The regulation of sodium transport in the kidney is important for maintenance of extracellular fluid volume and arterial blood-pressure regulation. The major sodium transporters and channels in individual renal tubule segments have been identified via physiological techniques, and complementary DNAs for all of the key sodium transporters and channels expressed along the renal tubule have been cloned. Complementary DNA probes and antibodies are now being used to investigate the molecular basis of renal tubule sodium-transport regulation. This review summarizes some of the major observations made in the past year that are relevant to the regulation of the major sodium transporters in the proximal tubule (the type 3 sodium-hydrogen exchanger, NHE3), the thick ascending limb of Henle (the bumetanide-sensitive sodium-potassium-chloride cotransporter, NKCC2), and the distal convoluted tubule (the thiazide-sensitive sodium-chloride cotransporter, NCC).
Previously, the only known blockers of water permeability through aquaporin-1 (AQP1) water channels were mercurial reagents such as HgCl(2). For AQP1, inhibition by mercury has been attributed to the formation of a mercaptide bond with cysteine residue 189 found in the putative pore-forming region loop E. Here we show that the nonmercurial compound, tetraethylammonium (TEA) chloride, reduces the water permeability of human AQP1 channels expressed in Xenopus oocytes. After preincubation of the oocytes for 15 min with 100 microM TEA, AQP1 water permeability was reduced by 20 to 40%, a degree of partial block similar to that obtained with 15 min of incubation in 100 microM HgCl(2). The reduction of water permeability was dose-dependent for tested concentrations up to 10 mM TEA. TEA blocks the Shaker potassium channel by interacting with a tyrosine residue in the outer pore region. We tested whether an analogous tyrosine residue in loop E of AQP1 could be involved in the binding of TEA. Using polymerase chain reaction, tyrosine 186 in AQP1, selected for its proximity to the mercury-binding site, was mutated to phenylalanine (Y186F), alanine (Y186A), or asparagine (Y186N). Oocyte expression of the mutant AQP1 channels showed that the water permeability of Y186F was equivalent to that of wild-type AQP1; the other mutant channels did not conduct water. However, in contrast to wild-type AQP1, the water permeability of Y186F was not reduced with 100 microM TEA. These results suggest that TEA reduces AQP1 water permeability by interacting with loop E.
To identify novel gene targets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48-h water restriction protocol. In this study, 4,625 genes of the possible approximately 12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts, and the decreases were confirmed for 7 transcripts. In addition, we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; aquaporin-2 (AQP2), AQP3, Pax-8, and alpha- and beta-Na-K-ATPase subunits were all significantly increased in abundance; the beta- and gamma-subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3beta-hydroxysteroid dehydrogenase 4 (3betaHSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized the expression of this protein to AQP2-expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion.
Oxidative stress and mitochondrial dysfunction exacerbate acute kidney injury (AKI) but their role in any associated progress to chronic kidney disease (CKD) remains unclear. Antioxidant therapies often benefit AKI but their benefits in CKD are controversial since clinical and pre-clinical investigations often conflict. Here we examined the influence of the antioxidant, N-acetyl cysteine (NAC) on oxidative stress and mitochondrial function during AKI (20-minute bilateral renal ischemia plus reperfusion/IR) and progression to chronic kidney pathologies in mice. NAC (5% in diet) was given to mice 7 days prior and up to 21 days post-IR (21d-IR). NAC treatment: prevented proximal tubular epithelial cell apoptosis at early IR (40-min post-ischemia), yet enhanced interstitial cell proliferation at 21d-IR; increased Transforming growth factor-β1 expression independent of IR time; and significantly dampened nuclear factor-like 2-initiated cytoprotective signalling at early IR. Long-term, NAC enhanced cellular metabolic impairment demonstrated by increased peroxisome proliferator activator-gamma serine-112 phosphorylation at 21d-IR. Intravital multiphoton microscopy revealed increased endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) in cortical tubular epithelial cells during ischemia and at 21d-IR that was not attenuated with NAC. Fluorescence lifetime imaging microscopy demonstrated persistent metabolic impairment by increased free/bound NADH in the cortex at 21d-IR that was enhanced by NAC. Increased mitochondrial dysfunction in remnant tubular cells was demonstrated at 21d-IR by tetramethylrhodamine methyl ester fluorimetry. In summary, NAC enhanced progression to CKD following AKI not only by dampening endogenous cellular antioxidant responses at time of injury but also enhancing persistent kidney mitochondrial and metabolic dysfunction.
TonEBP is a transcription factor that, when activated by hypertonicity, increases transcription of genes, including those involved in organic osmolyte accumulation. Surprisingly, it is expressed in virtually all tissues, including many never normally exposed to hypertonicity. We measured TonEBP mRNA (real-time PCR) and protein (Western blot analysis) in tissues of control (plasma osmolality 294 +/- 1 mosmol/kgH2O) and hyposmotic (dDAVP infusion plus water loading for 3 days, 241 +/- 2 mosmol/kgH2O) rats to test whether the ubiquitous expression of TonEBP mRNA is osmotically regulated around the normal plasma osmolality. TonEBP protein is reduced by hyposmolality in thymus and liver, but not in brain, and is not detected in heart and skeletal muscle. TonEBP mRNA decreases in brain and liver but is unchanged in other tissues. There are no general changes in mRNA of TonEBP-mediated genes: aldose reductase (AR) does not change in any tissue, betaine transporter (BGT1) decreases only in liver, taurine transporter (TauT) only in brain and thymus, and inositol transporter (SMIT) only in skeletal muscle and liver. Heat shock protein (Hsp)70-1 and Hsp70-2 mRNA increase greatly in most tissues, which cannot be attributed to decreased TonEBP activity. The conclusions are as follows: 1) TonEBP protein or mRNA expression is reduced by hyposmolality in thymus, liver, and brain. 2) TonEBP protein and mRNA expression are differentially regulated in some tissues. 3) Although AR, SMIT, BGT1, and TauT are regulated by TonEBP in renal medullary cells, other sources of regulation may predominate in other tissues. 4) TonEBP abundance and activity are regulated by factors other than tonicity in some tissues.
