Cardiac infarcts increase sodium transporter transcripts (rBSC1) in the thick ascending limb of Henle.

BACKGROUND: Enhanced expression of the kidney-specific sodium transporter, rBSC1, in the thick ascending limb of Henle (TAL) and of the renal water channel, aquaporin-2 (AQP2), in collecting duct has been identified in rats with congestive heart failure (CHF) as a cause for enhanced sodium and water retention in this condition. However, the mechanism of impaired urinary sodium excretion observed even in rats with mild cardiac dysfunction remains unknown. METHODS: Male Sprague-Dawley rats with myocardial infarctions measuring 15 to 30% of the left ventricular circumference with no overt CHF were prepared. We measured the amount of rBSC1 or AQP2 mRNA using competitive polymerase chain reaction (PCR) by inducing a point mutation at the middle of the PCR product for rBSC1 or by deleting 180 bp from the 760 bp PCR product for AQP2, respectively. The results were confirmed by in situ hybridization. rBSC1 protein expression was examined by immunohistochemistry and Western blot analysis using a specific antibody against rBSC1. RESULTS: Although plasma renin activity was slightly elevated in rats with myocardial infarction (MI), no significant differences in lung weight or plasma concentrations for aldosterone and atrial natriuretic peptide were observed between control rats and MI rats. Competitive PCR showed a significant increase in rBSC1 mRNA in the renal outer medulla and cortex of MI rats, which was confirmed by in situ hybridization. However, the AQP2 mRNA of these rats remained unchanged throughout the kidney. Renin-angiotensin II blockade by oral captopril administration did not influence the alteration in rBSC1 mRNA induced by myocardial infarction. Immunohistochemistry and Western blots showed the enhanced expression of rBSC1 protein in TAL of rats with small to moderate cardiac infarcts. CONCLUSIONS: rBSC1 is up-regulated even in rats with small to moderate myocardial infarctions, which may enhance the sodium transport in the TAL in this pathophysiologic condition.

Topographic distribution of aquaporin 2 mRNA in the kidney of dehydrated rats.

BACKGROUND: Stimulation of arginine vasopressin results in an immediate redistribution of water channels (aquaporin 2; AQP2) in the apical membrane of the collecting ducts, leading to water reabsorption. Water restriction for >/=24 h increases AQP2 proteins in the whole collecting duct which is highest in the inner medulla of the kidney, indicating that dehydration enhances synthesis of this protein. Although increased expression of AQP2 mRNA under this condition has been reported, the increased ratio of mRNA expression in the three regions of the kidney, cortex, outer medulla, and inner medulla, during the dehydration is still unclear. METHODS: We investigated the AQP2 transcripts using male Sprague-Dawley rats deprived of water for 24 h. Mimic cDNA for competitive polymerase chain reaction (PCR) was constructed by deleting 180 bp at the middle of a 780-bp partial PCR product for rat AQP2 cDNA. In situ hybridization of the kidney and Northern blotting of inner medulla were performed using a 60-bp oligo-cDNA probe which identified rat AQP2 transcripts in the collecting duct. RESULTS: Dehydration resulted in a significant increase in plasma osmolality and arginine vasopressin concentration and urinary osmolality. Competitive PCR demonstrated that dehydration increased AQP2 transcripts in all parts of the kidney, but was highest in the inner medulla. Northern blotting confirmed the high increased rate of AQP2 transcription in the inner medulla. In situ hybridization showed markedly intensified signals in the inner medulla of dehydrated rats. CONCLUSIONS: Our data indicate that dehydration increases the abundance of AQP2 transcripts which may be closely associated with enhancement in AQP2 protein synthesis reported previously. This topographically variable increase in transcription is considered to be one of the mechanisms involved in long-term regulation of water permeability in the collecting duct.

Differential upregulation of rat Na-K-Cl cotransporter, rBSC1, mRNA in the thick ascending limb of Henle in different pathological conditions.

BACKGROUND: Na-Cl cotransport across the apical membrane of epithelial cells in the thick ascending limb of the loop of Henle (TAL) plays a major role in salt accumulation for hypertonic medullary interstitium. The electroneutral, rat bumetanide-sensitive sodium transporter, rBSC1, is involved in this process. We studied the level of rBSC1 mRNA in dehydration and cardiac failure, since sodium transport in TAL may be enhanced in both conditions in spite of the difference in extracellular fluid accumulation. METHODS: Male Sprague-Dawley rats were deprived of water for 24 hours and myocardial infarction of about 40% of left ventricular circumference was induced in another group of rats that later developed congestive heart failure (CHF). Digoxigenin-labeled cRNA probe for rBSC1 was constructed using polymerase chain reaction (PCR), and Northern blot analysis was performed using RNAs from renal outer medulla. By inducing a point mutation at the middle of PCR product, we compared the amount of rBSC1 transcripts in the renal cortex using competitive PCR, since TAL represents a small fraction of the total cortical tissue. RESULTS: Northern analysis showed a significant increase in rBSC1 mRNA in the renal outer medulla of both dehydrated and CHF rats. In the renal cortex, however, the increase was noted only in CHF by competitive PCR. In situ hybridization using the riboprobe for northern analysis demonstrated that the transcript signal in dehydrated rats was intensified segmentally in TAL located in the inner stripe of outer medulla. Western analysis and immunohistochemistry using a specific antibody against rBSC1 confirmed the distinct segmental enhancement of apical protein expression in dehydration and diffuse enhanced expression in CHF. CONCLUSIONS: rBSC1 is differentially upregulated in different pathological conditions.

Induction of apoptosis in ischemia-reperfusion model of mouse kidney: possible involvement of Fas.

Although ischemia-reperfusion of mouse kidney is known to cause severe renal failure due to tubular cell death, the exact cellular mechanism responsible for this phenomenon is not clear. To investigate the spatial and temporal development of renal cell death and the role of Fas/APO-1/CD95 (Fas) in this process, the left renal vessels were occluded in a group of mice for 30, 60, or 120 min followed by reperfusion for 24 h (n = 4 for each group). Analysis of the isolated DNA in agarose-gel electrophoresis revealed a typical ladder pattern of bands consisting of multiples of 180 to 200 bp, considered the hallmark of apoptosis. The intensity of the bands increased proportionately with the duration of ischemia. Histochemical analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling showed the presence of nuclei with DNA double-strand breaks specifically in distal renal tubules of the outer medulla. The presence of apoptosis was also confirmed by electron microscopy. Analysis of total RNA by Northern blotting revealed one appropriate-sized band for Fas mRNA in the normal kidney, which intensified in the ischemia-reperfused kidney. Moreover, nonradioactive in situ hybridization revealed that distal renal tubular epithelial cells were positive for Fas mRNA in the outer medulla. Fas antigen was also localized to the renal tubular epithelial cells of the outer medulla by immunohistochemistry. The number of apoptotic cells in the ischemia-reperfusion kidney of the lpr/lpr mouse was low. These findings strongly indicate that ischemia-reperfusion of the kidney induces apoptosis of a specific area of tubular epithelial cells in the outer medulla through the Fas system.