Renal Tubular Transport Research Articles

Oxidative stress upregulates renal Angiotensin II type I receptors via activation of transcription factor nuclear factor‐kappaB

Regulation of redox homeostasis is associated with normal functioning of cells, whereas oxidative stress is seen with various cardiovascular disorders like hypertension. Angiotensin II (Ang II), acting via Ang II type I (AT1) receptors, has known effects on the renal proximal tubular sodium transport, thus plays an important role in regulating blood pressure. Oxidative stress is also known to activate redox sensitive transcription factor NF‐kappaB and evidences indicate that AT1 receptor gene posses binding site for this transcription factor. Herein, we aim to investigate if oxidative stress upregulates renal AT1 receptors through NF‐kappaB activation. Human proximal tubular (HK‐2) cells were incubated with a pro oxidant L‐buthionine sulfoximine (BSO), an inhibitor of glutathione, for 48 hrs.ResultsCompared to control, there was an increase in levels of protein nitrotyrosination indicating oxidative stress in BSO treated HK‐2 cells. There was also activation of NF‐kappaB, upregulation of AT1 receptor protein, and increased Ang II‐induced Ca2+ signaling. Furthermore, treatment with pyrrolidine dithiocarbamate, a NF‐kappaB inihibitor, blocked NF‐kappaB activation, abolished AT1 receptor upregulation and exaggerated Ang II induced Ca2+ signaling.ConclusionThese results suggest that oxidative stress upregulates renal AT1 receptors via activation of transcription factor NF‐kappaB.Grant: AHA‐SDG‐0835428N

Effects of extracellular nucleotides on renal tubular solute transport

A range of P2 receptor subtypes has been identified along the renal tubule, in both apical and basolateral membranes. Furthermore, it has been shown that nucleotides are released from renal tubular cells, and that ectonucleotidases are present in several nephron segments. These findings suggest an autocrine/paracrine role for nucleotides in regulating tubular function. The present review catalogues the known actions of extracellular nucleotides on tubular solute transport. In the proximal tubule, there is firm evidence that stimulation of apical P2Y(1) receptors inhibits bicarbonate reabsorption, whilst basolaterally applied ATP has the opposite effect. Clearance studies suggest that systemic diadenosine polyphosphates profoundly reduce proximal tubular fluid transport, through as yet unidentified P2 receptors. To date, only circumstantial evidence is available for an action of nucleotides on transport in the loop of Henle; and no studies have been made on native distal tubules, though observations in cell lines suggest an inhibitory effect on sodium, calcium and magnesium transport. The nephron segment most studied is the collecting duct. Apically applied nucleotides inhibit the activity of small-conductance K(+) channels in mouse collecting duct, apparently through stimulation of P2Y(2) receptors. There is also evidence, from cell lines and native tissue, that apically (and in some cases basolaterally) applied nucleotides inhibit sodium reabsorption. In mice pharmacological profiling implicates P2Y(2) receptors; but in rats, the receptor subtype(s) responsible is/are unclear. Recent patch-clamp studies in rat collecting ducts implicate apical P2Y and P2X subtypes, with evidence for both inhibitory and stimulatory effects. Despite considerable progress, clarification of the physiological role of the tubular P2 receptor system remains some way off.

A molecularly guided tour along the nephron

The kidney is an intriguingly multifaceted organ controlling many vital body functions. Therefore, the kidney orchestrates a large set of tasks including the maintenance of the body fluid balance and thereby the blood pressure, the reabsorption of glucose, amino acids and other nutrients, the excretion of sodium, potassium, calcium, magnesium, and other electrolytes, and the control of the acid/base balance. In addition, the kidney produces hormones and proteins exerting important general physiological roles. For instance, the biological active form of vitamin D is mainly produced in the kidney through the conversion of 25hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 by the renal enzyme 25-hydroxyvitamin D-1-α-hydroxylase. In addition, the antiaging hormone klotho is primarily produced in the kidney and controls overall mineral metabolism. Also, the glycoprotein erythropoietin is generated here and enhances the oxygen-carrying capacity of the blood by activating the production of erythrocytes by the bone marrow. The importance of these renal hormones is sadly demonstrated by the severe symptoms accompanying chronic kidney diseases. The kidney is an anatomically complex structure consisting of a large number of nephrons, the functional units of the kidney. Each nephron begins with a glomerulus that filters blood entering the kidney. This filtrate then flows along the length of the nephron. The major function of the epithelial cells lining the nephron is the reabsorption of water and small molecules from the filtrate into the blood and the secretion of wastes from the blood into the urine. To this end, the kidney is equipped with a host of transport proteins including ion channels, electrolyte transporters, and pumps within specific epithelial cells along the various tubule segments. In recent years, our understanding of the molecular nature of these transport proteins has made major progress through the application of functional expression cloning techniques using Xenopus laevis oocytes and the identification of gene defects in inherited renal tubular transport disorders. The regulation of these transport processes is constantly challenged by a demanding and greatly variable environment including an acidic luminal pH, a high medullary osmolarity, and a variability in blood and urinary flow as well as in the composition of the prourine [4]. The activity of these transporters can basically be controlled at several discrete levels. First, the expression level of the particular transporter can be regulated by specific hormones. Second, trafficking of the transporter to the designated plasma membrane can be controlled by intracellular signaling processes. Third, the activity of the transport protein present at the plasma membrane can be subject to various extra-en intracellular regulatory mechanisms. Ample timely physiological studies have addressed these various regulatory aspects of renal transport processes. New key physiological mechanisms have emerged from these recent investigations particularly through a multidisciplinary approach using model cells and organisms. The complexity in structure and function of the kidney is increasingly revealed by these investigations. To do justice to this fact, a standard nomenclature has been disseminated to facilitate the renal physiologist [14]. Consequently, this special issue has been divided according to the main parts of the nephron including the proximal tubule, the loop of Henle, the distal convoluted tubule, the connecting tubule, and the collecting duct. In this guided tour along the nephron, world-renowned renal physiologists highlight the Pflugers Arch Eur J Physiol (2009) 458:1–3 DOI 10.1007/s00424-009-0661-3

Protective effect of 20-HETE analogues in experimental renal ischemia reperfusion injury

While it is known that the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemic injury in the heart and brain, its role in kidney injury is unclear. Here we determined the effects on ischemia-reperfusion injury of the 20-HETE analogues, 20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid (5,14-20-HEDE), and N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-20-HEDGE), and of the inhibitor of 20-HETE synthesis N-hydroxy-N-(4-butyl-2 methylphenyl) formamidine (HET0016). Using Sprague-Dawley rats we found that while treatment with the inhibitor exacerbated renal injury, infusion of both 5,14-20-HEDE and 5,14-20-HEDGE significantly attenuated injury when compared to vehicle or inhibitor-treated rats. Medullary blood flow, measured by laser-Doppler flowmetry, decreased to half of the baseline one hour after reperfusion in the control rats, but 5,14-20-HEDGE completely prevented this. Treatment of control animals with 5,14-20-HEDGE increased urine output and sodium excretion without altering their mean arterial pressure or glomerular filtration rate. Our results suggest that 20-HETE analogues protect the kidney from ischemia-reperfusion injury by inhibiting renal tubular sodium transport and preventing the post-ischemic fall in medullary blood flow. Analogues of 20-HETE may be useful in the treatment of acute ischemic kidney injury.

Molecular Pathophysiology of Renal Tubular Acidosis

Renal tubular acidosis (RTA) is characterized by metabolic acidosis due to renal impaired acid excretion. Hyperchloremic acidosis with normal anion gap and normal or minimally affected glomerular filtration rate defines this disorder. RTA can also present with hypokalemia, medullary nephrocalcinosis and nephrolitiasis, as well as growth retardation and rickets in children, or short stature and osteomalacia in adults. In the past decade, remarkable progress has been made in our understanding of the molecular pathogenesis of RTA and the fundamental molecular physiology of renal tubular transport processes. This review summarizes hereditary diseases caused by mutations in genes encoding transporter or channel proteins operating along the renal tubule. Review of the molecular basis of hereditary tubulopathies reveals various loss-of-function or gain-of-function mutations in genes encoding cotransporter, exchanger, or channel proteins, which are located in the luminal, basolateral, or endosomal membranes of the tubular cell or in paracellular tight junctions. These gene mutations result in a variety of functional defects in transporter/channel proteins, including decreased activity, impaired gating, defective trafficking, impaired endocytosis and degradation, or defective assembly of channel subunits. Further molecular studies of inherited tubular transport disorders may shed more light on the molecular pathophysiology of these diseases and may significantly improve our understanding of the mechanisms underlying renal salt homeostasis, urinary mineral excretion, and blood pressure regulation in health and disease. The identification of the molecular defects in inherited tubulopathies may provide a basis for future design of targeted therapeutic interventions and, possibly, strategies for gene therapy of these complex disorders.

SGK1-sensitive renal tubular glucose reabsorption in diabetes

The hyperglycemia of diabetes mellitus increases the filtered glucose load beyond the maximal tubular transport rate and thus leads to glucosuria. Sustained hyperglycemia, however, may gradually increase the maximal renal tubular transport rate and thereby blunt the increase of urinary glucose excretion. The mechanisms accounting for the increase of renal tubular glucose transport have remained ill-defined. A candidate is the serum- and glucocorticoid-inducible kinase SGK1. The kinase has been shown to stimulate Na(+)-coupled glucose transport in vitro and mediate the stimulation of electrogenic intestinal glucose transport by glucocorticoids in vivo. SGK1 expression is confined to glomerula and distal nephron in intact kidneys but may extend to the proximal tubule in diabetic nephropathy. To explore whether SGK1 modifies glucose transport in diabetic kidneys, Akita mice (akita(+/-)), which develop spontaneous diabetes, have been crossbred with gene-targeted mice lacking SGK1 on one allele (sgk1(+/-)) to eventually generate either akita(+/-)/sgk1(-/-) or akita(+/-)/sgk1(+/+) mice. Both akita(+/-)/sgk1(-/-) and akita(+/-)/sgk1(+/+) mice developed profound hyperglycemia (>20 mM) within approximately 6 wk. Body weight and plasma glucose concentrations were not significantly different between these two genotypes. However, urinary excretion of glucose and urinary excretion of fluid, Na(+), and K(+), as well as plasma aldosterone concentrations, were significantly higher in akita(+/-)/sgk1(-/-) than in akita(+/-)/sgk1(+/+) mice. Studies in isolated perfused proximal tubules revealed that the electrogenic glucose transport was significantly lower in akita(+/-)/sgk1(-/-) than in akita(+/-)/sgk1(+/+) mice. The data provide the first evidence that SGK1 participates in the stimulation of renal tubular glucose transport in diabetic kidneys.

AVP-stimulated nucleotide secretion in perfused mouse medullary thick ascending limb and cortical collecting duct

Extracellular nucleotides are local, short-lived signaling molecules that inhibit renal tubular transport via both luminal and basolateral P2 receptors (1, 2). Apparently, the renal epithelium itself is able to release nucleotides (3, 4). The mechanism and circumstances under which epithelia nucleotide release is stimulated remains elusive (5, 6). Here, we investigate the phenomenon of nucleotide secretion in intact perfused mouse medullary thick ascending limb (mTAL) and cortical collecting duct (CCD). The nucleotide secretion was monitored by a biosensor cell placed to register nucleotides in the tubular out-flow. [Ca(2+)](i) was measured simultaneously in the biosensor cells and the renal tubule with fluo-4. We were able to identify spontaneous tubular nucleotide secretion in resting perfused mTAL. This was seen as lively [Ca(2+)](i) oscillations in the nucleotide biosensor cells when the tubular outflow fluid engulfed the sensing cells. In mouse mTAL 10 nM AVP and dDAVP induced robust [Ca(2+)](i) oscillations, whereas AVP in the CCD induced large, slow and transient [Ca(2+)](i) elevations. Importantly, we identify that AVP/dDAVP triggers tubular secretion of nucleotides in mTAL. After addition of AVP/dDAVP the biosensor cells registered bursts of nucleotides originating from the tubular perfusate. The approximated tubular nucleotide concentration reached peak values of approximately 0.2-0.3 microM. A very similar response was observed after AVP stimulation of CCDs. Thus, AVP stimulated tubular secretion of nucleotides in a burst like pattern with peak tubular nucleotide concentrations in the low micromolar range. Luminal nucleotides are prone to activate luminal P2 receptors which in turn are well described to inhibit AVP-augmented aquaporin-2-dependent water absorption or ENaC-mediated Na(+) transport (8). Therefore, we speculate that local nucleotide signaling is an intrinsic feed-back element of hormonal control of renal tubular transport.

Claudin Tight Junction Proteins: Novel Aspects in Paracellular Transport

Claudins are essential components of the intercellular tight junction and major determinants of paracellular solute fluxes across epithelia and endothelia. Many members of this family display a distinct charge or size specificity, whereas others render the epithelium impermeable to transport. Due to intercellular localization, claudin-mediated transport processes are passive and driven by an electrochemical gradient. In epithelial tissues, claudins exhibit a temporal-spatial expression pattern corresponding with regional and local solute transport profiles. Whereas paracellular transport mechanisms in organs such as intestine and kidney have been extensively investigated, little is known about the molecular mechanisms determining solute transport in the peritoneum, and thus the determinants of peritoneal dialysis. Given the ubiquitous expression of claudins in endothelia and epithelia, it is predictable that claudins also contribute to pore formation and determination in the peritoneum, and that they are involved in solute flux. Therefore, we review the basic characteristics of claudin family members and their function as exemplified in renal tubular transport and give an outlook to what extent claudin family members might be of importance for solute reabsorption across the peritoneal membrane.

Genetic variants of organic cation transporter 2 (OCT2) significantly reduce metformin uptake in oocytes

1. The authors sought to evaluate the contribution of organic cation transporters (OCTs) to the renal tubular transport of metformin using LLC-PK1 cells as an in vitro model for the renal proximal tubule, and to investigate the effects of three non-synonymous genetic variants of OCT2 on the transport activity of metformin in vitro using an oocyte over-expression system.2. The basolateral-to-apical transport of metformin was significantly greater than the apical-to-basolateral transport and showed concentration dependency with the kinetic parameters: maximum transport rate (Vmax), 922 pmol min−1 per 5 × 105 cells; Michaelis–Menten constant (Km), 393 µM; intrinsic clearance (CLint), 2.35 µl min−1 per 5 × 105 cells; and diffusion constant (Kd), 0.33 µl min−1 per 5 × 105 cells. The basolateral-to-apical transport of metformin was inhibited by phenoxybenzamine, an inhibitor of OCTs, but not by cyclosporine A, MK571, or fumitremorgin C, which are inhibitors of P-glycoprotein, multidrug resistance proteins (MRPs), and breast cancer resistance protein (BCRP), respectively, suggesting that OCTs play a role in renal tubular secretion of metformin.3. Metformin uptake was much greater in oocytes expressing OCT2-wild type (OCT2-WT) than OCT1-WT compared with uptake in water-injected oocytes. Uptake was significantly decreased in oocytes expressing OCT2-T199I, -T201M, and -A270S compared with that in OCT2-WT, suggesting that metformin is a better substrate for OCT2 than for OCT1 and that the amino acid-substituted variants of OCT2 cause a functional decrease in metformin uptake.4. In conclusion, the genetic variants of OCT2 (OCT2-T199I, -T201M, and -A270S) decreased the transport activity of metformin and thus may contribute to the inter-individual variation in metformin disposition as OCT2 plays a pivotal role in renal excretion, the major disposition route of metformin.

Endothelin Receptor Antagonism

The endothelins are a family (ET-1, ET-2, and ET-3) of naturally occurring polypeptides, with ET-1 being the most clinically pertinent isoform. ET-1 was formally identified in 1988, at which time it was viewed mainly as a potent vasoconstrictor; however, the biological understanding of endothelin has since rapidly evolved, and it is now appreciated that ET-1 is not only a potent vasoconstrictor but also that it has a prominent role in fibrogenesis, inflammatory states, oxidative stress, atherosclerosis, salt and water balance, and pulmonary artery hypertension.1–3 ET-1 binds to 2 receptors: ETA and ETB. In general, stimulation of these receptors has opposing actions. For example, ETA stimulation is vasoconstricting, as well as being profibrotic and proinflammatory, whereas ETB stimulation is vasodilatory, antiproliferative, and natriuretic. The ETB receptor also subserves a clearance role for ET-1. The varying distribution of the endothelin receptors in different tissues is responsible for the mixture of actions attributed to endothelin. For example, the renal medulla contains the highest concentration of endothelin receptors in the body.4 The renal cortex is also heavily populated with endothelin receptors (50% of the density of the medulla). The ETB receptor predominates in the kidney, composing 70% of the receptors in both locations. Thus, the important roles of ET-1 and the ETB receptor are in regulating renal hemodynamics and tubular transport processes, as well as mesangial and vascular smooth muscle cell proliferation and mitogenesis.3 Likewise, ETA and ETB receptors are highly expressed in smooth muscle cells and foamy macrophages in atherosclerotic models. Stimulation of receptors in these locations is the basis of the proinflammatory and (pro)atherosclerotic actions of …

Adenosine and renal tubular function

Intrarenal adenosine is present in the cytoplasm of renal epithelial cells and in the extracellular space. Adenosine is generated at high levels in response to imbalance between energy demand and supply (e.g. increased tubular sodium chloride transport or hypoxia) and activates cell membrane adenosine receptors to affect renal vascular and tubular functions. Adenosine regulates renal sodium and water excretion via a myriad of effects on renal hemodynamic, glomerular filtration rate, renin secretion and direct effects on the renal tubule epithelium. This review examines the direct effects of adenosine on renal tubular epithelial transport in light of the most recent evidence and discusses some physiologic and pathophysiologic implications. Intrarenal adenosine affects proximal fluid and solute transport in a biphasic fashion. Under physiological conditions adenosine stimulates proximal tubular re-absorption, thus reducing the load delivered to the distal nephron. A supra-physiologic increase in adenosine such as in ischemia reduces reabsorption in the proximal tubule, thus reducing renal oxygen consumption. Intrarenal adenosine and its receptors have important regulatory functions in the renal epithelium. A complete understanding of this autocrine/paracrine system holds great potential for novel therapeutic strategies, such as the use of nucleoside analogues for reno-protection in renal ischemia.

Novel NaPi-2c mutations that cause mistargeting of NaPi-2c protein and uncoupling of Na-Picotransport cause HHRH

hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is an autosomal recessive inherited disorder of mineral and bone metabolism. It is characterized by hypophosphatemia, rickets, and increased serum 1,25-dihydroxyvitamin D concentration, resulting in secondary absortive hypercalciuria and

Programming of hypertension: the nervous kidney

two decades after Barker and coworkers ([2][1]) described an inverse epidemiological relationship between birth weight and risk of adult cardiovascular morbidity, including hypertension, the pathophysiology of prenatally programmed hypertension remains incompletely understood. Experimental models

Effect of renal denervation on prenatal programming of hypertension and renal tubular transporter abundance

Prenatal glucocorticoids are often administered to pregnant women to accelerate pulmonary maturation. We have demonstrated that administration of dexamethasone during specific periods of pregnancy in the rat causes hypertension in the offspring when they are studied as adults. The purpose of the present study was to determine whether the hypertension due to prenatal dexamethasone was mediated by renal nerves. We administered dexamethasone to rats daily for 4 days between days 15 and 18 of gestation. Rats underwent bilateral renal denervation or sham operation at 6 wk of age, and blood pressure was measured at 8 wk of age. Prenatal dexamethasone in the sham operation group resulted in an increase in blood pressure compared with vehicle-treated sham controls (134 +/- 3 vs. 145 +/- 3 mmHg, P < 0.05). Renal denervation did not affect blood pressure significantly in the prenatal vehicle-treated control group but resulted in normalization in blood pressure in the prenatal dexamethasone group and (130 +/- 3 and 128 +/- 5 mmHg, respectively). Prenatal dexamethasone increased type 3 Na+/H+ exchanger (NHE3), Na+K+-2Cl(-) cotransporter (NKCC2), and Na+-Cl(-) cotransporter (NCC), but not alpha-, beta-, and gamma-epithelial Na+ channel (ENaC) protein abundance compared with controls. The increase in NHE3, NKCC2, and NCC protein abundance by prenatal dexamethasone was not seen in 8-wk-old rats 2 wk after renal denervation. Renal denervation did not affect NHE3, NKCC2, and NCC protein abundance in prenatal vehicle-treated animals. This study is consistent with renal nerves playing a role in mediating the hypertension by prenatal programming by dexamethasone.

Oral Delivery of 1,3‐Dicyclohexylurea Nanosuspension Enhances Exposure and Lowers Blood Pressure in Hypertensive Rats

Cytochrome P450-derived epoxyeicosatrienoic acids (EET) are biologically active metabolites of arachidonic acid that have potent effects on renal vascular reactivity and tubular ion transport and have been implicated in the control of blood pressure. EETs are hydrolyzed to their less active diols, dihydroxyeicosatrienoic acids (DHET), by the enzyme soluble epoxide hydrolase (sEH). 1,3-dicyclohexylurea (DCU), a potent sEH inhibitor, lowers systemic blood pressure in spontaneously hypertensive rats when dosed intraperitoneally. However, DCU has poor aqueous solubility, posing a challenge for in vivo oral delivery. To overcome this limitation, we formulated DCU in a nanosuspension using wet milling. Milling reduced particle size, increasing the total surface area by approximately 40-fold. In rats chronically infused with angiotensin II, the DCU nanosuspension administered orally twice daily for 4 days produced plasma exposures an order of magnitude greater than unmilled DCU and lowered blood pressure by nearly 30 mmHg. Consistent with the mechanism of sEH inhibition, DCU increased plasma 14,15-EET and decreased plasma 14,15-DHET levels. These data confirm the antihypertensive effect of sEH inhibition and demonstrate that greatly enhanced exposure of a low-solubility compound is achievable by oral delivery using a nanoparticle drug delivery system.

The Primary Hyperoxalurias

The primary hyperoxalurias (PHs) are rare autosomal-recessive inborn errors of metabolism. In the most severe form (type 1), recurrent kidney stones and progressive nephrocalcinosis lead to the loss of kidney function, accompanied by systemic oxalosis, and often requires dialysis and/or transplantation. The variety of genetic mutations leading to PH increasingly are being defined, resulting in the ability to diagnose most patients accurately via minimally invasive means. During and after definitive diagnosis, supportive therapies with pyridoxine supplementation, urinary crystallization inhibitors, and hydration should be used, but have varying success. Emerging information about the renal tubular and intestinal transport of oxalate is leading to increasing evidence to support the use of oxalate-degrading bacteria (probiotics) and enzymes in the treatment of PH. Organ transplantation historically has offered the only potential cure for PH, and may include kidney-alone, combined liver-kidney, or pre-emptive liver-alone transplantation. Exciting new approaches in the treatment of type 1 PH, however, are under investigation. These include the restoration of defective enzymatic activity through the use of chemical chaperones, hepatocyte cell transplantation, or enzyme replacement by recombinant gene therapy. These novel approaches illustrate the goal for the ideal treatment of PH: correcting the genetic defect without exposing patients to the life-long risks associated with organ transplantation.

Adaptive Changes of Sodium and Potassium Transporters in CRF Rat Kidneys

In chronic renal failure (CRF), residual nephrons can magnify their excretion of sodium and potassium. However, long‐term adaptive mechanism of renal tubular transporters in CRF has not been fully evaluated. We performed immunoblotting and immunocytochemistry, thus investigating the sequential changes of expression of major renal sodium and potassium transporters in rats from 4 weeks to 12 weeks after 5/6 nephrectomy (Nx). At 4 weeks, the density of NKCC2, NCC, ENaC‐α, and ENaC‐γ in CRF rats significantly increased (477%, 222%, 451%, and 435% of control, respectively) although the expression of NHE3 and SGLT1 did not altered. In contrast, expression of NKCC2 and NCC markedly decreased at 12 weeks (55.4% and 30.8%, respectively), and NHE3 and SGLT1 also decreased at 12 weeks (48.4% and 24.6%, respectively). The protein abundance of ENaC‐α significantly increased during the whole period. Urinary potassium excretion rate was well‐correlated with urinary urea or sodium excretion rate, although immunoblotting of ROMK and Na‐K‐ATPase showed a gradual decrease in the late stages of CRF. We suggest that this sequential decrease of major renal sodium transporters may be a main mechanism for increased urinary sodium excretion, and that increased urea excretion rates or distal sodium delivery may be a major explanation for increased urinary potassium excretion in CRF rats.

Molecular variants of the thiazide-sensitive Na+-Cl− cotransporter in hypertensive families

The thiazide-sensitive Na-Cl cotransporter (TSC) is involved in the fine regulation of sodium excretion by the kidney, and an increase in its activity causes salt-sensitive hypertension and hypercalciuria. To explore the possibility that activating mutations in the TSC gene may be involved in the pathogenesis of primary hypertension, we screened genomic DNA of 68 individuals from hypertensive families of patients with hypercalciuria for variations using single-strand conformation polymorphism and investigated the functional properties using the Xenopus laevis oocyte expression system. We identified 18 variants in the TSC and, of those, four were nonsynonymous. The A728T, R904Q and R919C variants were introduced into the human TSC cDNA and expressed in Xenopus oocytes to study their effect on Na transport. Although T728 and Q904 did not show any difference in Na uptake, the R919C mutant showed a 60% increase compared to wild-type human TSC and an exaggerated inhibitory action of hydrochlorothiazide. Immunocytochemical analysis revealed no difference in cell surface expression of R919C mutants compared to wild-type hTSC. There was no significant difference in the inhibitory effect of the carboxyl terminus of the serine-threonine kinase WNK4 on the R919C mutant and wild-type hTSC. Our results show that the substitution of arginine for cysteine at position 919 of TSC increases Na transport function, and provide support for the hypothesis that mutations in renal tubular sodium transporters may contribute to the development of primary hypertension, a polygenic disorder, by increasing renal sodium reabsorption.

Regulation of renal ion transport by the calcium-sensing receptor: an update

Extracellular calcium has profound effects on renal tubular transport, presumably via the calcium-sensing receptor, which is expressed in all nephron segments, but its effects in specific segments and the mechanism of regulation of transport are not fully understood. Recognition that activating calcium-sensing receptor mutations result in a Bartter-like syndrome demonstrate that the transport effects of extracellular calcium are mediated by the calcium-sensing receptor. Its presence in the gills and solute and water-transporting organs of fish coupled with appropriate calcium-sensing receptor kinetics indicate that the calcium-sensing receptor was originally involved in the regulation of sodium chloride, calcium and magnesium transport. Based on its physiological effects on tubular transport and biochemical and genetic data, the calcium-sensing receptor appears to act by mechanisms that distinguish it from other G protein-coupled receptors. The calcium-sensing receptor mediates the effects of extracellular calcium on the kidney, is an essential control point in the regulation of calcium balance and possibly the physiological regulation of sodium chloride balance. The thick ascending limb of Henle and distal convoluted tubule appear to be the nephron segments most responsible for the effects of the calcium-sensing receptor, although its mechanisms of action are not fully established.

Possible involvement of organic anion and cation transporters in renal excretion of xanthine derivatives, 3-methylxanthine and enprofylline

Whether organic anion and cation transporters are involved in the renal excretion of xanthine derivatives, 3-methylxanthie and enprofylline, remains unclear. In this study, we have investigated the effects of typically predominant substrates for organic anion and cation transporters on the tubular secretion of 3-methylxanthine and enprofylline in rats. In the renal clearance experiments using typical substrates for organic anion transporters, probenecid and p-aminohippurate, probenecid (20 mg/kg), but not p-aminohippurate (100 mg/kg), significantly decreased the renal clearance and clearance ratio of 3-methylxanthine and enprofylline. The typical substrates for organic cation transport systems, tetraethylammonium (30.6 mg/kg) and cimetidine (50 or 100 mg/kg), significantly decreased the renal clearance and clearance ratio of 3-methylxanthine and enprofylline. These results suggest that the renal secretory transport of 3-methylxanthine and enprofylline are mediated by probenecid-, cimetidine- and tetraethylammonium-sensitive transport systems. Uric acid, an organic anion, significantly inhibited the renal secretion of 3-methylxanthine, but not enprofylline, suggesting that the renal tubular transport of 3-methylxanthine is also mediated via uric acid-sensitive transport system. These findings suggest the possibility that both organic anion and cation transporters are, at least, involved in the renal tubular transport of 3-methylxanthine and enprofylline in rats.