Cyclophilin B Interacts with Sodium-Potassium ATPase and Is Required for Pump Activity in Proximal Tubule Cells of the Kidney

Cyclophilins (Cyps), the intracellular receptors for Cyclosporine A (CsA), are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1) that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA

Identification of Copy Number Variants Defining Genomic Differences among Major Human Groups

BACKGROUND:Understanding the genetic contribution to phenotype variation of human groups is necessary to elucidate differences in disease predisposition and response to pharmaceutical treatments in different human populations. METHODOLOGY/PRINCIPAL FINDINGS:We have investigated the genome-wide profile of structural variation on pooled samples from the three populations studied in the HapMap project by comparative genome hybridization (CGH) in different array platforms. We have identified and experimentally validated 33 genomic loci that show significant copy number differences from one population to the other. Interestingly, we found an enrichment of genes related to environment adaptation (immune response, lipid metabolism and extracellular space) within these regions and the study of expression data revealed that more than half of the copy number variants (CNVs) translate into gene-expression differences among populations, suggesting that they could have functional consequences. In addition, the identification of single nucleotide polymorphisms (SNPs) that are in linkage disequilibrium with the copy number alleles allowed us to detect evidences of population differentiation and recent selection at the nucleotide variation level. CONCLUSIONS:Overall, our results provide a comprehensive view of relevant copy number changes that might play a role in phenotypic differences among major human populations, and generate a list of interesting candidates for future studies

Effects of Losartan Pretreatment in an Experimental Model of Ischemic Acute Kidney Injury

Background/Aims: Contributions to the understanding of acute renal failure (ARF) pathogenesis have not been translated into an effective clinical therapy. We studied the effects of pretreatment with the angiotensin II type 1 (AT1) receptor blocker, losartan, on renal function, tissue injury, inflammatory response and serum aldosterone levels in a model of ischemic ARF. Methods: Rats underwent unilateral renal ischemia followed by 24 h of reperfusion (IR), and were pretreated or not with 8 (IRL8) or 80 (IRL80) mg/kg/day of losartan for 3 days. Results: IR kidneys showed marked renal dysfunction, epithelial damage, capillary congestion, increased myeloperoxidase (MPO) activity and increased TNF-alpha, IL1-beta and IL-6 mRNA levels. IRL80 kidneys showed protection against dysfunction and tissue injury, associated with normal MPO activity and cytokine mRNA levels. The lower dose was not able to achieve the same degree of functional renoprotection and could not prevent an increase of MPO or pro-inflammatory cytokine mRNA levels. The high losartan dose completely prevented an increase of serum aldosterone levels induced by IR. Conclusion: Renoprotection of the high losartan dose would be mainly mediated by its anti-inflammatory actions. Our results show a potential pathophysiological role of AT1 activation in promoting renal dysfunction, structural injury, inflammation and aldosterone elevation after IR injury. Copyright (C) 2009 S. Karger AG, Base

Challenges in array comparative genomic hybridization for the analysis of cancer samples

PURPOSE: To address some of the challenges facing the incorporation of array comparative genomic hybridization technology as a clinical tool, including archived tumor tissue, tumor heterogeneity, DNA quality and quantity, and array comparative genomic hybridization platform selection and performance. METHODS: Experiments were designed to assess the impact of DNA source (e.g., archival material), quantity, and amplification on array comparative genomic hybridization results. Two microdissection methods were used to isolate tumor cells to minimize heterogeneity. These data and other data sets were used in a further performance comparison of two commonly used array comparative genomic hybridization platforms: bacterial artificial chromosome (Roswell Park Cancer Institute) and oligonucleotide (Agilent Technologies, Santa Clara, CA). RESULTS: Array comparative genomic hybridization data from as few as 100 formalin-fixed, paraffin-embedded cells isolated by laser capture microdissection and amplified were remarkably similar to array comparative genomic hybridization copy number alterations detected in the bulk (unamplified) population. Manual microdissection from frozen sections provided a rapid and inexpensive means to isolate tumor from adjacent DNA for amplification and array comparative genomic hybridization. Whole genome amplification introduced no appreciable allele bias on array comparative genomic hybridization. The array comparative genomic hybridization results provided by the bacterial artificial chromosome and Agilent platforms were concordant in general, but bacterial artificial chromosome array comparative genomic hybridization showed far fewer outliers and overall less technical noise, which could adversely affect the statistical interpretation of the data. CONCLUSIONS: This study demonstrates that copy number alterations can be robustly and reproducibly detected by array comparative genomic hybridization in DNA isolated from challenging tumor types and sources, including archival materials, low DNA yield, and heterogeneous tissues. Furthermore, bacterial artificial chromosome array comparative genomic hybridization offers the advantage over the Agilent oligonucleotide platform of presenting fewer outliers, which could affect data interpretation. ©2007The American College of Medical Genetics