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Gerardo Gamba

Publications and source records attributed to Gerardo Gamba.

At least 19 recordsLinked to original sources
PubMed2006

WNK4 kinase is a negative regulator of K+-Cl- cotransporters.

WNK kinases [with no lysine (K) kinase] are emerging as regulators of several membrane transport proteins in which WNKs act as molecular switches that coordinate the activity of several players. Members of the cation-coupled chloride cotransporters family (solute carrier family number 12) are one of the main targets. WNK3 activates the Na(+)-driven cotransporters NCC, NKCC1, and NKCC2 and inhibits the K(+)-driven cotransporters KCC1 to KCC4. WNK4 inhibits the activity of NCC and NKCC1, while in the presence of the STE20-related proline-alanine-rich kinase SPAK activates NKCC1. Nothing is known, however, regarding the effect of WNK4 on the K(+)-Cl(-) cotransporters. Using the heterologous expression system of Xenopus laevis oocytes, here we show that WNK4 inhibits the activity of the K(+)-Cl(-) cotransporters KCC1, KCC3, and KCC4 under cell swelling, a condition in which these cotransporters are maximally active. The effect of WNK4 requires its catalytic activity because it was lost by the substitution of aspartate 318 for alanine (WNK4-D318A) that renders WNK4 catalytically inactive. In contrast, three different WNK4 missense mutations that cause pseudohypoaldosteronism type II do not affect the WNK4-induced inhibition of KCC4. Finally, we observed that catalytically inactive WNK4-D318A is able to bypass the tonicity requirements for KCC2 and KCC3 activation in isotonic conditions. This effect is enhanced by the presence of catalytically inactive SPAK, was prevented by the presence of protein phosphatase inhibitors, and was not present in KCC1 and KCC4. Our results reveal that WNK4 regulates the activity of the K(+)-Cl(-) cotransporters expressed in the kidney.

Animals↗
PubMed2006

The Na+:Cl- cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion.

The renal Na(+):Cl(-) cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl(-) influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl(-) efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr(53) and Thr(58). Elimination of these threonines together with serine residue Ser(71) completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation.

Amino Acid Sequence↗
PubMed2006

Mineralocorticoid receptor blockade confers renoprotection in preexisting chronic cyclosporine nephrotoxicity.

Recent studies from our laboratory have shown that the mineralocorticoid receptor (MR) blockade with spironolactone (Sp) prevented renal dysfunction and reduced renal injury in both acute and chronic cyclosporine (CsA) nephrotoxicity. This study was designed to evaluate whether Sp administration reduces functional and structural renal damage associated in the setting of preexisting chronic CsA nephrotoxicity. Twenty eight male Wistar rats were fed a low-sodium diet. Fourteen received vehicle (V) and the others were treated with CsA (15 mg/kg sc). After 18 days one half of each group received Sp (20 mg/kg po) for the subsequent 18 days. Creatinine clearance, arteriolopathy, tubulointerstitial fibrosis, arteriolar thickening, glomerular diameter, apoptosis index and TGF-beta, procaspase-3, and kidney injury molecule 1 (Kim-1) mRNA levels as well as Kim-1 shedding in urine were evaluated. Sp reduced the progression of renal dysfunction and tubulointerstitial fibrosis in preexisting chronic CsA nephrotoxicity. There was a significant reduction of arteriolar thickening in the CsA+Sp group that was associated with greater glomerular diameter and reduction of apoptosis index. These renoprotective effects were associated with reduction of TGF-beta, procaspase-3, and Kim-1 mRNA levels as well as Kim-1 shedding into the urine. In conclusion, MR blockade with Sp prevented the progression of renal injury in preexisting chronic CsA nephropathy. These results suggest that Sp may reduce CsA-induced established nephrotoxicity in patients.

Animals↗
PubMed2006

Affinity-defining domains in the Na-Cl cotransporter: a different location for Cl- and thiazide binding.

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. Little is known regarding the structure-function relationship in this cotransporter. Previous studies from our group reveal that mammalian NCC exhibits higher affinity for ions and thiazides than teleost NCC and suggest a role for glycosylation upon thiazide affinity. Here we have constructed a series of chimeric and mutant cDNAs between rat and flounder NCC to define the role of glycosylation status, the amino-terminal domain, the carboxyl-terminal domain, the extracellular glycosylated loop, and the transmembrane segments upon affinity for Na+, Cl-, and metolazone. Xenopus laevis oocytes were used as the heterologous expression system. We observed that elimination of glycosylation sites in flounder NCC did not affect the affinity of the cotransporter for metolazone. Also, swapping the amino-terminal domain, the carboxyl-terminal domain, the glycosylation sites, or the entire extracellular glycosylation loop between rat and flounder NCC had no effect upon ions or metolazone affinity. In contrast, interchanging transmembrane regions between rat and flounder NCC revealed that affinity-modifying residues for chloride are located within the transmembrane 1-7 region and for thiazides are located within the transmembrane 8-12 region, whereas both segments seem to be implicated in defining sodium affinity. These observations strongly suggest that binding sites for chloride and thiazide in NCC are different.

Amino Acid Sequence↗
PubMed2006

WNK3 bypasses the tonicity requirement for K-Cl cotransporter activation via a phosphatase-dependent pathway.

SLC12A cation/Cl- cotransporters are mutated in human disease, are targets of diuretics, and are collectively involved in the regulation of cell volume, neuronal excitability, and blood pressure. This gene family has two major branches with different physiological functions and inverse regulation: K-Cl cotransporters (KCC1-KCC4) mediate cellular Cl- efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-Cl cotransporters (NCC and NKCC1/2) mediate cellular Cl- influx and are activated by phosphorylation. A single kinase/phosphatase pathway is thought to coordinate the activities of these cotransporters in a given cell; however, the mechanisms involved are as yet unknown. We previously demonstrated that WNK3, a paralog of serine-threonine kinases mutated in hereditary hypertension, is coexpressed with several cation/Cl- cotransporters and regulates their activity. Here, we show that WNK3 completely prevents the cell swelling-induced activation of KCC1-KCC4 in Xenopus oocytes. In contrast, catalytically inactive WNK3 abolishes the cell shrinkage-induced inhibition of KCC1-KCC4, resulting in a >100-fold stimulation of K-Cl cotransport during conditions in which transport is normally inactive. This activation is completely abolished by calyculin A and cyclosporine A, inhibitors of protein phosphatase 1 and 2B, respectively. Wild-type WNK3 activates Na-(K)-Cl cotransporters by increasing their phosphorylation, and catalytically inactive kinase inhibits Na-(K)-Cl cotransporters by decreasing their phosphorylation, such that our data suggest that WNK3 is a crucial component of the kinase/phosphatase signaling pathway that coordinately regulates the Cl- influx and efflux branches of the SLC12A cotransporter family.

Animals↗
PubMed2006

Identification and functional characterization of EhClC-A, an Entamoeba histolytica ClC chloride channel located at plasma membrane.

ClC chloride channels perform a wide variety of physiological functions and they had been characterized in animals, yeast, plants and bacteria but not in protozoa. By blast search we found in Entamoeba histolytica, the protozoan responsible for human amoebiasis, two genes (Ehclc-A and Ehclc-B) encoding for putative polypeptides with 25-30% identity to ClC chloride channels of several organisms. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that both genes are transcribed in trophozoites. Phylogenetic analysis revealed that EhClC-A and EhClC-B polypeptides belong to the eukaryotic branch of plasma membrane ClCs. Specific antibodies against EhClC-A confirmed that it is located at the trophozoite plasma membrane. Xenopus laevis oocytes microinjected with Ehclc-A cRNA elicited anion currents not detected in oocytes microinjected with water. Induced currents were inwardly rectifying and had a permeability sequence of Cl->Br->I->F->>NO3-. The chloride channel blocker 4-acetamido-4'isothiocyanostilbene-2, 2'-disulphonic acid (SITS) strongly inhibited the oocytes anion currents and trophozoites growth. Experiments at diverse pHs suggested that EhClC-A is not a Cl-/H+ exchanger, but it is an ion channel that could be involved in pH regulation. EhClC-A may also participate in cell volume regulation. As far as we know, EhClC-A is the first chloride channel characterized in protozoa.

Animals↗
PubMed2006

WNK protein kinases modulate cellular Cl- flux by altering the phosphorylation state of the Na-K-Cl and K-Cl cotransporters.

Precise control of cellular Cl(-) transport is necessary for many fundamental physiological processes. For example, the intracellular concentration of Cl(-), fine-tuned through the coordinated action of cellular Cl(-) influx and efflux mechanisms, determines whether a neuron's response to GABA is excitatory or inhibitory. In epithelia, synchrony between apical and basolateral Cl(-) flux, and transcellular and paracellular Cl(-) transport, is necessary for efficient transepithelial Cl(-) reabsorption or secretion. In cells throughout the body, coordination of Cl(-) entry and exit mechanisms help defend against changes in cell volume. The Na-K-Cl and K-Cl cotransporters of the SLC12 gene family are important molecular determinants of Cl(-) entry and exit, respectively, in these systems. The WNK serine-threonine kinase family, members of which are mutated in an inherited form of human hypertension, are components of a signaling pathway that coordinates Cl(-) influx and efflux through SLC12 cotransporters to dynamically regulate intracellular Cl(-) activity.

Biological Transport↗
PubMed2005

Activity of the renal Na+-K+-2Cl- cotransporter is reduced by mutagenesis of N-glycosylation sites: role for protein surface charge in Cl- transport.

The renal-specific Na(+)-K(+)-2Cl(-) cotransporter NKCC2 belongs to the SLC12 gene family; it is the target for loop diuretics and the cause of type I Bartter's syndrome. Because the NKCC2 sequence contains two putative N-linked glycosylation sites, one of which is conserved with the renal Na(+)-Cl(-) cotransporter in which glycosylation affects thiazide affinity, we assessed the role of glycosylation on NKCC2 functional properties. One (N442Q or N452Q) or both (N442,452Q) N-glycosylation sites were eliminated by site-directed mutagenesis. Wild-type NKCC2 and mutant clones were expressed in Xenopus laevis oocytes and analyzed by (86)Rb(+) influx, Western blotting, and confocal microscopy. Inhibition of glycosylation with tunicamycin in wild-type NKCC2-injected oocytes resulted in an 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in N442,452Q-injected oocytes. Functional activity was reduced by 50% in N442Q- and N452Q-injected oocytes and by 80% in oocytes injected with N442,452Q, whereas confocal microscopy of oocytes injected with wild-type or mutant enhanced green fluorescent protein-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced approximately 20% in single mutants and 50% in the double mutant. Ion transport kinetic analyses revealed no changes in cation affinity and a small increase in Cl(-) affinity by N442Q and N442,452Q. However, a slight decrease in bumetanide affinity was observed. Our data demonstrate that NKCC2 is glycosylated and suggest that prevention of glycosylation reduces its functional expression by affecting insertion into the plasma membrane and the intrinsic activity of the cotransporter.

Animals↗
PubMed2005

WNK3 kinase is a positive regulator of NKCC2 and NCC, renal cation-Cl- cotransporters required for normal blood pressure homeostasis.

WNK1 and WNK4 [WNK, with no lysine (K)] are serine-threonine kinases that function as molecular switches, eliciting coordinated effects on diverse ion transport pathways to maintain homeostasis during physiological perturbation. Gain-of-function mutations in either of these genes cause an inherited syndrome featuring hypertension and hyperkalemia due to increased renal NaCl reabsorption and decreased K(+) secretion. Here, we reveal unique biochemical and functional properties of WNK3, a related member of the WNK kinase family. Unlike WNK1 and WNK4, WNK3 is expressed throughout the nephron, predominantly at intercellular junctions. Because WNK4 is a potent inhibitor of members of the cation-cotransporter SLC12A family, we used coexpression studies in Xenopus oocytes to investigate the effect of WNK3 on NCC and NKCC2, related kidney-specific transporters that mediate apical NaCl reabsorption in the thick ascending limb and distal convoluted tubule, respectively. In contrast to WNK4's inhibitory activity, kinase-active WNK3 is a potent activator of both NKCC2 and NCC-mediated transport. Conversely, in its kinase-inactive state, WNK3 is a potent inhibitor of NKCC2 and NCC activity. WNK3 regulates the activity of these transporters by altering their expression at the plasma membrane. Wild-type WNK3 increases and kinase-inactive WNK3 decreases NKCC2 phosphorylation at Thr-184 and Thr-189, sites required for the vasopressin-mediated plasmalemmal translocation and activation of NKCC2 in vivo. The effects of WNK3 on these transporters and their coexpression in renal epithelia implicate WNK3 in NaCl, water, and blood pressure homeostasis, perhaps via signaling downstream of vasopressin.

Animals↗
PubMed2005

WNK3 modulates transport of Cl- in and out of cells: implications for control of cell volume and neuronal excitability.

The regulation of Cl(-) transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion cotransporters: Cl(-) influx is mediated by the Na-K-2Cl cotransporter NKCC1 and Cl(-) efflux via K-Cl cotransporters, KCC1 or KCC2. A Cl(-)/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl(-)-transporting epithelia and in neurons expressing ionotropic GABA(A) receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl(-) influx via NKCC1, and that it inhibits Cl(-) exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl(-) via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl(-)/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission.

Animals↗
PubMed2005

WNK lies upstream of kinases involved in regulation of ion transporters.

Two members of a recently discovered family of protein kinases {WNK1 and WNK4 [with no K (lysine) kinases-1 and -4]} are the cause of an inherited disease known as pseudohypoaldosteronism type II that features arterial hypertension. The family is known as WNK due to a lack of the invariant catalytic lysine in kinase subdomain II. The mechanisms by which WNKs regulate blood pressure are beginning to be understood at the physiological level from recent studies showing effects of WNK4 on several plasma membrane co-transporters and ion channels. However, little is known about the function of WNKs at the biochemical level. In this issue of the Biochemical Journal, Vitari et al. have shown that WNK1 and WNK4 interact with other kinases, SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress response kinase-1), which are involved in the regulation of ion transporters. WNK1 and WNK4 phosphorylate SPAK and OSR1, which in turn phosphorylate the N-terminal domain of the basolateral Na+-K+-2Cl- co-transporter, NKCCl. The phosphorylation site involved in SPAK or OSR1 activation is identified as a threonine residue within the T-loop.

Hypertension↗
PubMed2005

NH2-terminal heterogeneity in the KCC3 K+-Cl- cotransporter.

The SLC12A6 gene encoding the K(+)-Cl(-) cotransporter KCC3 is expressed in multiple tissues, including kidney. Here, we report the molecular characterization of several NH(2)-terminal isoforms of human and mouse KCC3, along with intrarenal localization and functional characterization in Xenopus laevis oocytes. Two major isoforms, KCC3a and KCC3b, are generated by transcriptional initiation 5' of two distinct first coding exons. Northern blot analysis of mouse tissues indicates that KCC3b expression is particularly robust in the kidney, which also expresses KCC3a. Western blotting of mouse tissue using an exon 3-specific antibody reveals that the kidney is also unique in expressing immunoreactive protein of a lower mass, suggestive evidence that the shorter KCC3b protein predominates in kidney. Immunofluorescence reveals basolateral expression of KCC3 protein along the entire length of the proximal tubule, in both the mouse and rat. Removal of the 15-residue exon 2 by alternative splicing generates the KCC3a-x2M and KCC3b-x2M isoforms; other splicing events at an alternative acceptor site within exon 1a generate the KCC3a-S isoform, which is 60 residues shorter than KCC3a. This variation in sequence of NH(2)-terminal cytoplasmic domains occurs proximal to a stretch of highly conserved residues and affects the content of putative phosphorylation sites. Kinetic characterization of KCC3a in X. laevis oocytes reveals apparent K(m)s for Rb(+) and Cl(-) of 10.7 +/- 2.5 and 7.3 +/- 1.2 mM, respectively, with an anion selectivity of Br(-) > Cl(-) > PO(4) = I(-) = SCN(-) = gluconate. All five NH(2)-terminal isoforms are activated by cell swelling (hypotonic conditions), with no activity under isotonic conditions. Although the isoforms do not differ in the osmotic set point of swelling activation, this activation is more rapid for the KCC3a-x2M and KCC3a-S proteins. In summary, there is significant NH(2)-terminal heterogeneity of KCC3, with particularly robust expression of KCC3b in the kidney. Basolateral swelling-activated K(+)-Cl(-) cotransport mediated by KCC3 likely functions in cell volume regulation during the transepithelial transport of both salt and solutes by the proximal tubule.

Amino Acid Sequence↗
PubMed2005

Renocortical mRNA expression of vasoactive factors during spironolactone protective effect in chronic cyclosporine nephrotoxicity.

We showed that spironolactone reduced structural damage and prevented renal dysfunction in chronic cyclosporine (CsA) nephrotoxicity. These findings evidenced an aldosterone renal vascular effect under this condition. To investigate aldosterone's role in modulating renal vascular tone, renocortical vasoactive pathways mRNA levels in chronic CsA nephrotoxicity as well as spironolactone's effect on renal function in acute CsA nephrotoxicity were evaluated. Two experimental sets were designed. For chronic nephrotoxicity, rats fed with low-sodium diet were divided into groups receiving vehicle, spironolactone (Sp), CsA, and CsA+Sp, for 21 days. Creatinine clearance, survival percentage, and renocortical mRNA levels of pro-renin, angiotensinogen (Ang), angiotensin receptors (AT(1A), AT(1B), and AT(2)), preproendothelin, endothelin receptors (ET(A), ET(B)), cyclooxygenase-2 (COX-2), and adenosine receptors (Ad(1), Ad(2A), Ad(2B), and Ad(3)) were analyzed. For acute nephrotoxicity, similar groups fed with a standard chow diet for 7 days were included. Serum potassium and sodium, glomerular filtration rate (GFR), and renal blood flow (RBF) were determined. In chronic model, CsA produced pro-renin and ET upregulation, altered adenosine receptors expression, and reduced Ang, AT(1A), AT(1B), ET(B), and COX-2 mRNA levels. Spironolactone protective effect in chronic nephrotoxicity was associated with prevention of pro-renin upregulation and increased AT(2), together with ET(B) reduction. In acute nephrotoxicity, spironolactone completely prevented GFR and RBF reduction induced by CsA. Our results suggest that aldosterone contributes to renal vasoconstriction observed in CsA nephrotoxicity and that renoprotection conferred by spironolactone was related to modification of renocortical vasoactive pathways expression, in which pro-renin normalization was the most evident change in chronic nephropathy. Finally, our data point to spironolactone as a potential treatment to reduce CsA nephrotoxicity in transplant patients.

Aldosterone↗
PubMed2005

Low calorie commercial sugar is a sensitive marker of glomerular filtration rate.

BACKGROUND: Glomerular filtration rate (GFR) in humans and animals might be determined with precision by measuring the clearance of an ideal marker, such as inulin. However, the use of inutest, an inulin analog, is limited by its cost and accessibility. The present study tested whether low calorie commercial sugar (LC sugar) can be used to measure GFR during normal and renal dysfunction. METHODS: Two groups of 6 male Wistar rats weighing 300 to 350 g were included. One group was treated with a daily dose of cyclosporine (CsA) 30 mg/kg subcutaneously for 7 days and the other group was formed by nontreated control rats. In one half of each group, GFR was evaluated by using inutest and in the other half by using LC sugar. GFR was also evaluated by using a wide LC sugar plasma concentration range in an additional group. RESULTS: In nontreated rats, the mean GFR evaluated with LC sugar was 2.2 +/- 0.1 mL/min. This value is equal to that obtained with inutest: 2.3 +/- 0.1 mL/min. CsA administration produced a significant reduction of renal blood flow and renal function. The GFR reduction induced by CsA was similarly determined by both LC sugar and inutest to be at 1.0 +/- 0.2 and 1.1 +/- 0.2 mL/min (P= NS), respectively. In addition, GFR did not change when LC sugar plasma concentration gradually increased. CONCLUSION: Our results show that in both normal and pathophysiologic conditions, LC sugar is a good marker of GFR similar to the gold standard inutest.

Animals↗
PubMed2005

Role of WNK kinases in regulating tubular salt and potassium transport and in the development of hypertension.

A recently discovered family of protein kinases is responsible for an autosomal-dominant disease known as Gordon's syndrome or pseudohypoaldosteronism type II (PHA-II) that features hyperkalemia and hyperchloremic metabolic acidosis, accompanied by hypertension and hypercalciuria. Four genes have been described in this kinase family, which has been named WNK, due to the absence of a key lysine in kinase subdomain II (with no K kinases). Two of these genes, WNK1 and WNK4 located in human chromosomes 12 and 17, respectively, are responsible for PHA-II. Immunohystochemical analysis revealed that WNK1 and WNK4 are predominantly expressed in the distal convoluted tubule and collecting duct. The physiological studies have shown that WNK4 downregulates the activity of ion transport pathways expressed in these nephron segments, such as the apical thiazide-sensitive Na+-Cl- cotransporter and apical secretory K+ channel ROMK, as well as upregulates paracellular chloride transport and phosphorylation of tight junction proteins such as claudins. In addition, WNK4 downregulates other Cl- influx pathways such as the basolateral Na+-K+-2Cl- cotransporter and Cl-/HCO3- exchanger. WNK4 mutations behave as a loss of function for the Na+-Cl- cotransporter and a gain of function when it comes to ROMK and claudins. These dual effects of WNK4 mutations fit with proposed mechanisms for developing electrolyte abnormalities and hypertension in PHA-II and point to WNK4 as a multifunctional regulator of diverse ion transporters.

Down-Regulation↗
PubMed2005

Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters.

Electroneutral cation-Cl(-) cotransporters compose a family of solute carriers in which cation (Na(+) or K(+)) movement through the plasma membrane is always accompanied by Cl(-) in a 1:1 stoichiometry. Seven well-characterized members include one gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter, two genes encoding loop diuretic-sensitive Na(+)-K(+)-2Cl(-) cotransporters, and four genes encoding K(+)-Cl(-) cotransporters. These membrane proteins are involved in several physiological activities including transepithelial ion absorption and secretion, cell volume regulation, and setting intracellular Cl(-) concentration below or above its electrochemical potential equilibrium. In addition, members of this family play an important role in cardiovascular and neuronal pharmacology and pathophysiology. Some of these cotransporters serve as targets for loop diuretics and thiazide-type diuretics, which are among the most commonly prescribed drugs in the world, and inactivating mutations of three members of the family cause inherited diseases such as Bartter's, Gitelman's, and Anderman's diseases. Major advances have been made in the past decade as consequences of molecular identification of all members in this family. This work is a comprehensive review of the knowledge that has evolved in this area and includes molecular biology of each gene, functional properties of identified cotransporters, structure-function relationships, and physiological and pathophysiological roles of each cotransporter.

Animals↗
PubMed2004

Renal protection by a soy diet in obese Zucker rats is associated with restoration of nitric oxide generation.

The obese Zucker rat is a valuable model for studying kidney disease associated with obesity and diabetes. Previous studies have shown that substitution of animal protein with soy ameliorates the progression of renal disease. To explore the participation of nitric oxide (NO) and caveolin-1 in this protective effect, we evaluated proteinuria, creatinine clearance, renal structural lesions, nitrites and nitrates urinary excretion (UNO(2)(-)/NO(3)V), and mRNA and protein levels of neuronal NO synthase (nNOS), endothelial NOS (eNOS), and caveolin-1 in lean and fatty Zucker rats fed with 20% casein or soy protein diet. After 160 days of feeding with casein, fatty Zucker rats developed renal insufficiency, progressive proteinuria, and renal structural lesions; these alterations were associated with an important fall of UNO(2)(-)/NO(3)V, changes in nNOS and eNOS mRNA levels, together with increased amount of eNOS and caveolin-1 present in plasma membrane proteins of the kidney. In fatty Zucker rats fed with soy, we observed that soy diet improved renal function, UNO(2)(-)/NO(3)V, and proteinuria and reduced glomerulosclerosis, tubular dilation, intersticial fibrosis, and extracapilar proliferation. Renal protection was associated with reduction of caveolin-1 and eNOS in renal plasma membrane proteins. In conclusion, our results suggest that renal protective effect of soy protein appears to be mediated by improvement of NO generation and pointed out to caveolin-1 overexpression as a potential pathophysiological mechanism in renal disease.

Animals↗