Renal medullary gene expression in aquaporin-1 null mice

Abstract

Mice that lack the aquaporin-1 gene (AQP1) lack a functional countercurrent multiplier mechanism, fail to concentrate the inner medullary (IM) interstitium, and present with a urinary concentrating defect. In this study, we use DNA microarrays to identify the gene expression profile of the IM of AQP1 null mice and corresponding changes in gene expression resulting from a loss of a hypertonic medullary interstitium. An ANOVA analysis model, CARMA, was used to isolate the knockout effect while taking into account experimental variability associated with microarray studies. In this study 5,701 genes of the possible approximately 12,000 genes on the array were included in the ANOVA; 531 genes were identified as demonstrating a >1.5-fold up- or downregulation between the wild-type and knockout groups. We randomly selected 35 genes for confirmation by real-time PCR, and 29 of the 35 genes were confirmed using this method. The overall pattern of gene expression in the AQP1 null mice was one of downregulation compared with gene expression in the renal medullas of the wild-type mice. Heat shock proteins 105 and 94, aldose reductase, adenylate kinase 2, aldolase B, aldehyde reductase 6, and p8 were decreased in the AQP1 null mice. Carboxylesterase 3, matrilin 2, lipocalin 2, and transforming growth factor-alpha were increased in IM of AQP1 null mice. In addition, we observed a loss of vasopressin type 2 receptor mRNA expression in renal medullas of the AQP1 null mice. Thus the loss of the hyperosmotic renal interstitium, due to a loss of the concentrating mechanism, drastically altered not only the phenotype of these animals but also their renal medullary gene expression profile.

Effects of water restriction on gene expression in mouse renal medulla: identification of 3βHSD4 as a collecting duct protein

“…Total RNA was isolated from the inner medulla of control (samples C1, C2, C3) and water-restricted mice (samples E1, E2, and E3) for microarray analysis of gene expression using our previously published methods (21). Total RNA from each sample was then amplified before being labeled in the cDNA reaction and each sample from an individual mouse was hybridized to an array four times (see MIAME document, supplemental data).…”

“…Total RNA from each sample was then amplified before being labeled in the cDNA reaction and each sample from an individual mouse was hybridized to an array four times (see MIAME document, supplemental data). Data were analyzed and transformed as previously described (21). The ANOVA was performed on a gene by gene basis and was limited to genes that were measured confidently on a minimum of three of the four hybridizations, for at least one sample.…”

“…Full methodology for the RNA purification, amplification, and cDNA production has been previously published (21). Briefly, RNA samples isolated from individual mouse inner medullas were labeled C1-C3 for control mice and E1-E3 for water-restricted mice.…”

“…Microarrays for our study were prepared within the Genomic Research Laboratory (GRL) at the University of Arizona using the NIA mouse 15K clone set http://lgsun.grc.nia.nih.gov/cDNA/15k.html. Full methodology for the production of the microarrays and the hybridization protocols has been published previously (21). Labeled modified cDNA in hybridization buffer was loaded onto a slide and set to hybridize at 47°C for a minimum of 16 h. After completion, a short wash was run in the hybridization station after which the slide was removed and dipped in 0.05ϫ SSC to remove any residual nonhybridized cDNA.…”

NKCC2 Surface Expression in Mammalian Cells

“…By contrast, it has been generally assumed until recently that the expression of aldolase B in the kidney is restricted to the cortex and more precisely to the proximal tubules (44 -46). However, recent studies revealed that aldolase B is also expressed in the renal medulla (47). Most interestingly, Lu et al (25) indicated that in mouse kidney, aldolase B protein is detected in the cytoplasm of the thick ascending limb and in the Bowman capsule.…”