denaturing polyacrylamide gel electrophoresis (PAGE). Individual genomic DNA samples from 80 unrelated dogs (53 males plus 27 females for a total of 107 X chromosomes) contained 10–12 CAGs in the 5′ repeats and 10–13 CAGs in the 3′ repeats. Thirteen distinct androgen receptor genotypes were identified. Eleven (or 41%) of the 27 unrelated females were heterozygous in one or both repeat regions, whereas all male samples produced single bands as expected for X chromosome markers. A total of seven distinct haplotypes contributed to the 13 genotypes. The ‘polymorphism information content’ or PIC for this seven-allele X chromosome marker was 0.67.     

Novel mutations of the PKD1 gene in Korean patients with autosomal dominant polycystic kidney disease

The gene for the most common form of autosomal dominant polycystic kidney disease (ADPKD), PKD1, has recently been characterized and shown to encode an integral membrane protein, polycystin-1, which is involved in cell-cell and cell-matrix interactions. Until now, approximately 30 mutations of the 3' single copy region of the PKD1 gene have been reported in European and American populations. However, there is no report of mutations in Asian populations. Using the polymerase chain reaction and single-strand conformation polymorphism (SSCP) analysis, 91 Korean patients with ADPKD were screened for mutation in the 3' single copy region of the PKD1 gene. As a result, we have identified and characterized six mutations: three frameshift mutations (11548del8bp, 11674insG and 12722delT), a nonsense mutation (Q4010X), and two missense mutations (R3752W and D3814N). Five mutations except for Q4010X are reported here for the first time. Our findings also indicate that many different mutations are likely to be responsible for ADPKD in the Korean population. The detection of additional disease-causing PKD1 mutations will help in identifying the location of the important functional regions of polycystin-1 and help us to better understand the pathophysiology of ADPKD.     

Role of the N-terminal polycystic kidney disease domain in chitin degradation by chitinase A from a marine bacterium, Alteromonas sp. strain O-7

AIMS: The aim of study was to clarify whether the polycystic kidney disease (PKD) domain of chitinase A (ChiA) participates in the hydrolysis of powdered chitin. METHODS AND RESULTS: Site-directed mutagenesis of the conserved aromatic residues of PKD domain was performed by PCR. The aromatic residues, W30, Y48, W64 and W67, were replaced by alanine, and single- and double-mutant chitinases were produced in Escherichia coli XL10 and purified with HisTrap column. Single mutations were not quite effective on the hydrolysing activities against chitinous substrates when compared with wild-type ChiA. However, mutations of W30 and W67 decreased the activities against powdered chitin by 87.6%. Wild-type and mutant PKD domains were produced in E. coli TOP10 and purified with glutathione-Sepharose 4B column. Wild-type PKD domain showed significant binding activity to powdered chitin, whereas mutations of W30 and W67 reduced the binding activity to powdered chitin drastically. These results suggest that PKD domain of ChiA is essential for effective hydrolysis of powdered chitin through the interaction between two aromatic residues and chitin molecule. CONCLUSIONS: PKD domain of ChiA participates in the effective hydrolysis of powdered chitin through the interaction between two aromatic residues (W30 and W67) and chitin molecule. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study provide important information on chitin degradation by microbial chitinases.     

A methionine-Mettl3-N6-methyladenosine axis promotes polycystic kidney disease

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Mouse models of polycystic kidney disease induced by defects of ciliary proteins

Polycystic kidney disease (PKD) is a common hereditary disorder which is characterized by fluid-filled cysts in the kidney. Mutation in either PKD1, encoding polycystin-1 (PC1), or PKD2, encoding polycystin-2 (PC2), are causative genes of PKD. Recent studies indicate that renal cilia, known as mechanosensors, detecting flow stimulation through renal tubules, have a critical function in maintaining homeostasis of renal epithelial cells. Because most proteins related to PKD are localized to renal cilia or have a function in ciliogenesis. PC1/PC2 heterodimer is localized to the cilia, playing a role in calcium channels. Also, disruptions of ciliary proteins, except for PC1 and PC2, could be involved in the induction of polycystic kidney disease. Based on these findings, various PKD mice models were produced to understand the roles of primary cilia defects in renal cyst formation. In this review, we will describe the general role of cilia in renal epithelial cells, and the relationship between ciliary defects and PKD. We also discuss mouse models of PKD related to ciliary defects based on recent studies. [BMB Reports 2013; 46(2): 73-79]     

Computational screening of disease associated mutations on NPC1 gene and its structural consequence in Niemann-Pick type-C1

Niemann-Pick disease type C1 （NPC1）, caused by mutations of NPC1 gene, is an inherited lysosomal lipid storage disorder. Loss of functional NPC1 causes the accumulation of free cholesterol （FC） in endocytic organelles that comprised the characteristics of late endosomes and/or lysosomes. In this study we analyzed the pathogenic effect of 103 nsSNPs reported in NPC1 using computational methods. Rl186C, S940L, R958Q and I1061T mutations were predicted as most deleterious and disease associated with NPC1 using SIFT, Polyphen 2.0, PANTHER, PhD-SNP, Pmut and MUTPred tools which were also endorsed with previous in vivo experimental studies. To understand the atomic arrangement in 3D space, the native and disease associated mutant （Rl186C, S940L, R958Q and I1061T） structures were modeled. Quantitative structural and flexibility analysis was conceded to observe the structural consequence of prioritized disease associated mutations （R1186C, S940L, R958Q and I1061T）. Accessible surface area （ASA）, free folding energy （FFE） and hydrogen bond （NH bond） showed more flexibility in 3D space in mutant structures. Based on the quantitative assessment and flexibility analysis of NPC1 variants, I1061T showed the most deleterious effect. Our analysis provides a clear clue to wet laboratory scientists to understand the structural and functional effect of NPCI gene upon mutation.     

Novel mutations in the 3' region of the polycystic kidney disease 1 (PKD1) gene

Mutation screening in 90 unrelated ADPKD1 patients was carried out on some of the exons in the single copy area (37, 38, 39, 44, 45) using genomic PCR and SSCP. Four novel mutations were found: a 15 bp in-frame deletion in exon 39 [nt11449 (del 15)], a 2 bp deletion in exon 44 [nt12252 (del 2)], a G insertion in exon 44 [nt12290 (Ins G)], and a GTT in-frame deletion in exon 45 [nt12601 (del 3)].     

Pax2在肾脏发育和肾疾病中的调控作用

配对盒基因2(Paired box2,Pax2)是肾脏发育中重要的转录因子,在前、中、后肾发育的全过程表达,集中分布在发育的各级小管和间充质成分,具有特定的时空特性。研究表明Pax2与多种调节肾脏发育的因子Gdnf、Ret、SHH、Wnt4及Fgf等相互作用,共同精准诱导生肾索形成,前/中肾管的形成及分化,输尿管芽的发生及分支,肾单位的诱导分化。Pax2的变异导致多种先天性肾脏及输尿管发育畸形,最易发生在肾-视神经盘缺损综合征。在肾细胞癌、Wilms瘤和多种肾小球及肾小管获得性疾病中存在Pax2的异常表达,其诊断和治疗价值将是今后研究的重点。文章主要对Pax2的分子结构、在肾脏发育和肾疾病的表达及调控进行了综述。