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Improved Structure and Function in Autosomal Recessive Polycystic Rat Kidneys with Renal Tubular Cell Therapy.

ABSTRACT: Autosomal recessive polycystic kidney disease is a truly catastrophic monogenetic disease, causing death and end stage renal disease in neonates and children. Using PCK female rats, an orthologous model of autosomal recessive polycystic kidney disease harboring mutant Pkhd1, we tested the hypothesis that intravenous renal cell transplantation with normal Sprague Dawley male kidney cells would improve the polycystic kidney disease phenotype. Cytotherapy with renal cells expressing wild type Pkhd1 and tubulogenic serum amyloid A1 had powerful and sustained beneficial effects on renal function and structure in the polycystic kidney disease model. Donor cell engraftment and both mutant and wild type Pkhd1 were found in treated but not control PCK kidneys 15 weeks after the final cell infusion. To examine the mechanisms of global protection with a small number of transplanted cells, we tested the hypothesis that exosomes derived from normal Sprague Dawley cells can limit the cystic phenotype of PCK recipient cells. We found that renal exosomes originating from normal Sprague Dawley cells carried and transferred wild type Pkhd1 mRNA to PCK cells in vivo and in vitro and restricted cyst formation by cultured PCK cells. The results indicate that transplantation with renal cells containing wild type Pkhd1 improves renal structure and function in autosomal recessive polycystic kidney disease and may provide an intra-renal supply of normal Pkhd1 mRNA.

SUBMITTER: Kelly KJ

PROVIDER: S-EPMC4489886 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Improved Structure and Function in Autosomal Recessive Polycystic Rat Kidneys with Renal Tubular Cell Therapy.

Kelly K J KJ Zhang Jizhong J Han Ling L Kamocka Malgorzata M Miller Caroline C Gattone Vincent H VH Dominguez Jesus H JH

PloS one 20150702 7

Autosomal recessive polycystic kidney disease is a truly catastrophic monogenetic disease, causing death and end stage renal disease in neonates and children. Using PCK female rats, an orthologous model of autosomal recessive polycystic kidney disease harboring mutant Pkhd1, we tested the hypothesis that intravenous renal cell transplantation with normal Sprague Dawley male kidney cells would improve the polycystic kidney disease phenotype. Cytotherapy with renal cells expressing wild type Pkhd1 ...[more]

PMID: 26136112

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Project description:Kidneys are enlarged by aberrant proliferation of tubule epithelial cells leading to the formation of numerous cysts, nephron loss, and interstitial fibrosis in polycystic kidney disease (PKD). Pioglitazone (PIO), a PPAR-M-NM-3 agonist, decreased cell proliferation, interstitial fibrosis and inflammation, and ameliorated PKD progression in PCK rats. To examine the genetic mechanisms in this efficacy, we analyzed changes in global gene expression. By Gene Set Enrichment Analysis of 30655 genes, 13 of the top 20 down-regulated gene ontology biological process gene sets, and six of the top 20 curated gene set canonical pathways identified to be down-regulated by PIO-treatment were related to cell cycle and proliferation, including EGF, PDGF and JNK pathways. Their relevant pathways were identified using the Kyoto Encyclopedia of Gene and Genomes database. Stearoyl-Coenzyme A desaturase 1 is a key enzyme in fatty acid metabolism found in the top 5 genes down-regulated by PIO treatment. Immunohistochemical analysis revealed that the gene product of this enzyme was highly expressed in PCK kidneys, and decreased by PIO. These data show that PIO alters the expression of genes involved in cell cycle progression, cell proliferation, and fatty acid metabolism. RNA was extracted from kidneys of rats with or without PIO treatment using a monophasic solution of phenol/guanidine isothiocyanate and TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA) in accordance with their manual, and the samples were incubated with RNase-free DNase I (Ambion, TX, USA). The quality and concentration of each sample was confirmed by spectrophotometry (NanoDropTM ND-1000; Asahi glass Co. Ltd., Tokyo, Japan). Total RNA obtained from five females were pooled in each PIO-treated or control vehicle-treated (CONT) group. Briefly, 500 ng aliquots of total RNA obtained from kidneys of three rats were labeled using an Quick Amp Labeling Kit, one-color (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturerM-bM-^@M-^Ys instructions. The pooled renal RNA of PIO- or vehicle-treated rats were labeled with the Cy3-fluorescence dye. After determination of labeling efficiency, 1.65 M-NM-<g aliquots of Cy3-labeled RNA were hybridized using the Gene Expression hybridization kit (Agilent Technologies) onto Rat Oligo Microarrays (Agilent Technologies, product no. G4130A) according to the manufacturerM-bM-^@M-^Ys hybridization protocol. The microarray slides were examined with an Agilent microarray scanner and software. Data analysis was performed with Agilent Feature Extraction software (version 10.7.3.1).

Project description:Background and objectivesAutosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the PKHD1 gene. The longest open reading frame comprises 66 exons encoding polyductin or fibrocystin, a type I transmembrane protein with 4074 amino acids. Functional investigations are considerably hampered by its large size and lack of expression in tissues that are usually available for analysis such as lymphocytes or fibroblasts.Design, setting, participants, & measurementsAllegedly strong and clear-cut genotype-phenotype correlations for the type of PKHD1 mutation could be established. Thus far, practically all patients with two truncating mutations showed perinatal or neonatal demise and at least one hypomorphic missense mutation is thought to be indispensable for survival. Mutation analysis of >500 ARPKD families was performed by conventional and next-generation sequencing techniques.ResultsThis study presents four unrelated patients with ARPKD with a nonlethal, moderate clinical course despite the burden of two PKHD1 mutations expected to lead to premature termination of translation. In line with parental consanguinity, all mutations occurred in the homozygous state and segregated with the disorder in these families. To try to unravel the mechanisms that underlie this obvious contradiction, these patients were further analyzed in detail by utilizing different methods. In all cases, complex transcriptional alterations were detected. Alternative splicing patterns might disrupt a critical stoichiometric and temporal balance between different protein products and may play a crucial role in defining the phenotype of these patients.ConclusionsAlthough these findings represent rare events, they are of importance for genetic counseling and illustrate that some caution is warranted in the interpretation of mutations and their clinical significance. The authors hypothesize that expression of a minimal amount of functional protein is needed for survival of the neonatal period in ARPKD.

Dataset Information

Improved Structure and Function in Autosomal Recessive Polycystic Rat Kidneys with Renal Tubular Cell Therapy.

Publications

Improved Structure and Function in Autosomal Recessive Polycystic Rat Kidneys with Renal Tubular Cell Therapy.

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