Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice.
It has been controversial whether high water permeability in the thin descending limb of Henle (TDLH) is required for formation of a concentrated urine by the kidney. Freeze-fracture electron microscopy (FFEM) of rat TDLH has shown an exceptionally high density of intramembrane particles (IMPs), which were proposed to consist of tetramers of aquaporin-1 (AQP1) water channels. In this study, transepithelial osmotic water permeability (Pf) was measured in isolated perfused segments (0.5-1 mm) of TDLH in wild-type (+/+), AQP1 heterozygous (+/-), and AQP1 null (-/-) mice. Pf was measured at 37 degrees C using a 100 mM bath-to-lumen osmotic gradient of raffinose, and fluorescein isothiocyanate (FITC)-dextran as the luminal volume marker. Pf was (in cm/s): 0.26 +/- 0.02 ([+/+]; SE, n = 9 tubules), 0.21 +/- 0.01 ([+/-]; n = 12), and 0.031 +/- 0.007 ([-/-]; n = 6) (P < 0.02, [+/+] vs. [+/-]; P < 0.0001, [+/+] vs. [-/-]). FFEM of kidney medulla showed remarkably fewer IMPs in TDLH from (-/-) vs. (+/+) and (+/-) mice. IMP densities were (in microm-2, SD, 5-12 micrographs): 5,880 +/- 238 (+/+); 5,780 +/- 450 (+/-); and 877 +/- 420 (-/-). IMP size distribution analysis revealed mean IMP diameters of 8.4 nm ([+/+] and [+/-]) and 5.2 nm ([-/-]). These results demonstrate that AQP1 is the principal water channel in TDLH and support the view that osmotic equilibration along TDLH by water transport plays a key role in the renal countercurrent concentrating mechanism. The similar Pf and AQP1 expression in TDLH of (+/+) and (+/-) mice was an unexpected finding that probably accounts for the unimpaired urinary concentrating ability in (+/-) mice. (+info)
PST 2238: A new antihypertensive compound that modulates Na,K-ATPase in genetic hypertension.
A genetic alteration in the adducin genes is associated with hypertension and up-regulation of the expression of renal Na, K-ATPase in Milan-hypertensive (MHS) rats, in which increased ouabain-like factor (OLF) levels are also observed. PST 2238, a new antihypertensive compound that antagonizes the pressor effect of ouabain in vivo and normalizes ouabain-dependent up-regulation of the renal Na-K pump, was evaluated for its ability to lower blood pressure and regulate renal Na,K-ATPase activity in MHS genetic hypertension. In this study, we show that PST 2238, given orally at very low doses (1 and 10 microg/kg for 5-6 weeks), reduced the development of hypertension in MHS rats and normalized the increased renal Na,K-ATPase activity and mRNA levels, whereas it did not affect either blood pressure or Na,K-ATPase in Milan-normotensive (MNS) rats. In addition, a similar antihypertensive effect was observed in adult MHS rats after a short-term treatment. In cultured rat renal cells with increased Na-K pump activity at Vmax due to overexpression of the hypertensive variant of adducin, 5 days of incubation with PST 2238 (10(-10-)-10(-9) M) lowered the pump rate to the level of normal wild-type cells, which in turn were not affected by the drug. In conclusion, PST 2238 is a very potent compound that in MHS rats reduces blood pressure and normalizes Na-K pump alterations caused by a genetic alteration of the cytoskeletal adducin. Because adducin gene mutations have been associated with human essential hypertension, it is suggested that PST 2238 may display greater antihypertensive activity in those patients carrying such a genetic alteration. (+info)
Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity.
Hypertonicity (most often present as high salinity) is stressful to the cells of virtually all organisms. Cells survive in a hypertonic environment by increasing the transcription of genes whose products catalyze cellular accumulation of compatible osmolytes. In mammals, the kidney medulla is normally hypertonic because of the urinary concentrating mechanism. Cellular accumulation of compatible osmolytes in the renal medulla is catalyzed by the sodium/myo-inositol cotransporter (SMIT), the sodium/chloride/betaine cotransporter, and aldose reductase (synthesis of sorbitol). The importance of compatible osmolytes is underscored by the necrotic injury of the renal medulla and subsequent renal failure that results from the inhibition of SMIT in vivo by administration of a specific inhibitor. Tonicity-responsive enhancers (TonE) play a key role in hypertonicity-induced transcriptional stimulation of SMIT, sodium/chloride/betaine cotransporter, and aldose reductase. We report the cDNA cloning of human TonE binding protein (TonEBP), a transcription factor that stimulates transcription through its binding to TonE sequences via a Rel-like DNA binding domain. Western blot and immunohistochemical analyses of cells cultured in hypertonic medium reveal that exposure to hypertonicity elicits slow activation of TonEBP, which is the result of an increase in TonEBP amount and translocation to the nucleus. (+info)
Splicing of a retained intron within ROMK K+ channel RNA generates a novel set of isoforms in rat kidney.
The renal outer medulla K+ channel (ROMK) family of K+ channels may constitute a major pathway for K+ secretion in the distal nephron. To date, four main isoforms of this gene have been identified in the rat that differ only in their NH2-terminal amino acids and that share a common "core exon" that determines the remaining protein sequence. Using RT-PCR, we have identified a new set of ROMK isoforms in rat kidney that are generated by the deletion of a region within the ROMK core sequence that is identifiable as a typical mammalian intron. This splicing event was shown to be reproducible in vitro by detection of deleted ROMK mRNA in Madin-Darby canine kidney (MDCK) cells stably transfected with the gene for ROMK2. Translation of the deletion variant of ROMK2 was confirmed in vitro and visualized in MDCK cells following transient transfection with an enhanced green fluorescent protein tag. The deletion in this core region is predicted to generate hydrophilic proteins that are approximately one-third of the size of native ROMK and lack membrane-spanning domains. (+info)
Effect of acidification on the location of H+-ATPase in cultured inner medullary collecting duct cells.
In previous studies, our laboratory has utilized a cell line derived from the rat inner medullary collecting duct (IMCD) as a model system for mammalian renal epithelial cell acid secretion. We have provided evidence, from a physiological perspective, that acute cellular acidification stimulates apical exocytosis and elicits a rapid increase in proton secretion that is mediated by an H+-ATPase. The purpose of these experiments was to examine the effect of acute cellular acidification on the distribution of the vacuolar H+-ATPase in IMCD cells in vitro. We utilized the 31-kDa subunit of the H+-ATPase as a marker of the complete enzyme. The distribution of this subunit of the H+-ATPase was evaluated by immunohistochemical techniques (confocal and electron microscopy), and we found that there is a redistribution of these pumps from vesicles to the apical membrane. Immunoblot evaluation of isolated apical membrane revealed a 237 +/- 34% (P < 0.05, n = 9) increase in the 31-kDa subunit present in the membrane fraction 20 min after the induction of cellular acidification. Thus our results demonstrate the presence of this pump subunit in the IMCD cell line in vitro and that cell acidification regulates the shuttling of cytosolic vesicles containing the 31-kDa subunit into the apical membrane. (+info)
Role of renal medullary adenosine in the control of blood flow and sodium excretion.
This study determined the levels of adenosine in the renal medullary interstitium using microdialysis and fluorescence HPLC techniques and examined the role of endogenous adenosine in the control of medullary blood flow and sodium excretion by infusing the specific adenosine receptor antagonists or agonists into the renal medulla of anesthetized Sprague-Dawley rats. Renal cortical and medullary blood flows were measured using laser-Doppler flowmetry. Analysis of microdialyzed samples showed that the adenosine concentration in the renal medullary interstitial dialysate averaged 212 +/- 5.2 nM, which was significantly higher than 55.6 +/- 5.3 nM in the renal cortex (n = 9). Renal medullary interstitial infusion of a selective A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 300 pmol. kg-1. min-1, n = 8), did not alter renal blood flows, but increased urine flow by 37% and sodium excretion by 42%. In contrast, renal medullary infusion of the selective A2 receptor blocker 3, 7-dimethyl-1-propargylxanthine (DMPX; 150 pmol. kg-1. min-1, n = 9) decreased outer medullary blood flow (OMBF) by 28%, inner medullary blood flows (IMBF) by 21%, and sodium excretion by 35%. Renal medullary interstitial infusion of adenosine produced a dose-dependent increase in OMBF, IMBF, urine flow, and sodium excretion at doses from 3 to 300 pmol. kg-1. min-1 (n = 7). These effects of adenosine were markedly attenuated by the pretreatment of DMPX, but unaltered by DPCPX. Infusion of a selective A3 receptor agonist, N6-benzyl-5'-(N-ethylcarbonxamido)adenosine (300 pmol. kg-1. min-1, n = 6) into the renal medulla had no effect on medullary blood flows or renal function. Glomerular filtration rate and arterial pressure were not changed by medullary infusion of any drugs. Our results indicate that endogenous medullary adenosine at physiological concentrations serves to dilate medullary vessels via A2 receptors, resulting in a natriuretic response that overrides the tubular A1 receptor-mediated antinatriuretic effects. (+info)
Second messenger production in avian medullary nephron segments in response to peptide hormones.
We examined the sites of peptide hormone activation within medullary nephron segments of the house sparrow (Passer domesticus) kidney by measuring rates of hormone-induced generation of cyclic nucleotide second messenger. Thin descending limbs, thick ascending limbs, and collecting ducts had baseline activity of adenylyl cyclase that resulted in cAMP accumulation of 207 +/- 56, 147 +/- 31, and 151 +/- 41 fmol. mm-1. 30 min-1, respectively. In all segments, this activity increased 10- to 20-fold in response to forskolin. Activity of adenylyl cyclase in the thin descending limb was stimulated approximately twofold by parathyroid hormone (PTH) but not by any of the other hormones tested [arginine vasotocin (AVT), glucagon, atrial natriuretic peptide (ANP), or isoproterenol, each at 10(-6) M]. Thick ascending limb was stimulated two- to threefold by both AVT and PTH; however, glucagon and isoproterenol had no effect, and ANP stimulated neither cAMP nor cGMP accumulation. Adenylyl cyclase activity in the collecting duct was stimulated fourfold by AVT but not by the other hormones; likewise, ANP did not stimulate cGMP accumulation in this segment. These data support a tubular action of AVT and PTH in the avian renal medulla. (+info)
Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney.
BMP-7, a member of the bone morphogenic protein subfamily (BMPs) of the transforming growth factor-beta superfamily of secreted growth factors, is abundantly expressed in the fetal kidney. The precise role of this protein in renal physiology or pathology is unknown. A cDNA that encodes rat BMP-7 was cloned and used as a probe to localize BMP-7 mRNA expression by in situ hybridization in the adult rat kidney. The highest expression of BMP-7 mRNA could be seen in tubules of the outer medulla. In glomeruli, a few cells, mainly located at the periphery of the glomerular tuft, showed specific and strong signals. Also, high BMP-7 mRNA expression could be localized to the adventitia of renal arteries, as well as to the epithelial cell layer of the renal pelvis and the ureter. Preliminary evidence suggests that BMP-7 enhances recovery when infused into rats with ischemia-induced acute renal failure. We examined BMP-7 mRNA expression in kidneys with acute renal failure induced by unilateral renal artery clamping. BMP-7 mRNA abundance as analyzed by solution hybridization was reduced in ischemic kidneys after 6 and 16 h of reperfusion compared with the contralateral kidney. In situ hybridization in ischemic kidneys showed a marked decrease of BMP-7 mRNA in the outer medulla and in glomeruli. Utilizing rat metanephric mesenchymal cells in culture, we also demonstrate that BMP-7 induces epithelial cell differentiation. Taken together, these data suggest that BMP-7 is important in both stimulating and maintaining a healthy differentiated epithelial cell phenotype. (+info)