Stimulation of Na+-K+-2Cl- cotransport by arsenite in ferret erythrocytes.
(17/817)
1. Na+-K+-2Cl- cotransport activity was measured in ferret erythrocytes as the bumetanide-sensitive uptake of 86Rb. 2. The Na+-K+-2Cl- cotransport rate was stimulated by treating erythrocytes with sodium arsenite but not by sodium arsenate (up to 1 mM). Stimulation took an hour to develop fully. Arsenite had no effect on bumetanide-resistant 86Rb uptake. 3. In cells stored for 3 days or less, cotransport stimulation by arsenite could be described by assuming arsenite either acts at a single site (EC50, 60+/-14 microM, mean +/- S.E.M., n = 3) or that it acts at both high- (EC50, 35+/-9 microM, mean +/- S.E.M., n = 3) and low- (EC50 >2 mM) affinity sites. 4. Stimulation by 1 mM arsenite was greatest on the day of cell collection (rate about 3 times that of the control), even exceeding that produced by 20 nM calyculin A, and declined during cell storage. Addition of calyculin A to arsenite-stimulated cells resulted in further stimulation of Na+-K+-2Cl- cotransport, suggesting that arsenite and calyculin act synergistically. This was most apparent in stored cells. 5. Stimulation by 1 mM arsenite was not affected by treating cells with the mitogen-activated protein kinase inhibitors SB203580 (20 microM) and PD98059 (50 microM), but was both prevented and reversed by the kinase inhibitors staurosporine (2 microM), 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1, 50 microM) and genistein (0.3 mM), and with a combination of 10 microM A23187 and 2 mM EDTA (to reduce intracellular Mg2+ concentration). Only treatment with EDTA and A23187 prevented stimulation by the combination of 1 mM arsenite and 20 nM calyculin, whereas no treatment was able to fully reverse this stimulation once elicited. 6. Our data are consistent with arsenite stimulating (perhaps indirectly) a kinase that phosphorylates and activates the Na+-K+-2Cl- cotransporter. (+info)
Cyclooxygenase inhibitors increase Na-K-2Cl cotransporter abundance in thick ascending limb of Henle's loop.
(18/817)
Cyclooxygenase inhibitors, such as indomethacin and diclofenac, have well-described effects to enhance renal water reabsorption and urinary concentrating ability. Concentrating ability is regulated in part at the level of the thick ascending limb of Henle's loop, where active NaCl absorption drives the countercurrent multiplication mechanism. We used semiquantitative immunoblotting to test the effects of indomethacin and diclofenac, given over a 48-h period, on the expression levels of the ion transporters responsible for active NaCl transport in the thick ascending limb. Both agents strongly increased the expression level of the apical Na-K-2Cl cotransporter in both outer medulla and cortex. Neither agent significantly altered outer medullary expression levels of other thick ascending limb proteins, namely, the type 3 Na/H exchanger (NHE-3), Tamm-Horsfall protein, or alpha1- or beta1-subunits of the Na-K-ATPase. Administration of the EP3-selective PGE(2) analog, misoprostol, to indomethacin-treated rats reversed the stimulatory effect of indomethacin on Na-K-2Cl cotransporter expression. We conclude that cyclooxygenase inhibitors enhance urinary concentrating ability in part through effects to increase Na-K-2Cl cotransporter expression in the thick ascending limb of Henle's loop. This action is most likely due to elimination of an EP3-receptor-mediated tonic inhibitory effect of PGE(2) on cAMP production. (+info)
Decreased renal Na-K-2Cl cotransporter abundance in mice with heterozygous disruption of the G(s)alpha gene.
(19/817)
Transport processes along the nephron are regulated in part by hormone stimulation of adenylyl cyclases mediated by the heterotrimeric G protein G(s). To assess the role of this pathway in the regulation of Na-K-2Cl cotransporter abundance in the renal thick ascending limb (TAL), we studied mice with heterozygous disruption of the Gnas gene, which codes for the alpha-subunit of G(s). Outer medullary G(s)alpha protein abundance (as assessed by semiquantitative immunoblotting) and glucagon-stimulated cAMP production were significantly reduced in the heterozygous G(s)alpha knockout mice (GSKO) relative to their wild-type (WT) littermates. Furthermore, Na-K-2Cl cotransporter protein abundance in the outer medulla was significantly reduced (band density, 48% of WT). In addition, GSKO mice had a significantly reduced (72% of WT) urinary osmolality in response to a single injection of 1-deamino-[8-D-arginine]vasopressin (DDAVP), a vasopressin analog. In contrast, outer medullary protein expression of the type 3 Na/H exchanger (NHE-3) or Tamm-Horsfall protein did not differ between the GSKO mice and their WT littermates. However, abundance of type VI adenylyl cyclase was markedly decreased in the outer medullas of GSKO mice, suggesting a novel feed-forward regulatory mechanism. We conclude that expression of the Na-K-2Cl cotransporter of the TAL is dependent on G(s)alpha-mediated hormone stimulation, most likely due to long-term changes in cellular cAMP levels. (+info)
Altered expression of Na transporters NHE-3, NaPi-II, Na-K-ATPase, BSC-1, and TSC in CRF rat kidneys.
(20/817)
In chronic renal failure (CRF), reduction in renal mass leads to an increase in the filtration rates of the remaining nephrons and increased excretion of sodium per nephron. To address the mechanisms involved in the increased sodium excretion, we determined the total kidney levels and the densities per nephron of the major renal NaCl transporters in rats with experimental CRF. Two weeks after 5/6 nephrectomy (reducing the total number of nephrons to approximately 24 +/- 8%), the rats were azotemic and displayed increased Na excretion. Semiquantitative immunoblotting revealed significant reduction in the total kidney levels of the proximal tubule Na transporters NHE-3 (48% of control), NaPi-II (13%), and Na-K-ATPase (30%). However, the densities per nephron of NHE-3, NaPi-II, and Na-K-ATPase were not significantly altered in remnant kidneys, despite the extensive hypertrophy of remaining nephrons. Immunocytochemistry confirmed the reduction in NHE-3 and Na-K-ATPase labeling densities in the proximal tubule. In contrast, there was no significant reduction in the total kidney levels of the thick ascending limb and distal convoluted tubule NaCl transporters BSC-1 and TSC, respectively. This corresponded to a 3.6 and 2.5-fold increase in densities per nephron, respectively (confirmed by immunocytochemistry). In conclusion, in this rat CRF model: 1) increased fractional sodium excretion is associated with altered expression of proximal tubule Na transporter expression (NHE-3, NaPi-II, and Na-K-ATPase), consistent with glomerulotubular imbalance in the face of increased single-nephron glomerular filtration rate; and 2) compensatory increases in BSC-1 and TSC expression per nephron occur in distal segments. (+info)
Decreased abundance of collecting duct aquaporins in post-ischemic renal failure in rats.
(21/817)
Increased urine flow is often a feature of mild to moderate acute renal failure. This study examines the possible role of dysregulation of collecting duct aquaporins as a factor in this increase. In rats, the left renal pedicle was clamped for 45 min followed by contralateral nephrectomy. Control rats were identical except that the renal pedicle was not clamped. Rats were sacrificed and the kidneys were homogenized at various time points after release of the clamp for semiquantitative immunoblotting of collecting duct aquaporins, as well as the thick ascending limb Na-K-2Cl cotransporter and the proximal tubule water channel, aquaporin-1. Urinary flow rate was significantly increased 18 h after the ischemic insult and remained increased through 72 h. Whole kidney aquaporin-2 protein abundance was 45% of controls at 18 h, 55% of controls at 36 h, and returned to normal 72 h after ischemia. Whole kidney aquaporin-3 protein abundance was 37% of controls at 18 h, 13% of controls at 36 h, and 45% of controls at 72 h. The decline in aquaporin-2 and -3 was confirmed by immunocytochemistry. Abundance of the thick ascending limb Na-K-2Cl cotransporter protein was not significantly decreased. Aquaporin-1 protein abundance was not significantly decreased at 18 h after the ischemic insult, but was significantly reduced after 36 h. Thus, the post-ischemic state is associated with decreased levels of the collecting duct aquaporins, coinciding with an increase in water excretion. It is concluded that decreased aquaporin protein abundance in collecting duct cells is a contributing factor in the increased urine flow seen in moderate post-ischernic acute renal failure. (+info)
Decreased activity of the red blood cell ATPase-dependent Na+ pump in patients with cardiac syndrome X.
(22/817)
Marked Na(+)/Li(+) countertransport hyperactivity and post-load hyperinsulinaemia have been described in 93% of patients with cardiac syndrome X. We hypothesized that more complex abnormalities in Na(+) traffic across the cell membrane are present in these patients. The aim of the present study was to evaluate the activities of the two main transporters responsible for transmembrane Na(+) transport, i.e. the ATPase-dependent Na(+) pump and the Na(+)-K(+)-2Cl(-) co-transporter, in a selected group of patients with cardiac syndrome X. We evaluated 19 patients with cardiac syndrome X and 14 control subjects. The ATPase-dependent Na(+) pump and Na(+)-K(+)-2Cl(-) co-transport activities were assessed from Na(+)-loaded red blood cells by using nystatine, in the presence of furosemide and ouabain, as appropriate. Erythrocyte Na(+)/Li(+) countertransport activity, serum lipid and post-load (75 g of oral glucose) insulin levels were also evaluated. The V(max) of Na(+)/Li(+) countertransport (P=0.0001) and post-load insulin levels (120 min; P=0.001) were confirmed to be higher in patients with syndrome X than in controls. The V(max) of Na(+)-K(+)-2Cl(-) co-transport was similar in patients and controls. By contrast, the V(max) of the ATPase-dependent Na(+) pump was significantly lower (P=0.002) in syndrome X patients (3.13+/-0.87 mmol.h(-1).l(-1)) than in controls (4.28+/-1.10 mmol.h(-1).l(-1)). Serum total cholesterol and triacylglycerol concentrations were also higher in patients with syndrome X than in control subjects (P<0.0001). Thus decreased activity of the ATPase-dependent Na(+) pump was present in patients with cardiac syndrome X. Such an abnormality has the biological potential to augment microvascular tone and the response to constrictor stimuli via increased intracellular free Ca(2+). Of note, syndrome X patients also manifested Na(+)/Li(+) countertransport hyperactivity which, in turn, is known to induce peripheral insulin resistance and consequent abnormalities in insulin secretion and lipid turnover. Thus cardiac syndrome X appears as a multifaceted syndrome presenting with either metabolic or cardiovascular symptoms, or both, based on the expression of complex abnormalities in Na(+) traffic across the cell membrane. (+info)
The pathophysiological and molecular basis of Bartter's and Gitelman's syndromes.
(23/817)
Molecular defects affecting the transport of sodium, potassium and chloride in the nephron through the ROMK K+ channel, Na+/K+/2Cl- cotransporter, the Na+/Cl- cotransporter and chloride channel have been identified in patients with Bartter's and Gitelman's syndromes. Defects of the angiotensin II type I receptor and CFTR have also being described. These defects are simple (i.e., most are single amino acid substitutions) but affect key elements in tubular transport. The simplicity of the genetic defects may explain why the inheritance of these conditions remains unclear in most kindreds (i.e., not just recessive or dominant) and emphasises the crucial importance of the conformational structure of these channels. Application of this molecular information will allow the early genetic identification of patients with these syndromes and enable us to differentiate between the various disorders at a functional level. It may also identify a subgroup in which the heterozygous form may make patients potentially exquisitely sensitive to diuretics. (+info)
Renal expression of aquaporins in liver cirrhosis induced by chronic common bile duct ligation in rats.
(24/817)
Semiquantitative immunoblotting was used to investigate the expression levels of the four major renal aquaporins, the Na-K-2Cl cotransporter of the thick ascending limb, the type 3 Na-H exchanger, and the Na-K-ATPase in kidneys from rats with cirrhosis secondary to common bile duct ligation (CBDL). These rats had significant water retention and hyponatremia. In contrast to models of cirrhosis induced by carbon tetrachloride, aquaporin-2 expression in CBDL-induced cirrhosis was decreased. Thus, these results show that in the setting of extracellular fluid volume expansion, excessive water retention with hyponatremia can occur in the absence of increases in aquaporin-2 abundance. In addition, the expression levels of the two basolateral collecting duct aquaporins (aquaporin-3 and -4) were decreased in CBDL rats relative to sham-operated control rats. Similarly, the Na-K-2Cl cotransporter of the thick ascending limb and the type 3 Na-H exchanger showed decreases in expression. In contrast, the expression levels of aquaporin-1 and the all subunit of the Na-K-ATPase were not decreased. Thus, dysregulation of multiple water channels and ion transporters may play a role in water balance abnormalities associated with CBDL-induced cirrhosis in rats. (+info)