High salt differentially regulates surface NKCC2 expression in thick ascending limbs of Dahl salt-sensitive and salt-resistant rats (original) (raw)
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Hyperphosphorylation of Na-K-2Cl Cotransporter in Thick Ascending Limbs of Dahl Salt-Sensitive Rats
Hypertension, 2012
Salt-sensitive hypertension involves a renal defect preventing the kidney from eliminating excess NaCl. The thick ascending limb of Henle loop reabsorbs ≈30% of filtered NaCl via the apical Na-K-2Cl cotransporter (NKCC2). Higher NKCC2 activity and Cl reabsorption have been reported in the thick ascending limbs from Dahl salt-sensitive rats (DSS) fed normal salt. NKCC2 activity is primarily regulated by protein trafficking and phosphorylation at Thr 96 /Thr 101 via STE20- and SPS1-related proline and alanine-rich kinases and oxidative stress-responsive kinase 1. However, the mechanism for enhanced NKCC2 activity in DSS is unclear. We hypothesized that DSS exhibit enhanced NKCC2 trafficking and higher NKCC2 phosphorylation compared with Dahl salt-resistant rats on normal salt diet. We measured steady state surface NKCC2 expression and phosphorylation at Thr 96 and Thr 101 by surface biotinylation and Western blot. In DSS, the surface:total NKCC2 ratio was enhanced by 25% compared with...
Pflugers Archiv-european Journal of Physiology
The Milan hypertensive strain of rats (MHS) develops hypertension as a consequence of the increased tubular Na+ reabsorption sustained by enhanced expression and activity of the renal tubular Na–K–ATPase. To verify whether the Na–K–2Cl cotransporter (NKCC2) is involved in the maintenance of hypertension in MHS rats, we have analysed the phosphorylation state and the activation of NKCC2 in Milan rats. Western blotting and immunofluorescence experiments were performed using specific antibodies against the regulatory phospho-threonines in the NKCC2 N terminus (R5 antibody). The phosphorylation levels of NKCC2 were significantly increased in the kidney of MHS rats. Moreover, the administration of furosemide in vivo decreased the blood pressure and increased the urine output and natriuresis in MHS rats demonstrating the actual involvement of NKCC2 activity in the pathogenesis of hypertension in this strain of rats. The up-regulation of NKCC2 activity is most probably mediated by a STE20/SPS1-related proline/alanine-rich kinase (SPAK) phosphorylation at serine-325 since it was significantly increased in MHS rats. Interestingly, aldosterone treatment caused an increase in NKCC2 phosphorylation in NKCC2-expressing MDCK cells. In conclusion, we demonstrated an increase in the activity of NKCC2 along the TAL that significantly contributes to the increase in systemic blood pressure in MHS rats. The elevated plasma levels of aldosterone, found in MHS rats, may influence Na+ balance through a SPAK-dependent regulation of NKCC2 accounting for the maintenance of the hypertensive state in MHS rats.
AJP: Renal Physiology, 2008
We investigated which of the NaCl transporters are involved in the maintenance of salt sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 months after birth. In MHS, as compared to normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged both for Na + -H + isoform 3 (NHE3) and Na + -K + -2Cl -(NKCC2), but higher (119%, n=5, p<0.005) for Na + -Cl -(NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots which revealed: the renal tubule cells to reabsorb sodium (13). Therefore the importance of the Na + -K + -ATPase in the pathogenesis of hypertension is fully established (3).
Journal of Biological Chemistry, 2011
We have observed that, in renal proximal tubular cells, cardiotonic steroids such as ouabain in vitro signal through Na/K-ATPase, which results in inhibition of transepithelial 22 Na ؉ transport by redistributing Na/K-ATPase and NHE3. In the present study, we investigate the role of Na/K-ATPase signaling in renal sodium excretion and blood pressure regulation in vivo. In Sprague-Dawley rats, high salt diet activated c-Src and induced redistribution of Na/K-ATPase and NHE3 in renal proximal tubules. In Dahl salt sensitive (S) and resistant (R) rats given high dietary salt, we found different effects on blood pressure but, more interestingly, different effects on renal salt handling. These differences could be explained by different signaling through the proximal tubular Na/K-ATPase. Specifically, in Dahl R rats, high salt diet significantly stimulated phosphorylation of c-Src and ERK1/2, reduced Na/K-ATPase activity and NHE3 activity, and caused redistribution of Na/K-ATPase and NHE3. In contrast, these adaptations were either much less effective or not seen in the Dahl S rats. We also studied the primary culture of renal proximal tubule isolated from Dahl S and R rats fed a low salt diet. In this system, ouabain induced Na/K-ATPase/c-Src signaling and redistribution of Na/K-ATPase and NHE3 in the Dahl R rats, but not in the Dahl S rats. Our data suggested that impairment of Na/K-ATPase signaling and consequent regulation of Na/K-ATPase and NHE3 in renal proximal tubule may contribute to salt-induced hypertension in the Dahl S rat. . 2 The abbreviations used are: BP, blood pressure; CTS, cardiotonic steroids; EE, early endosome; ERK1/2, extracellular signal-regulated kinases 1 and 2; MBG, marinobufagenin; NHE3, sodium/hydrogen exchanger isoform 3; RPT, renal proximal tubule; HS, high salt diet; LS, low salt diet.
Nature Medicine, 2004
Hypertension is the most common chronic disease, and is the leading risk factor for death that is due to stroke, myocardial infarction or endstage renal failure 1,2 .The critical importance of excess salt intake in the pathogenesis of hypertension is widely recognized 3-6 ,but the mechanism by which excess salt intake elevates blood pressure has puzzled researchers. Recently discovered cardiotonic steroids (CTS), such as endogenous ouabain 7 , and other steroids 8-10 , including marinobufagenin, proscillaridin A and bufalin, have been proposed as candidate intermediaries. In humans, a chronic high-salt diet causes a rise in plasma CTS 11-13 .Moreover,∼50% of patients with essential hypertension have substantially elevated levels of endogenous ouabain . Plasma CTS are also high in several salt-dependent hypertensive animals 7,13,16 .Indeed, PST2238, a ouabain antagonist, lowers blood pressure in salt-dependent hypertensive rats and in certain patients with essential hypertension 17,18 .Generally, it is believed that CTS inhibit the plasma membrane Na + /K + ATPase, the 'sodium-potassium pump,' and lead to an increase in cytosolic Na + concentration ([Na + ] cyt ). Cellular Na + accumulation raises the cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) through the involvement of the Na + /Ca 2+ exchanger (NCX), and thereby increases contraction in vascular or heart muscle. This may lead to hypertension 19 ,but the hypothesis has not yet been critically tested because little is understood of the function of NCX in these processes.
High Na+ Salt Diet and Remodeling of Vascular Smooth Muscle and Endothelial Cells
Biomedicines
Our knowledge on essential hypertension is vast, and its treatment is well known. Not all hypertensives are salt-sensitive. The available evidence suggests that even normotensive individuals are at high cardiovascular risk and lower survival rate, as blood pressure eventually rises later in life with a high salt diet. In addition, little is known about high sodium (Na+) salt diet-sensitive hypertension. There is no doubt that direct and indirect Na+ transporters, such as the Na/Ca exchanger and the Na/H exchanger, and the Na/K pump could be implicated in the development of high salt-induced hypertension in humans. These mechanisms could be involved following the destruction of the cell membrane glycocalyx and changes in vascular endothelial and smooth muscle cells membranes’ permeability and osmolarity. Thus, it is vital to determine the membrane and intracellular mechanisms implicated in this type of hypertension and its treatment.
Physiology and pathophysiology of the renal Na-K-2Cl cotransporter (NKCC2
is located in the apical membrane of the epithelial cells of the thick ascending limb of the loop of Henle (TAL). NKCC2 facilitates 20-25% of the reuptake of the total filtered NaCl load. NKCC2 is therefore one of the transport proteins with the highest overall reabsorptive capacity in the kidney. Consequently, even subtle changes in NKCC2 transport activity considerably alter the renal reabsorptive capacity for NaCl and eventually lead to perturbations of the salt and water homoeostasis. In addition to facilitating the bulk reabsorption of NaCl in the TAL, NKCC2 transport activity in the macula densa cells of the TAL constitutes the initial step of the tubular-vascular communication within the juxtaglomerular apparatus (JGA); this communications allows the TAL to modulate the preglomerular resistance of the afferent arteriole and the renin secretion from the granular cells of the JGA. This review provides an overview of our current knowledge with respect to the general functions of NKCC2, the modulation of its transport activity by different regulatory mechanisms, and new developments in the pathophysiology of NKCC2-dependent renal NaCl transport. differential splicing; macula densa; NKCC2; Slc12a1; thick ascending limb HUMAN KIDNEYS FILTER APPROXIMATELY 1.5 kg of NaCl and 180 liters of water each day. The bulk of the filtered load is reabsorbed along the tubular system and the collecting ducts, which results in the formation of 1.5 liters of urine/day. In total, 20-25% of the filtered NaCl is reabsorbed along the thick ascending limb of the loop of Henle (TAL), whereas virtually no water is reabsorbed in this portion of the nephron due to the lack of paracellular and transcellular water permeability. The major apical entry pathway for NaCl is provided by the Na-K-2Cl cotransporter, NKCC2 (BSC1, bumetanide-sensitive cotrans-porter 1), which accounts for 80% of the total salt reabsorption of the TAL (52, 64). The driving force for NKCC2-dependent salt transport is provided by the activity of the basolateral Na-K-ATPase; basolateral chloride conductivity (clcnkb channels) and apical K recycling via ROMK channels complete the net trans-cellular transport of NaCl in the TAL (53). NKCC2 is encoded by a single gene, but differential splicing of its pre-mRNA gives rise to several splice isoforms, which differ markedly in their transport characteristics and in their localization along the TAL (46, 63). Three different isoforms, NKCC2B, NKCC2A, and NKCC2F, differ in the variable exon 4, which encodes the amino acids of the second transmembrane domain and parts of the adjacent intracellular loop of the cotransporter (96, 114). This specific portion of NKCC2 has been show to be crucial for chloride binding. In addition to these three full-length isoforms, truncated variants of NKCC2 have been reported; these variants differ in the C-terminal portion of the protein and increase the total number of NKCC2 isoforms to at least six (96, 114). In addition, isoforms with tandem repeats of exon 4, such as exon 4A/4F, 4B/4F, and 4B/4A, have been described (20, 43, 67, 161). Because of the high overall salt transport capacity of NKCC2 and its crucial role in the urinary concentrating mechanism , even subtle modulations in NKCC2 transport activity result in considerable changes in renal salt reabsorptive capacity (103). Thus inhibitors of NKCC2, known as loop diuretics, constitute the most potent class of diuretics. The profound impact of NKCC2 inhibition on renal salt reabsorption is further enhanced by the limited transport capacity of the portions of the nephron downstream of the TAL, such as the distal convoluted tubule or the collecting duct. The large amount of salt reabsorption mediated by NKCC2 is most likely responsible for the evolution of a complex regulatory network in the TAL; this network modulates NKCC2 expression, differential splicing of its pre-mRNA, surface trafficking, specific transport activity, and, ultimately, the TAL salt-reabsorptive capacity. The regulatory network that controls NKCC2 expression and activity comprises systemic hormones (such as angiotensin II, catecholamines, and vasopressin) and local paracrine/autocrine factors (such as nitric oxide), all of which modulate the complex intracellular signaling network of TAL epithelial cells (61). The NKCC2-dependent salt retrieval in the TAL substantially contributes to the overall salt-reabsorptive capacity of the kidney; in addition, NKCC2 transport activity initiates tubular-vascular cross talk within the kidney (81, 126). Thus NKCC2-dependent salt transport is the initial step that links the tubular chloride concentration at the macula densa (MD) to the control of the tone of the afferent arteriole and eventually the glomer-ular filtration rate (GFR) of the respective nephron (129). This mechanism is known as tubuloglomerular feedback (TGF)
Red cell sodium-proton exchange is increased in Dahl salt-sensitive hypertensive rats
Kidney International, 1992
Red cell sodium-proton exchange is increased in DahI salt-sensitive hypertensive rats. To investigate the relationship between red blood cell Na1H exchange (EXC) and genetic factors in hypertension, we studied the maximal rate of the antiporter (mmol/liter cell x hr; flux units = FU) in three strains of genetically hypertensive rats. Saltresistant Dahl rats (DR) were normotensive under low (0.02%) and high (8%) NaCI diets, while salt-sensitive DahI rats (DS) became markedly hypertensive after four weeks on the high-NaCI diet. Na1JH exchange did not differ between DR and DS rats when both were fed with the low-NaCI diet (mean SE, 31 3, N = 15, vs. 29 3 FU, N = 14). On the high-NaCI diet, the DR Strain did not exhibit significant changes in blood pressure and antiporter activity, but the DS rats significantly increased their blood pressure and Na/H exchange (57 4 FU, N 13) versus DR rats (38 3 FU, N = 15, P < 0.02). DS rats also significantly increased blood pressure and antiporter activity when fed with high-NaCl diet for one week. These data indicate that high NaCI intake per se does not increase Na/H EXC because the control DR strain did not exhibit transport and blood pressure alterations as observed in the DS strain. Milan hypertensive and spontaneously hypertensive rats (Charles River substrain) had higher blood pressures than Milan and Wystar-Kyoto normotensive rats when they were maintained for four weeks on a 1.5% NaC1 diet; however, no differences were seen among normotensive and hypertensive strains in Na/H exchange activity. When the four strains were fed for four weeks with a low-NaCI diet, blood pressure and Na/H exchange activity did not change in any of these strains. Na/H exchange activity in the three hypertensive strains did not correlate with previously reported measurements in kidney brush border membrane vesicles, a finding suggesting that measurements in intact cells reveal antiporter regulatory mechanisms. Our data indicate that elevated Na/H exchange is not due solely to hypertension or high-NaCI diet per se, since these alterations were not shared by all genetic rat models of hypertension. The development of hypertension with a high-NaCI intake in the DS strain followed by a stimulation of RBC Na/H exchange indicate that the antiporter is up-regulated by salt-sensitive elevation of blood pressure.
Renal Function in Mice with Targeted Disruption of the A Isoform of the Na-K-2Cl Co-Transporter
Journal of the American Society of Nephrology, 2007
Three different full-length splice isoforms of the Na-K-2Cl co-transporter (NKCC2/BSC1) are expressed along the thick ascending limb of Henle (TAL), designated NKCC2A, NKCC2B, and NKCC2F. NKCC2F is expressed in the medullary, NKCC2B mainly in the cortical, and NKCC2A in medullary and cortical portions of the TAL. NKCC2B and NKCC2A were shown to be coexpressed in the macula densa (MD) segment of the mouse TAL. The functional consequences of the existence of three different isoforms of NKCC2 are unclear. For studying the specific role of NKCC2A in kidney function, NKCC2A-/- mice were generated by homologous recombination. NKCC2A-/- mice were viable and showed no gross abnormalities. Ambient urine osmolarity was reduced significantly in NKCC2A-/- compared with wild-type mice, but water deprivation elevated urine osmolarity to similar levels in both genotypes. Baseline plasma renin concentration and the effects of a high- and a low-salt diet on plasma renin concentration were similar in NKCC2A+/+ and -/- mice. However, suppression of renin secretion by acute intravenous saline loading (5% of body weight), a measure of MD-dependent inhibition of renin secretion, was reduced markedly in NKCC2A-/- mice compared with wild-type mice. Cl and water absorption along microperfused loops of Henle of NKCC2A-/- mice were unchanged at normal flow rates but significantly reduced at supranormal flow. Tubuloglomerular feedback function curve as determined by stop flow pressure measurements was left-shifted in NKCC2A-/- compared with wild-type mice, with maximum responses being significantly diminished. In summary, NKCC2A activity seems to be required for MD salt sensing in the high Cl concentration range. Coexpression of both high- and low-affinity isoforms of NKCC2 may permit transport and Cl-dependent tubuloglomerular feedback regulation to occur over a wider Cl concentration range.