Exacerbated brain edema in a rat streptozotocin model of hyperglycemic ischemic stroke: Evidence for involvement of blood-brain barrier Na-K-Cl cotransport and Na/H exchange.

Cerebral edema is exacerbated in diabetic ischemic stroke through poorly understood mechanisms. We showed previously that blood-brain barrier (BBB) Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) are major contributors to edema formation in normoglycemic ischemic stroke. Here, we investigated whether hyperglycemia-exacerbated edema involves changes in BBB NKCC and NHE expression and/or activity and whether inhibition of NKCC or NHE effectively reduces edema and injury in a type I diabetic model of hyperglycemic stroke. Cerebral microvascular endothelial cell (CMEC) NKCC and NHE abundances and activities were determined by Western blot, radioisotopic flux and microspectrofluorometric methods. Cerebral edema and Na in rats subjected to middle cerebral artery occlusion (MCAO) were assessed by nuclear magnetic resonance methods. Hyperglycemia exposures of 1-7d significantly increased CMEC NKCC and NHE abundance and activity. Subsequent exposure to ischemic factors caused more robust increases in NKCC and NHE activities than in normoglycemic CMEC. MCAO-induced edema and brain Na uptake were greater in hyperglycemic rats. Intravenous bumetanide and HOE-642 significantly attenuated edema, brain Na uptake and ischemic injury. Our findings provide evidence that BBB NKCC and NHE contribute to increased edema in hyperglycemic stroke, suggesting that these Na transporters are promising therapeutic targets for reducing damage in diabetic stroke.

Altered expression of Na(+)/K(+)-ATPase and other osmoregulatory genes in the gills of euryhaline animals in response to salinity transfer: a meta-analysis of 59 quantitative PCR studies over 10 years.

Recent advances in molecular techniques have allowed gene expression in euryhaline animals to be quantified during salinity transfers. As these investigations transition from studying single genes to utilizing genomics-based methodologies, it is an appropriate time to summarize single gene studies. Therefore, a meta-analysis was performed on 59 published studies that used quantitative polymerase chain reaction (qPCR) to examine expression of osmoregulatory genes (the Na(+)/K(+)-ATPase, NKA; the Na(+)/K(+)/2Cl(-) cotransporter, NKCC; carbonic anhydrase, CA; the cystic fibrosis transmembrane regulator, CFTR; and the H(+)-ATPase, HAT) in response to salinity transfer. Based on 887 calculated effect sizes, NKA, NKCC, CA, and HAT are up-regulated after salinity transfer, while surprisingly, CFTR is unchanged. Meta-analysis also identified influential factors contributing to these changes. For example, expression was highest: 1) during transfers from higher to lower salinities comprising a physiological transition from osmoconformity to osmoregulation, 2) 1-3 days following transfer, 3) during dissimilar transfers, and 4) in crustaceans rather than teleosts. Methodological characteristics (e.g., types of controls) were not important. Experiments lacking in the current literature were also identified. Meta-analyses are powerful tools for quantitatively synthesizing a large body of literature, and this report serves as a template for their application in other areas of comparative physiology.

Na-K-2Cl cotransporter and aquaporin 1 in arachnoid granulations, meningiomas, and meningiomas invading dura.

Meningioma invasion of the dura may contribute to the high rate of recurrence. Recently, ion channels that affect cell shape and movement have been implicated in cancer invasion. Combined Na-K-2Cl cotransporter (NKCC1) and aquaporin 1 (AQP1) expression in arachnoid granulations and meningiomas with and without dural invasion has not been characterized. Arachnoid granulations associated with dura were collected from 10 adult formalin-fixed dura/leptomeninges. Thirty-four frozen meningiomas were evaluated by Western blot. An additional 58 formalin-fixed, paraffin-embedded meningiomas including 36 World Health Organization grade I, 15 grade II, and 7 grade III meningiomas were evaluated by immunohistochemistry. By Western blot, NKCC1 was found in 17 (100%) of 17 World Health Organization grade I, 13 (87%) of 15 grade II, and both grade III meningiomas. Distinct AQP1 was not detected in the meningioma lysates tested. By immunohistochemistry, extensive NKCC1 but no AQP1 immunoreactivity was detected in the arachnoid granulation cells. Extensive NKCC1 was detected in meningioma cells in 56 and in capillaries in 43 by immunohistochemistry. In those tumors with dural or bone/soft tissue invasion, NKCC1 immunoreactivity was seen in invading cells in all cases and in their capillaries in the majority. AQP1 was detected in meningioma cells in 29 and in capillaries in all. AQP1 was also detected in cells and capillaries invading the dura or bone in 10 and 18 of 18, respectively. This was extensive in all subtypes of meningiomas studied. These findings suggest that NKCC1 and AQP1 participate in meningioma biology and invasion. NKCC1 might be targeted by FDA-approved NKCC1 inhibitors.

Effect of estradiol on the expression of renal sodium transporters in rats.

BACKGROUND: Although estradiol has been thought to perform an important role in blood pressure regulation, the effects of estradiol on the expression of renal sodium transporters are not fully understood. METHODS: Female Sprague-Dawley rats were treated with 17ß-estradiol or vehicle for 10 days after ovariectomy, and after both ovariectomy and adrenalectomy to eliminate the effect of aldosterone. RESULTS: In the ovariectomized (OVX) rats, estradiol decreased the abundance of the Na-K-2Cl cotransporter (NKCC2) (31.5% of control (OVX), p < 0.01), Na-Cl cotransporter (NCC) proteins (40.5% of control (OVX), p < 0.01) and α- and γ-subunits of the epithelial sodium channel (ENaC) (44.7% and 11.0% of control (OVX), p < 0.01). Estradiol also reduced plasma aldosterone levels (OVX + 17ß-estradiol vs. OVX, 116.3 ± 44.4 vs. 184.2 ± 33.4 pmol/l, p < 0.05) and systolic blood pressure (OVX + 17ß-estradiol vs. OVX, 115 ± 4 vs. 132 ± 2 mmHg, p < 0.05). In rats having undergone adrenalectomy and ovariectomy, estradiol did not reduce systolic blood pressure, or the expression of sodium transporters. CONCLUSION: Estradiol decreased systolic blood pressure, plasma aldosterone levels, and the expression of renal sodium transporters. After aldosterone was eliminated, estradiol did not affect blood pressure or the expression of sodium transporters, which indicates that the effect of estradiol on the renal sodium transporters is at least partly influenced by aldosterone.

Expression of heme oxygenase-1 in thick ascending loop of henle attenuates angiotensin II-dependent hypertension.

Kidney-specific induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II) -dependent hypertension, but the relative contribution of vascular versus tubular induction of HO-1 is unknown. To determine the specific contribution of thick ascending loop of Henle (TALH) -derived HO-1, we generated a transgenic mouse in which the uromodulin promoter controlled expression of human HO-1. Quantitative RT-PCR and confocal microscopy confirmed successful localization of the HO-1 transgene to TALH tubule segments. Medullary HO activity, but not cortical HO activity, was significantly higher in transgenic mice than control mice. Enhanced TALH HO-1 attenuated the hypertension induced by Ang II delivered by an osmotic minipump for 10 days (139 ± 3 versus 153 ±2 mmHg in the transgenic and control mice, respectively; P<0.05). The lower blood pressure in transgenic mice associated with a 60% decrease in medullary NKCC2 transporter expression determined by Western blot. Transgenic mice also exhibited a 36% decrease in ouabain-sensitive sodium reabsorption and a significantly attenuated response to furosemide in isolated TALH segments. In summary, these results show that increased levels of HO-1 in the TALH can lower blood pressure by a mechanism that may include alterations in NKCC2-dependent sodium reabsorption.

Rare mutations in SLC12A1 and SLC12A3 protect against hypertension by reducing the activity of renal salt cotransporters.

OBJECTIVES: Screening for variants in SLC12A1 and SLC12A3 genes, encoding the renal Na:Cl (NCC) and Na:K:2Cl (NKCC2) cotransporters, respectively, in 3125 members of the Framingham Heart Study (FHS) revealed that carrying a rare mutation in one of these genes was associated with a significant reduction in blood pressure, in the risk of arterial hypertension, and of death due to cardiovascular disease. Because near 60% of the rare mutations identified have not been related to Bartter's or Gitelman's disease, the consequence of such mutations on cotransporter activity is unknown. METHODS: We used the heterologous expression system of Xenopus laevis oocytes, microinjected with wild-type or mutant NCC or NKCC2 cRNAs, to examine the effect of these inferred NCC and NKCC2 mutations on the cotransporters' functional properties. Cotransporter activity was defined as the diuretic-sensitive radioactive tracer uptake and response to known modulators was assessed. RESULTS: Basal NCC activity was significantly reduced in all NCC mutants and, excluding NCC-S186F, response to WNK3, WNK4, or intracellular chloride depletion was conserved. Similarly, basal activity was reduced in six out of nine NKCC2 mutants and response to WNK3 was maintained. No effect on protein expression was seen, except for NCC-S186F, which was significantly reduced. CONCLUSIONS: The rare NCC or NKCC2 mutations found in the FHS significantly reduced the basal activity of the cotransporters. This observation supports that even a small, but chronic reduction of NCC or NKCC2 function results in a lower blood pressure and decreased risk of hypertension in otherwise healthy individuals in the general population.

Calcium-dependent diuretic system in preascitic liver cirrhosis.

BACKGROUND & AIMS: Extracellular Ca(++) activates cell membrane calcium-sensing receptors (CaRs), leading to renal tubule production of prostaglandins E(2) (PGE(2)), which decrease both sodium reabsorption in the thick ascending limb of Henle's loop and free-water reabsorption in collecting ducts. AIMS & METHODS: To assess the activity of this diuretic system in experimental cirrhosis, we evaluated renal function, hormonal status, PGE(2) urinary excretion, and renal tissue concentrations of Na(+)-K(+)-2Cl(-) co-transporters (BSC-1) and CaRs in three groups of rats: one group of controls receiving 5% glucose solution (vehicle) intravenously and two groups of rats with CCl(4)-induced preascitic cirrhosis receiving either vehicle or 0.5mg i.v. Poly-l-Arginine (PolyAg), a CaR-selective agonist. RESULTS: Compared to controls, cirrhotic rats showed reduced urine volume and sodium excretion (p<0.05). Western blot analysis revealed reduced CaRs and increased BSC-1 protein content in kidneys of cirrhotic rats compared with controls (all p<0.01). PolyAg-treated cirrhotic rats had their urine and sodium excretion returned to normal; PolyAg also increased renal plasma flow, PGE(2) urinary excretion, and free-water clearance in cirrhotic rats (all p<0.01 v. untreated cirrhotic animals). CONCLUSIONS: In preascitic cirrhosis, sodium retention may be linked to down-regulation of renal CaRs and up-regulation of tubular sodium-retaining channels. Calcimimetic drugs normalize preascitic sodium retention.

The detailed localization pattern of Na+/K+/2Cl- cotransporter type 2 and its related ion transport system in the rat endolymphatic sac.

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na(+)/K(+)/2Cl(-) cotransporter type 2 (NKCC-2) and Na(+)/K(+)-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na(+)/K(+)-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na(+)/K(+)-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules.

Expression of aquaporin1, 3, and 4, NKCC1, and NKCC2 in the human endolymphatic sac.

OBJECTIVE: To locate aquaporin (AQP) 1, 3, and 4, Na-K-2Cl cotransporter (NKCC) 1 and 2 in the human endolymphatic sac (ES). METHODS: A sample of human ES was harvested during the removal of vestibular schwannoma via the translabyrinthine approach. The sample was immediately fixed in 4% paraformaldehyde and embedded in OCT compound. Immunohistochemistry was performed with AQP1, 3, and 4, NKCC1, and NKCC2 polyclonal antibodies. RESULTS: AQP1, AQP3, and NKCC2 were strongly expressed in the epithelial layer of the ES. AQP4 and NKCC1 were weakly expressed in the epithelial layer of the ES. CONCLUSIONS: As it is impossible to perform quantitative analysis based on the fluorescence intensity of each immunoreactivity, we have presented the existence of AQP1, 3, and 4, NKCC1, and NKCC2 in the ES. The expression of NKCC1 and 2 indicated that the ES may have both secretory and adsorptive functions to maintain the homeostasis of endolymph.

Reversal of renal tubule transporter downregulation during severe leptospirosis with antimicrobial therapy.

Tubular dysfunction is a hallmark of severe leptospirosis. Antimicrobial therapy is thought to interfere on renal involvement. We evaluated the expression of a proximal tubule type-3 Na+/H+ exchanger (NHE3) and a thick ascending limb Na+-K+-2Cl(-) cotransporter (NKCC2) in controls and treated hamsters. Animals infected by a serovar Copenhageni isolate, were treated or not with ampicillin (AMP) and/or N-acetylcysteine (NAC). Leptospiral antigen(s) and expression of renal transporters were evaluated by immunohistochemistry, and serum thiobarbituric acid (TBARS) was quantified. Infected hamsters had high amounts of detectable leptospiral antigen(s) in target tissues while renal expression of NHE3 and NKCC2 decreased. Ampicillin treatment was associated with minimal or no detection of leptospiral antigens, normal expression of NHE3 and NKCC2 transporters, and reduced levels of TBARS. NAC effect was restricted to lowering TBARS. Early and late AMP treatment rescued tubular defects in severe leptospirosis disease, and there was no evidence of benefit from antioxidant therapy.

Late-onset manifestation of antenatal Bartter syndrome as a result of residual function of the mutated renal Na+-K+-2Cl- co-transporter.

Genetic defects of the Na+-K+-2Cl- (NKCC2) sodium potassium chloride co-transporter result in severe, prenatal-onset renal salt wasting accompanied by polyhydramnios, prematurity, and life-threatening hypovolemia of the neonate (antenatal Bartter syndrome or hyperprostaglandin E syndrome). Herein are described two brothers who presented with hyperuricemia, mild metabolic alkalosis, low serum potassium levels, and bilateral medullary nephrocalcinosis at the ages of 13 and 15 yr. Impaired function of sodium chloride reabsorption along the thick ascending limb of Henle's loop was deduced from a reduced increase in diuresis and urinary chloride excretion upon application of furosemide. Molecular genetic analysis revealed that the brothers were compound heterozygotes for mutations in the SLC12A1 gene coding for the NKCC2 co-transporter. Functional analysis of the mutated rat NKCC2 protein by tracer-flux assays after heterologous expression in Xenopus oocytes revealed significant residual transport activity of the NKCC2 p.F177Y mutant construct in contrast to no activity of the NKCC2-D918fs frameshift mutant construct. However, coexpression of the two mutants was not significantly different from that of NKCC2-F177Y alone or wild type. Membrane expression of NKCC2-F177Y as determined by luminometric surface quantification was not significantly different from wild-type protein, pointing to an intrinsic partial transport defect caused by the p.F177Y mutation. The partial function of NKCC2-F177Y, which is not negatively affected by NKCC2-D918fs, therefore explains a mild and late-onset phenotype and for the first time establishes a mild phenotype-associated SLC12A1 gene mutation.

Aminoglycoside ototoxicity in three murine strains and effects on NKCC1 of stria vascularis.

BACKGROUND: After establishing a murine model of aminoglycoside antibiotic (AmAn) induced ototoxicity, the sensitivity of AmAn induced ototoxicity in three murine strains and the effect of kanamycin on the expression of Na-K-2Cl cotransporter-1 (NKCC1) in stria vascularis were investigated. METHODS: C57BL/6J, CBA/CaJ, NKCC1(+/-) mice (24 of each strain) were randomly divided into four experimental groups: A: kanamycin alone; B: kanamycin plus 2, 3-dihydroxybenzoate; C: 2, 3-dihydroxybenzoate alone; and D: control group. Mice were injected with kanamycin or/and 2, 3-dihydroxybenzoate twice daily for 14 days. Auditory brainstem response (ABR) was measured and morphology of cochlea delineated with succinate dehydrogenase staining. Expression of NKCC1 in stria vascularis was detected immunohistochemically. RESULTS: All three strains in groups A and B developed significant ABR threshold shifts (P < 0.01), which were accompanied by outer hair cell loss. NKCC1 expression in stria vascularis was the weakest in group A (A cf D, P < 0.01) and the strongest in groups C and D (P < 0.05). CBA/CaJ mice had the highest sensitivity to AmAn. CONCLUSIONS: Administration of kanamycin established AmAn induced ototoxicity. Kanamycin inhibited the expression of NKCC1 in stria vascularis. 2, 3-dihydroxybenzoate attenuated AmAn induced ototoxicity-possibly by enhancing the expression of NKCC1. Age related hearing loss did not show additional sensitivity to AmAn induced ototoxicity in murine model.

Na+-K+-2Cl- cotransporters and Cl- channels regulate citric acid cough in guinea pigs.

Loop diuretics have been shown to inhibit cough and other airway defensive reflexes via poorly defined mechanisms. We test the hypothesis that the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC1) is expressed by sensory nerve fibers innervating the airways where it plays an important role in regulating sensory neural activity. NKCC1 immunoreactivity was present on the cell membranes of most nodose and jugular ganglia neurons projecting to the trachea, and it was present on the peripheral terminals of putative mechanosensory nerve fibers in the airways. In urethane-anesthetized, spontaneously breathing guinea pigs, bolus application of citric acid (1 mM to 2 M) to an isolated and perfused segment of the tracheal mucosa evoked coughing and respiratory slowing. Removal of Cl- from the tracheal perfusate evoked spontaneous coughing and significantly potentiated cough and respiratory slowing reflexes evoked by citric acid. The NKCC1 inhibitor furosemide (10-100 microM) significantly reduced both the number of coughs evoked by citric acid and the degree of acid-evoked respiratory slowing (P < 0.05). Localized tracheal pretreatment with the Cl- channel inhibitors DIDS or niflumic acid (100 microM) also significantly reduced cough, whereas the GABAA receptor agonist muscimol potentiated acid-evoked responses. These data suggest that vagal sensory neurons may accumulate Cl- due to the expression of the furosemide-sensitive Cl- transporter, NKCC1. Efflux of intracellular Cl-, in part through calcium-activated Cl- channels, may play an important role in regulating airway afferent neuron activity.

Mice heterozygous for beta-ENaC deletion have defective potassium excretion.

The present studies were designed to determine whether mice heterozygous for deletion of beta-ENaC exhibited defects in Na+/K+ transport and blood pressure regulation. In response to an acute KCl infusion, +/-mice developed higher serum [K+] and excreted only 40% of the K+ excreted by +/+mice. After 6 days on a low (0.01%)-Na+ diet, the cumulative Na+ excretion from days 3-6 was greater for +/-mice. This low-Na+ diet caused higher serum [K+] and lower K+ excretion rates in +/-mice than in +/+mice, but the rectal potential differences were not different. Analyses of mRNA from mice on this diet showed the expected approximately 50% reduction of beta-ENaC in kidney and colon of +/-mice. Unexpectedly, the level of gamma-ENaC mRNA was similarly reduced. NHE3 mRNA was approximately 30% higher in +/-mice whereas mRNA of the Na-K-2Cl cotransporter was not different. Also unexpectedly, the amount of beta-ENaC proteins was similar in both groups of mice but there was a reduction of one form of gamma-ENaC in +/-mice. These experiments demonstrate that mice heterozygous for beta-ENaC have a small but detectable defect in their ability to conserve Na+ and a more readily apparent defect in the ability to secrete K+.

17-beta Estradiol attenuates streptozotocin-induced diabetes and regulates the expression of renal sodium transporters.

Diabetes mellitus is associated with natriuresis, whereas estrogen has been shown to be renoprotective in diabetic nephropathy and may independently regulate renal sodium reabsorption. The aim of this study was to determine the effects of 17-beta estradiol (E(2)) replacement to diabetic, ovariectomized (OVX) female rats on the expression of major renal sodium transporters. Female, Sprague-Dawley rats (210 g) were randomized into four groups: (1) OVX; (2) OVX+E(2); (3) diabetic+ovariectomized (D+OVX); and (4) diabetic+ovariectomized+estrogen (D+OVX+E(2)). Diabetes was induced by a single intraperitoneal injection of streptozotocin (55 mg/kg.body weight (bw)). Rats received phytoestrogen-free diet and water ad libitum for 12 weeks. E(2) attenuated hyperglycemia, hyperalbuminuria, and hyperaldosteronism in D rats, as well as the diabetes-induced changes in renal protein abundances for the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), and the alpha- and beta-subunits of the epithelial sodium channel (ENaC), that is, E(2) decreased NKCC2, but increased alpha- and beta-ENaC abundances. In nondiabetic rats, E(2) decreased plasma K(+) and increased urine K(+)/Na(+) ratio, as well as decreased the abundance of NKCC2, beta-ENaC, and alpha-1-Na-K-adenosine triphosphate (ATP)ase in the outer medulla. Finally, the diabetic, E(2) rats had measurably lower final circulating levels of E(2) than the nondiabetic E(2) rats, despite an identical replacement protocol, suggesting a shorter biological half-life of E(2) with diabetes. Therefore, E(2) attenuated diabetes and preserved renal sodium handling and related transporter expression levels. In addition, E(2) had diabetes-independent effects on renal electrolyte handling and associated proteins.

Renal ENaC subunit, Na-K-2Cl and Na-Cl cotransporter abundances in aged, water-restricted F344 x Brown Norway rats.

Renal sodium reabsorption is a key determinant of final urine concentration. Our aim was to determine whether differences existed between aged and young rats in their response to water restriction with regard to the regulation of abundance of any of the major distal renal sodium transporter proteins. Male Fisher 344 x Brown Norway (F344 x BN) rats of 3-, 10-, 24-, or 31 months of age (3M, 10M, 24M, or 31M) were either water restricted (WR) for 5 days or control (ad libitum water). Major renal sodium transporters and channel subunits were evaluated by immunoblotting and immunohistochemistry. Age did not significantly affect plasma arginine vasopressin or aldosterone levels, but renin activity was only 8% in 31M-WR rats relative to 3M-WR (P<0.05). Extreme aging (31M) led to decreased outer medullary abundance of the bumetanide-sensitive Na-K-2Cl cotransporter and decreased cortical abundance of the beta- and gamma-subunits (70-kDa band) of the epithelial sodium channel (ENaC) (P<0.05). Water restriction significantly (P<0.05) increased the abundance of Na-K-2Cl cotransporter (NKCC2) and Na-Cl cotransporter (NCC) across ages. However, these increases were significantly blunted as rats aged. Mean band densities were increased in WR rats (relative to age controls) by 54 and 106% at 3M, but only 25 and 29% at 24M and 0 and 6% at 31M for NKCC2 and NCC, respectively. Aged F344 x BN rats have reduced basal distal tubular renal sodium transporter abundances and blunted upregulation during water restriction, which may contribute to decreased urinary concentrating capacity.

Biphasic changes of epithelial sodium channel abundance and trafficking in common bile duct ligation-induced liver cirrhosis.

We hypothesize that dysregulation of the epithelial sodium channel (ENaC) may be responsible for the increased sodium retention in liver cirrhosis. Liver cirrhosis was induced by common bile duct ligation (CBDL). We examined the abundance of ENaC subunits and type 2 isoform of 11beta-hydroxysteroid dehydrogenase (11betaHSD2) in the kidney by immunoblotting and immunohistochemistry at 6 or 8 weeks after operation. At 6 weeks, cirrhotic rats had developed ascites and displayed a positive sodium balance. The urinary sodium excretion and fractional excretion of sodium were decreased, while plasma aldosterone was unchanged. The abundance of ENaC subunits was not changed in the cortex and outer stripe of the outer medulla (OSOM). In contrast, immunoperoxidase microscopy revealed an increased apical targeting of alpha-, beta- and gammaENaC in late distal convoluted tubule, connecting tubule and collecting duct. Moreover, 11betaHSD2 abundance was decreased in the cortex/OSOM and inner stripe of the outer medulla. At 8 weeks, urinary sodium excretion and fractional excretion of sodium were not changed, while the plasma aldosterone level was decreased. The expression of ENaC subunits was decreased in the cortex/OSOM. Immunoperoxidase microscopy confirmed decreased expression of ENaC subunits, whereas subcellular localization was not changed. These results suggest that increased apical targeting of ENaC subunits and diminished abundance of 11betaHSD2 may contribute to promote sodium retention in the sodium-retaining stage of liver cirrhosis (at 6 weeks). The subsequent decreased expression and reduced targeting of ENaC subunits may play a role in promoting sodium excretion in the later stage of liver cirrhosis (at 8 weeks).

Renal and endocrine changes in rats with inherited stress-induced arterial hypertension (ISIAH).

Hypertensive inbred rats (ISIAH; inherited stress-induced arterial hypertension) present with baseline hypertension (>170 mmHg in adult rats), but attain substantially higher values upon mild emotional stress. We aimed to characterize key parameters related to hypertension in ISIAH. Kidneys, adrenals, and systemic endocrine parameters were studied in ISIAH of different ages and compared to normotensive Wistar albino Glaxo (WAG) rats. Native organs were obtained for Western and PCR analysis. Perfusion-fixed organs were prepared for histopathology and quantitative histochemistry. Plasma renin and adrenal hormones were measured. Renal morphology was unaltered in ISIAH. The hypothalamic-pituitary-adrenocortical (HPA) axis was constitutively upregulated with enlarged adrenal cortices and enhanced plasma corticosterone levels. Plasma renin activity was not different between groups, whereas aldosterone levels were in part reduced. Juxtaglomerular NO synthase type 1, cyclooxygenase type 2, and renin expression were significantly reduced, whereas tubular gene products related to sodium transport (bumetanide-sensitive Na, K, 2Cl cotransporter type 2; thiazide-sensitive Na, Cl cotransporter; epithelial Na channel-alpha; 11beta-hydroxysteroid dehydrogenase type 2) were increased. These data suggest enhanced volume conservation by the kidney. Our data define ISIAH as an attractive model for the renal components determining salt and water homeostasis in hypertension. The specific condition of a basally stimulated HPA axis is highlighted, including the option to study effects superimposed by emotional stress.

The fine-structural distribution of G-protein receptor kinase 3, beta-arrestin-2, Ca2+/calmodulin-dependent protein kinase II and phosphodiesterase PDE1C2, and a Cl(-)-cotransporter in rodent olfactory epithelia.

The sequentially activated molecules of olfactory signal-onset are mostly concentrated in the long, thin distal parts of olfactory epithelial receptor cell cilia. Is this also true for molecules of olfactory signal-termination and -regulation? G-protein receptor kinase 3 (GRK3) supposedly aids in signal desensitization at the level of odor receptors, whereas beta-arrestin-2, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and phosphodiesterase (PDE) PDE1C2 are thought to do so at the level of the adenylyl cyclase, ACIII. The Na+, K(+)-2Cl(-)-cotransporter NKCC1 regulates Cl(-)-channel activity. In an attempt to localize the subcellular sites olfactory signal-termination and -regulation we used four antibodies to GRK3, two to beta-arrestin-2, five to CaMKII (one to both the alpha and beta form, and two each specific to CaMKII alpha and beta), two to PDE1C2, and three to Cl(-)-cotransporters. Only antibodies to Cl(-)-cotransporters labeled cytoplasmic compartments of, especially, supporting cells but also those of receptor cells. For all other antibodies, immunoreactivity was mostly restricted to the olfactory epithelial luminal border, confirming light microscopic studies that had shown that antibodies to GRK3, beta- arrestin-2, CaMKII, and PDE1C2 labeled this region. Labeling did indeed include receptor cell cilia but occurred in microvilli of neighboring supporting cells as well. Apical parts of microvillous cells that are distinct from supporting cells, and also of ciliated respiratory cells, immunoreacted slightly with most antibodies. When peptides were available, antibody preabsorption with an excess of peptide reduced labeling intensities. Though some of the antibodies did label apices and microvilli of vomeronasal (VNO) supporting cells, none immunoreacted with VNO sensory structures.

A novel splice site mutation of the thiazide-sensitive NaCl cotransporter gene in a Japanese patient with Gitelman syndrome.

Gitelman syndrome (GS, MIM 263800) is an inherited disorder characterized by metabolic alkalosis with hypokalemia, hypomagnesemia, and hypocalciuria. The genetic abnormalities causing GS are known to lie in the thiazide-sensitive NaCl cotransporter (TSC), which is expressed in the distal tubule of the kidney. The TSC gene, located at chromosome 16, consists of 26 exons and encodes the protein containing 12 putative transmembrane domains with long intracellular amino and carboxy termini. Most of the abnormalities identified in GS were missense mutations, distributed throughout the TSC gene without a hot spot. A 42-year-old Japanese man was introduced for close examination of hypokalemia. In renal clearance studies using furosemide or thiazide, chloride clearance was increased after furosemide but not after thiazide administration. Furthermore, the distal fractional chloride reabsorption was dramatically decreased by furosemide but not thiazide administration, suggesting a defect in the distal tubule. We then analyzed the TSC gene to confirm the diagnosis of GS, and identified a novel G to T mutation at the acceptor splice site preceding exon 14, resulting in disruption of a conventional 3'AG consensus splice site. Abnormal splicing by this mutation is predicted to cause the formation of truncated TSC with a partial deletion of the transmembrane domain, which will loose the function of transporter. In conclusion, we have identified a unique novel splice site mutation of the TSC gene in GS. The predicted structure of this mutant TSC can conceivably cause an impairment of the transporter activity and thereby be responsible for the development of GS in our patient.