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Author(s): Ella C Perrin [sup.1] , Andrew M South [sup.2] [sup.3] [sup.4] [sup.5] Author Affiliations: (1) grid.241167.7, 0000 0001 2185 3318, Wake Forest School of Medicine, , Winston Salem, NC, [...], Background Urine sodium and potassium are used as surrogate markers for dietary consumption in adults with hypertension, but their role in youth with hypertension and their association with components of the renin-angiotensin-aldosterone system (RAAS) are incompletely characterized. Some individuals with hypertension may have an abnormal RAAS response to dietary sodium and potassium intake, though this is incompletely described. Our objective was to investigate if plasma renin activity and serum aldosterone are associated with urine sodium and potassium in youth referred for hypertensive disorders. Methods This pilot study was a cross-sectional analysis of baseline data from 44 youth evaluated for hypertensive disorders in a Hypertension Clinic. We recorded urine sodium and potassium concentrations normalized to urine creatinine, plasma renin activity, and serum aldosterone and calculated the sodium/potassium (UNaK) and aldosterone/renin ratios. We used multivariable generalized linear models to estimate the associations of renin and aldosterone with urine sodium and potassium. Results Our cohort was diverse (37% non-Hispanic Black, 14% Hispanic), 66% were male, and median age was 15.3 years; 77% had obesity and 9% had a secondary etiology. Aldosterone was associated inversely with urine sodium/creatinine ([beta]: -0.34, 95% CI -0.62 to -0.06) and UNaK ([beta]: -0.09, 95% CI -0.16 to -0.03), and adjusted for estimated glomerular filtration rate and serum potassium. Conclusions Higher serum aldosterone levels, but not plasma renin activity, were associated with lower urine sodium/creatinine and UNaK at baseline in youth referred for hypertensive disorders. Further characterization of the RAAS could help define hypertension phenotypes and guide management. Graphical abstract

Aldosterone and corticosterone bind to mineralocorticoid (MR) and glucocorticoid receptors (GR), which, upon ligand binding, are thought to translocate to the cell nucleus to act as transcription factors. Mineralocorticoid selectivity is achieved by the 11 [beta]-hydroxysteroid dehydrogenase type 2 (11[beta]-HSD2) that inactivates 1l[beta]-hydroxy glucocorticoids. High expression levels of 11[beta]-HSD2 characterize the aldosterone-sensitive distal nephron (ASDN), which comprises the segment-specific cells of late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct (CD). We used MR- and GR-specific antibodies to study localization and regulation of MR and GR in kidneys of rats with altered plasma aldosterone and corticosterone levels. In control rats, MR and GR were found in cell nuclei of thick ascending limb (TAL), DCT, CNT, CD cells, and intercalated cells (IC). GR was also abundant in cell nuclei and the subapical compartment of proximal tubule (PT) cells. Dietary NaCI loading, which lowers plasma aldosterone, caused a selective removal of GR from cell nuclei of 11[beta]-HSD2-positive ASDN. The nuclear localization of MR was unaffected. Adrenalectomy (ADX) resulted in removal of MR and GR from the cell nuclei of all epithelial cells. Aldosterone replacement rapidly relocated the receptors in the cell nuclei. In ASDN cells, low-dose corticosterone replacement caused nuclear localization of MR, but not of GR. The GR was redistributed to the nucleus only in PT, TAL, early DCT, and IC that express no or very little 11 [beta]-HSD2. In ASDN cells, nuclear GR localization was only achieved when corticosterone was replaced at high doses. Thus ligand-induced nuclear translocation of MR and GR are part of MR and GR regulation in the kidney and show remarkable segment- and cell type-specific characteristics. Differential regulation of MR and GR may alter the level of heterodimerization of the receptors and hence may contribute to the complexity of corticosteroid effects on ASDN function. aldosterone; corticosterone; 11[beta]-hydroxysteroid dehydrogenase type 2 doi: 10.1152/ajprenal.00437.2010

The renal thiamin reabsorption process plays an important role in regulating thiamin body homeostasis and involves both thiamin transporters-1 and -2 (THTR1 and THTR2). Chronic alcohol use is associated with thiamin deficiency. Although a variety of factors contribute to the development of this deficiency, effects of chronic alcohol use on renal thiamin transport have not been thoroughly examined. We addressed this issue by examining the effect of chronic alcohol feeding of rats with liquid diet on physiological and molecular parameters of renal thiamin transport. Chronic alcohol feeding caused a significant inhibition in carrier-mediated thiamin transport across the renal brush-border membrane and was evident as early as 2 wk after initiation of alcohol feeding. Similarly, thiamin transport across the renal basolateral membrane was significantly inhibited by chronic alcohol feeding. The inhibition in renal thiamin transport was associated with a marked decrease in the level of expression of THTR1 and -2 proteins, mRNAs, and heterogeneous nuclear RNAs. Chronic alcohol feeding also caused a significant reduction in the level of expression of thiamin pyrophosphokinase but not that of the mitochondrial thiamin pyrophosphate transporter. These studies show that chronic alcohol feeding inhibits the entry and exit of thiamin in the polarized renal epithelial cells and that the effect is, at least in part, mediated at the transcriptional level. These findings also suggest that chronic alcohol feeding interferes with the normal homeostasis of thiamin in renal epithelial cells. transporter; vitamin B1; kidney; thiamin transporter-l; thiamin transporter-2 doi: 10.1152/ajprenal.00140.2010.

Our recent study (Prathapasinghe GA, Siow YL, O K. Am J Physiol Renal Physiol 292: F1354-F1363, 2007) indicates that homocysteine (Hcy) plays a detrimental role in ischemia-reperfusion-induced renal injury. Elevation of renal Hcy concentration during ischemia-reperfusion is attributed to reduced activity of cystathionine-[beta]-synthase (CBS) that catalyzes the rate-limiting step in the transsulfuration pathway for the metabolism of the majority of Hcy in the kidney. However, the mechanisms of impaired CBS activity in the kidney are unknown. The aim of this study was to investigate the effects of pH and nitric oxide (NO) on the CBS activity in the kidney during ischemia-reperfusion. The left kidney of a Sprague-Dawley rat was subjected to ischemia-reperfusion. The CBS activity was significantly reduced in kidneys subjected to ischemia alone (15-60 rain) or subjected to ischemia followed by reperfusion for 1-24 h. The pH was markedly reduced in kidneys upon ischemia. Injection of alkaline solution into the kidney partially restored the CBS activity during ischemia. Further analysis revealed that reduction of CBS activity during reperfusion was accompanied by an elevation of NO metabolites (nitrate and nitrite) in the kidney tissue. Injection of a NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline- 1-oxyl-3-oxide (PTIO), restored the CBS activity in the kidneys subjected to ischemia-reperfusion. Treatment with PTIO could abolish ischemiareperfusion-induced lipid peroxidation and prevent cell death in the kidney. These results suggested that metabolic acidosis during ischemia and accumulation of NO metabolites during reperfusion contributed, in part, to reduced CBS activity leading to an elevation of renal Hcy levels, which in turn, played a detrimental role in the kidney. homocysteine; oxidative stress; apoptosis

Impedance of renal vascular smooth muscle cells (VSMCs) cultured on microelectrodes was measured by electric cell-substrate impedance sensing. Changes in measured impedance as a function of frequency were compared with the calculated values obtained from an extended cell-electrode model to estimate the junctional resistance, distance between the ventral cell surface and the substratum, and apical and basolateral membrane capacitances of renal VSMCs. This cell-electrode model was derived to accommodate the slender and rectangular shape of VSMCs. The calculated changes in impedance ([Z.sub.cal]) based on the model agreed well with the experimental measurement ([Z.sub.exp]), and the percentage error defined as |([Z.sub.cal] - [Z.sub.exp])/[Z.sub.exp]| was 1.0%. To test the sensitivity of the new model for capturing changes in cell-cell and cell-substrate interactions induced by changes in cellular environment, we then applied this model to analyze timpedance changes induced by an integrin binding peptide in renal VSMCs. Our result demonstrates that integrin binding peptide decreases junctional resistance between cells, increases the distance between the basolateral cell surface and substratum, and increases the apical membrane capacitance, whereas the basolateral membrane capacitance stays relatively stable. This model provides a generic approach for impedance analysis of cell layers composed of slender, rectangular cells. electric cell-substrate impedance sensing; cell attachment; cell adhesion; extracellular matrix; integrin

Roles of basolateral solute uptake via NKCC1 and of myosin II in vasopressin-induced cell swelling in inner medullary collecting duct. AM J Physiol Renal Physiol 292: F192-F201, 2008. Fist published April 16, 2008: doi:10.1152/ajprenal.00011-2008.--Collecting duct cells swell when exposed to argininge vasopressin (AVP) in the presence of a transepithelial osmolality gradient. We investigated the mechanisms of AVP-induced cell swelling in isolated , perfused rate inner medullary collecting ducts (IMCDs) using quantitative video microscopy and fluorescence-based measurements of transepithelial water transport. We tested the roles of transepithelial water flow, basolateral solute entry, and the cytoskeleton (actomysosin). Whe a transepithelial osmolality gradient was imposed by addition of NaCl to the bath, AVP significantly increased both water flux and cell height. When the osmolality gradient was imposed by addition of mannitol, AVP increased water flux but not cell height, suggesting that AVP-induced cell swelling requires a NaCl gradien and is not merely dependent on the associated water flux. Bumetanide (Na-K-2Cl contransporter inhibitor) added to the bath markedly diminished the AVP-induced cell height increase. AVP-induced cell swelling was absent in IMCDs form NKCC1-knockout mice. In rat IMCDs, replacement of Na, K, or Cl in the peritubular bath cased significant cell shrinkage, consistent with a basolateral solute transport pathway dependent on all three ions. Immunocytochemistry using an antibody to NKCC1 confirmed basolateral expression in IMCD cells. The conventional nonmuscle myosin II inhibitor blebbistatin also diminished the AVP-induced cell height increase and cell shape change, consistent with a role for the actin cytoskeleton and myosin II. We conclude that the AVP-induced cell height increase is dependent on basolateral solute uptake via NKCC1 and changes in actin organization via myosin II, but is not dependent specifically on increased apical water entry. cell volume; blebbistatin; cytoskeleton; kidney; inner medulla

Volume depletion due to persistent glucosuria-induced osmotic diuresis is a significant problem in uncontrolled diabetes mellitus (DM). Angiotensin II receptor blockers (ARBs), such as candesartan, slow the progression of chronic kidney disease in patients with DM. However, mice with genetic knockout of components of the renin-angiotensin system have urine concentrating defects, suggesting that ARBs may exacerbate the volume depletion. Therefore, the effect of candesartan on UT-A1, UT-A3, NKCC2, and aquaporin-2 (AQP2) protein abundances was determined in control and 3-wk DM rats. Aldosterone levels in control rats (0.36 [+ or -] 0.06 nM) and candesartan-treated rats (0.34 [+ or -] 0.14 nM) were the same. DM rats had higher aldosterone levels (1.48 [+ or -] 0.37 nM) that were decreased by candesartan (0.97 [+ or -] 0.26 nM). Western analysis showed that UT-A1 expression was increased in DM rats compared with controls in inner medullary (IM) tip (158 [+ or -] 13%) and base (120 [+ or -] 25%). UT-A3 abundance was increased in IM tip (123 [+ or -] 11%) and base (146 [+ or -] 17%) of DM rats vs. controls. UT-A3 was unchanged in candesartan-treated control rats. In candesartan-treated DM rats, UT-A3 increased in IM tip (160 [+ or -] 14%) and base (210 [+ or -] 19%). Candesartan-treated DM rats had slightly higher AQP2 in IM (46%, P < 0.05) vs. control rats. NKCC2/BSC1 was increased 145 [+ or -] 10% in outer medulla of DM vs. control rats. We conclude that candesartan augments compensatory changes in medullary transport proteins, reducing the losses of solute and water during uncontrolled DM. These changes may represent a previously unrecognized beneficial effect of type 1 ARBs in DM. angiotensin II; UT-A urea transporter; aquaporin; AQP2; NKCC2

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on basolateral 18-pS K channels in the principal cell of the cortical collecting duct (CCD) of the rat kidney. Application of AA inhibited the 18-pS K channels in a dose-dependent manner and 10 [micro]M AA caused a maximal inhibition. The effect of AA on the 18-pS K channel was specific because application of 11,14,17-eicosatrienoic acid had no effect on channel activity. Also, the inhibitory effect of AA on the 18-pS K channels was abolished by blocking cytochrome P-450 (CYP) epoxygenase with N-methyl-sulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) but was not affected by inhibiting CYP [omega]-hydroxylase or cyclooxygenase. The notion that the inhibitory effect of AA was mediated by CYP epoxygenase-dependent metabolites was further supported by the observation that application of 100 nM 11,12-epoxyeicosatrienoic acid (EET) mimicked the effect of AA and inhibited the basolateral 18-pS K channels. In contrast, addition of either 5,6-, 8,9-, or 14, 15-EET failed to inhibit the 18-pS K channels. Moreover, application of 11,12-EET was still able to inhibit the 18-pS K channels in the presence of MS-PPOH. This suggests that 11,12-EET is a mediator for the AA-induced inhibition of the 18-pS K channels. We conclude that AA inhibits basolateral 18-pS K channels by a CYP epoxygenase-dependent pathway and that 11,12-EET is a mediator for the effect of AA on basolateral K channels in the CCD. eicosatrienoic acid; 11,12-epoxyeicosatrienoic acid; basolateral potassium conductance; sodium-potassium-adenosinetriphosphate and sodium transport

A transient ID mathematical model of whole-organ renal autoregulation in the rat is presented, examining the myogenic response on multiple levels of the renal vasculature. Morphological data derived from micro-CT imaging were employed to divide the vasculature via a Strahler ordering scheme. A previously published model of the myogenic response based on wall tension is expanded and adapted to fit the response of each level, corresponding to a distally dominant resistance distribution with the highest contributions localized to the afferent arterioles and interlobular arteries. The mathematical model was further developed to include the effects of in vivo viscosity variation and flow-induced dilation via endothelial nitric oxide production. Computer simulations of the autoregulatory response to pressure perturbations were examined and compared with experimental data. The model supports the hypothesis that change in circumferential wall tension is the catalyst for the myogenic response. The model provides a basis for examining the steady state and transient characteristics of the whole-organ renal myogenic response in the rat, as well as the modulatory influences of metabolic and hemodynamic factors. renal; myogenic response

N-methyl-D-aspartate (NMDA) receptor activated by glutamate/glycine is located in the kidneys. The NMDA receptor subunit NRI is increased in damaged renal tissue. This study explored the role of NMDA receptors in ischemia-reperfusion-induced renal dysfunction in rats. With Western blot analysis and renal functional assay, NMDA receptor expression was evaluated, as well as its functional role in female Wistar rat kidneys after 45 min of unilateral ischemia followed by 24 h of reperfusion. The effects of intrarenal NMDA receptor agonist and antagonist on renal blood flow (RBF), glomerular filtration rate (GFR), urine volume (UV), sodium (UNaV), and potassium (UKV) excretion were determined. NMDA NR1 was present in the glomeruli, brush-border membrane, and outer medulla but not in the cortex and inner medulla. Homogenous distribution of non-NMDA GluR2/3, sparse kainate KAI, and undetectable group I of metabotropic glutamate receptor were noted in the control kidneys. Ischemia-reperfusion kidneys showed enhanced renal NR1, but not NR2C and GluR2/3 expression, and were associated with decreased GFR/RBF and natriuretic/diuretic responses. Intrarenal NMDA agonists significantly reduced GFR, UV, UNaV, and UKV but had no effect on blood pressure and RBF in sham control and ischemia-reperfusion kidneys. NMDA antagonist D-2-amino-5-phosphonopentanoic acid (D-AP-5) treatment completely abolished NMDA-induced renal dysfunction. D-AP-5 treatment significantly ameliorated ischemia-reperfusion-induced glomerular and tubular dysfunction by restoring decreased GFR, UV, and [U.sub.Na]V levels. Ischemia-reperfusion upregulates renal NMDA NR1 receptor expression, leading to reduced glomerular and tubular function in the kidneys. The NMDA antagonist can ameliorate ischemia-reperfusion-induced renal dysfunction. ischemia-reperfusion; kidney; glutamate; renal function

Enhancing perinatal nitric oxide (NO) availability persistently reduces blood pressure in spontaneously hypertensive rats. We hypothesize that this approach can be generalized to other models of genetic hypertension, for instance those associated with renal injury. Perinatal exposure to the NO donor molsidomine was studied in fawn-hooded hypertensive (FHH) rats, a model of mild hypertension, impaired preglomerular resistance, and progressive renal injury. Perinatal molsidomine increased urinary NO metabolite excretion at 8 wk of age, i.e., 4 wk after treatment was stopped (P < 0.05). Systolic blood pressure was persistently reduced after molsidomine (42-wk females: 118 [+ or -] 3 vs. 141 [+ or -] 5 and 36-wk males: 139 [+ or -] 4 vs. 158 [+ or -] 4 mmHg; both P < 0.001). Perinatal treatment decreased glomerular filtration rate (P < 0.05) and renal blood flow (P < 0.01) and increased renal vascular resistance (P < 0.05), without affecting filtration fraction, suggesting persistently increased preglomerular resistance. At 4 wk of age natriuresis was transiently increased by molsidomine (P < 0.05). Molsidomine decreased glomerulosclerosis (P < 0.05). Renal blood flow correlated positively with glomerulosclerosis in control (P < 0.001) but not in perinatally treated FHH rats. NO dependency of renal vascular resistance was increased by perinatal molsidomine. Perinatal enhancement of NO availability can ameliorate development of hypertension and renal injury in FHH rats. Paradoxically, glomerular protection by perinatal exposure to the NO donor molsidomine may be due to persistently increased preglomerular resistance. The mechanisms by which increased perinatal NO availability can persistently reprogram kidney function and ameliorate hypertension deserve further study. proteinuria; renal hemodynamics; glomerulosclerosis

A low expression of angiotensinogen in the heart has been construed as indicating a circulating uptake mechanism to explain the local effects of angiotensin II on tissues. The recent identification of angiotensin(1-12) in an array of rat organs suggests this propeptide may be an alternate substrate for local angiotensin production. To test this hypothesis, tissues from 11-wk-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (n = 14) were stained with purified antibodies directed to the COOH terminus of angiotensin-(112). Robust angiotensin-(1-12) staining was predominantly found in ventricular myocytes with less staining found in the medial layer of intracoronary arteries and vascular endothelium. In addition, angiotensin-(1-12) immunoreactivity was present in the proximal, distal, and collecting renal tubules within the deep cortical and outer medullary zones in both strains. Preadsorption of the antibody with angiotensin-(1-12) abolished staining in both tissues. Corresponding tissue measurements by radioimmunoassay showed 47% higher levels of angiotensin-(l-12) in the heart of SHR compared with WKY rats (P < 0.05). In contrast, renal angiotensin-(1-12) levels were 16.5% lower in SHR compared with the WKY rats (P < 0.05). This study shows for first time the localization of angiotensin-(l-12) in both cardiac myocytes and renal tubular components of WKY and SHR. In addition, we show that increased cardiac angiotensin-(l-12) concentrations in SHR is associated with a small, but statistically significant, reduction in renal angiotensin-(1-12) levels. angiotensinogen; angiotensin I; angiotensin II; hypertension; renin

Significant morbidity and mortality among patients with diabetes mellitus result largely from a greatly increased incidence of microvascular complications. Proliferative diabetic retinopathy (PDR) and end stage renal disease (ESRD) are two of the most common and severe microvascular complications of diabetes. A high concordance exists in the development of PDR and ESRD in diabetic patients, as well as strong familial aggregation of these complications, suggesting a common underlying genetic mechanism. However, the precise gene(s) and genetic variant(s) involved remain largely unknown. Erythropoietin (EPO) is a potent angiogenic factor observed in the diabetic human and mouse eye. By a combination of case-control association and functional studies, we demonstrate that the T allele of SNP rs1617640 in the promoter of the EPO gene is significantly associated with PDR and ESRD in three European-American cohorts [Utah: P = 1.91 x [10.sup.-3]; Genetics of Kidneys in Diabetes (GoKinD) Study: P = 2.66 x 10-8; and Boston: P = 2.1 x [10.sup.-2]]. The EPO concentration in human vitreous body was 7.5-fold higher in normal subjects with the TT risk genotype than in those with the GG genotype. Computational analysis suggests that the risk allele (T) of rs1617640 creates a matrix match with the EVI1/MEL1 or AP1 binding site, accounting for an observed 25-fold enhancement of luciferase reporter expression as compared with the G allele. These results suggest that rs1617640 in the EPO promoter is significantly associated with PDR and ESRD. This study identifies a disease risk-associated gene and potential pathway mediating severe diabetic microvascular complications. diabetic microvascular complication | end stage renal disease | proliferative diabetic retinopathy | SNP | association

During exercise, muscle mechanoreflex-mediated sympathoexcitation evokes renal vasoconstriction. Animal studies suggest that prostaglandins generated within the contracting muscle sensitize muscle mechanoreflexes. Thus we hypothesized that local prostaglandin blockade would attenuate renal vasoconstriction during ischemic muscle stretch. Eleven healthy subjects performed static handgrip before and after local prostaglandin blockade (6 mg ketorolac tromethamine infused into the exercising forearm) via Bier block. Renal blood flow velocity (RBV; Duplex Ultrasound), mean arterial pressure (MAP; Finapres), and heart rate (HR; ECG) were obtained during handgrip, post-handgrip muscle ischemia (PHGMI) followed by PHGMI with passive forearm muscle stretch (PHGMI + stretch). Renal vascular resistance (RVR, calculated as MAP/RBV) was increased from baseline during all paradigms except during PHGMI + stretch after the ketorolac Bier block trial where RVR did not change from baseline. Before Bier block, RVR rose more during PHGMI + stretch than during PHGMI alone (P < .01). Similar results were found after a saline Bier block trial (A53 [+ or -] 13% vs. A35 [+ or -] 10%; P < 0.01). However, after ketorolac Bier block, RVR was not greater during PHGMI + stretch than during PHGMI alone [A39 [+ or -] 8% vs. A40 [+ or -] 12%; P = not significant (NS)]. HR and MAP responses were similar during PHGMI and PHGMI + stretch (P = NS). Passive muscle stretch during ischemia augments renal vasoconstriction, suggesting that ischemia sensitizes mechanically sensitive afferents. Inhibition of prostaglandin synthesis eliminates this mechanoreceptor sensitization-mediated constrictor responses. Thus mechanoreceptor sensitization in humans is linked to the production of prostaglandins. kidney; exercise; nervous system; sympathetic; muscle metabolite

Previous studies have yielded conflicting results as to whether extracellular adenosine generation and signaling contributes to hypoxia-induced increases in renal erythropoietin (EPO) secretion. In this study, we combined pharmacological and genetic approaches to elucidate a potential contribution of extracellular adenosine to renal EPO release in mice. To stimulate EPO secretion, we used routine carbon monoxide exposure (400 and 750 parts per million CO, 4 h), ambient hypoxia (8% oxygen, 4 h), or arterial hemodilution. Because the ecto-5-nucleotidase (CD73, conversion of AMP to adenosine) is considered the pacemaker of extracellular adenosine generation, we first tested the effect of blocking extracellular adenosine generation with the specific CD73-inhibitor adenosine 5'-([alpha],[beta]-methylene) diphosphate (APCP) or by gene-targeted deletion of cd73. These studies showed that neither APCP-treatment nor targeted deletion of cd73 resulted in changes of stimulated EPO mRNA or serum levels, although the increases of adenosine levels in the kidney following CO exposure were attenuated in mice with APCP treatment or in [cd73.sup.-/-] mice. Moreover, pharmacological studies using specific inhibitors of individual adenosine receptors ([A.sub.1]AR, DPCPX; [A.sub.2A]AR, DMPX; [A.sub.2B]AR, PSB 1115; [A.sub.3]AR, MRS 1191) showed no effect on stimulated increases of EPO mRNA or serum levels. Finally, stimulated EPO secretion was not attenuated in gene-targeted mice lacking [A.sub.1][AR.sup.-/-], [A.sub.2A][AR.sup.-/-], [A.sub.2B][AR.sup.-/-] , or [A.sub.3][AR.sup.-/-]. Together, these studies combine genetic and pharmacological in vivo evidence that increases of EPO secretion during limited oxygen availability are not affected by extracellular adenosine generation or signaling. adenosine receptors; ecto-5'-nucleotidase; CD73; carbon monoxide

Recent studies have shown that interleukin 6 (IL-6) acts on the cellular proliferation-activating transduction signals during cellular regeneration. Therefore, this study examined the effect of IL-6 on the activation of [Na.sup.+]/glucose cotransporters (SGLTs) and its related signaling pathways in primary cultured renal proximal tubule cells (PTCs). IL-6 increased the level of [alpha]-methyl-D-[[sup.14]C]glucopyranoside ([alpha]-MG) uptake in time- and dose-dependent manners. IL-6 also increased SGLT1 plus SGLT2 mRNA and protein expression level. The IL-6 receptors (IL-6R[alpha] and gp130) were expressed in PTCs. In addition, genistein and herbimycin A completely blocked the IL-6-induced increases in [alpha]-MG uptake and the protein expression level of SGLTs. On the other hand, IL-6 increased the level of 5-(and-6)-chloromethyl-2',7'-dichlorodihy-drofluorescein diacetate-sensitive cellular reactive oxygen species (ROS), and IL-6-induced increases in [alpha]-MG uptake and the protein expression level of SGLTs were blocked by ascorbic acid or taurine (antioxidants). IL-6 also increased the phosphorylation of signal transducer and activator of transcription-3 (STAT3), phosphoinositide-3 kinase (PI3K)/Akt, and mitogen-activated protein kinases (MAPKs) in a time-dependent manner. A pretreatment with STAT3 inhibitor LY 294002, an Akt inhibitor, or MAPK inhibitors significantly blocked the IL-6-induced increase in [alpha]-MG uptake. In addition, IL-6 increased the level of nuclear factor-[kappa]B (NF-[kappa]B) phosphorylation. A pretreatment with SN50 or BAY 11-7082 also blocked the IL-6-induced increase in [alpha]-MG uptake. In conclusion, IL-6 increases the SGLT activity through ROS, and its action in renal PTCs is associated with the STAT3, PI3K/Akt, MAPKs, and NF-[kappa]B signaling pathways. [Na.sup.+]/glucose cotransporters; kidney

Sodium supplementation given for 1 wk to nonpregnant rats induces changes that are adequate to maintain renal and circulatory homeostasis as well as arterial blood pressure. However, in pregnant rats, proteinuria, fetal growth restriction, and placental oxidative stress are observed. Moreover, the decrease in blood pressure and expansion of circulatory volume, normally associated with pregnancy, are prevented by high-sodium intake. We hypothesized that, in these pregnant rats, a loss of the balance between prooxidation and antioxidation, particularly in kidneys and heart, disturbs the normal course of pregnancy and leads to manifestations such as gestational hypertension. We thus investigated the presence of oxidative/nitrosative stress in heart and kidneys following high-sodium intake in pregnant rats. Markers of this stress [8-isoprostaglandin [F.sub.2[alpha]] (8-iso-[PGF.sub.2[alpha]]) and nitrotyrosine], producer of nitric oxide [nitric oxide synthases (NOSs)], and antioxidants [superoxide dismutase (SOD) and catalase] were measured. Then, molecules ([Na.sup.+]-[K.sup.+]-ATPase and aconitase) or process [apoptosis (Bax and Bcl-2), inflammation (monocyte chemoattractant protein-l, connective tissue growth factor, and TNF-[alpha])] susceptible to free radicals was determined. In kidneys from pregnant rats on 1.8% NaCl-water, NOSs, apoptotic index, and nitrotyrosine expression were increased, whereas [Na.sup.+]-[K.sup.+]-ATPase mRNA and activity were decreased. In the left cardiac ventricle of these rats, heightened nitrotyrosine, 8-iso-[PGF.sub.2[alpha]], and catalase activity together with reduced endothelial NOS protein expression and SOD and aconitase activities were observed. These findings suggest that oxidative/nitrosative stress in kidney and left cardiac ventricle destabilizes the normal course of pregnancy and could lead to gestational hypertension. [Na.sup.+]-[K.sup.+]-ATPase; rat model of preeclampsia; high-sodium supplementation

The desert-adapted spiny mouse has a significantly lower glomerular number, increased glomerular size, and a more densely packed renal papillae compared with the similar-sized laboratory-adapted C57BL/6 mouse. In the present study we examined the functional consequences of these structural differences in young adult male spiny and C57BL/6 mice and detailed the impact of 1 wk of a high-salt (10% wt/wt NaCl) diet. Basal food and water intake, urine and feces production, and urinary electrolyte concentrations were not different between species, although urinary urea concentrations were higher in spiny mice (P < 0.05). On normal salt, MAP of the anesthetized spiny mouse was ~18 mmHg lower, effective renal plasma flow (ERPF) was 40% lower (P < 0.001), and glomerular filtration rate (GFR) tended to be lower than in the C57BL/6 mouse. On the high-salt diet, both species had similar 24-h NaCl excretions; but C57BL/6 mice required a significantly increased amount of water (lower urine NaCl concentration) than the spiny mice. Filtration fraction was greater in both species on the high-salt diet. Spiny mice had greater GFR and ERPF after the high-salt diet, whereas the C57BL/6 mouse showed little change in GFR. The ability of the spiny mouse to tolerate a significantly higher plasma osmolality after salt, measured by a decreased drinking response, and the ability to increase ERPF at a lower MAP are features that allow this species to conserve water more efficiently than can be done in the C57BL/6 mouse. These features are important, particularly since the desert mouse has a smaller kidney, with fewer nephrons. kidney; Acomys cahirinus; high-salt diet

A medullary thick ascending limb (mTAL) cell line, termed raTAL, has been established from freshly isolated rat mTAL tubules and cultured continuously for up to 75 passages; it retains characteristics of mTAL cells even after retrieval from storage in liquid nitrogen for several months. The cells express Tamm-Horsfall glycoprotein (THP), a TAL-specific marker, grow to confluence, exhibit a polygonal morphology characteristic of epithelial cells, and form 'domes.' Detection of THP, [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC2), [Na.sup.+]-[K.sup.+]-ATPase, and renal outer medullary [K.sup.+] channel (ROMK) was achieved using indirect immunofluorescence and confocal microscopy. Western blot analysis of NKCC2 expression using two different antibodies revealed a band of ~160 kDa, and RT-PCR analysis demonstrated the presence of NKCC2 isoforms A and F, which was confirmed by DNA sequencing; transport of [Cl.sup.-] into raTAL cells was inhibited by furosemide. Ouabain- and bumetanide-sensitive oxygen consumption, an index of ion transport activity in the mTAL, was observed in raTAL cells, and the number of domes present was reduced significantly when cells were incubated in the presence of ouabain or bumetanide. The specific activity of [Na.sup.+]-[K.sup.+]-ATPase activity was determined in raTAL cells (0.67 [+ or -] 0.18 nmol [P.sub.i] x [micro]g [protein.sup.-1] x [min.sup.-1]), primary cultures of mTAL cells (0.39 [+ or -] 0.08 nmol [P.sub.i] x [micro]g [protein.sup.-1] x [min.sup.-1]), and freshly isolated mTAL tubules (1.10 [+ or -] 0.29 nmol [P.sub.1] x [micro]g [protein.sup.-1] x [min.sup.-1]), and ~30-50% of total cellular ATPase activity was inhibited by ouabain, in accord with other mTAL preparations. This cell line will be used in studies that address biochemical, molecular, and physiological mechanisms in the mTAL. medullary thick ascending limb; cell culture; renal epithelial cells; renal cell lines

Accurate determination of renal function in mice is a major impediment to the use of murine models in acute kidney injury. The purpose of this study was to determine whether early changes in renal function could be detected using dynamic gamma camera imaging in a mouse model of ischemia-reperfusion (I/R) injury. C57BL/6 mice (n = 5/group) underwent a right nephrectomy, followed by either 30 min of I/R injury or sham surgery of the remaining kidney. Dynamic renal studies (21 min, 10 s/frame) were conducted before surgery (baseline) and at 5, 24, and 48 h by injection of [sup.99m]Tc-mercaptoacetyltriglycine (MAG3; ~1.0 mCi/mouse) via the tail vein. The percentage of injected dose (%ID) in the kidney was calculated for each 10-s interval after MAG3 injection, using standard region of interest analyses. A defect in renal function in I/R-treated mice was detected as early as 5 h after surgery compared with sham-treated mice, identified by the increased %ID (at peak) in the I/R-treated kidneys at 100 s (P < 0.01) that remained significantly higher than sham-treated mice for the duration of the scan until 600 s (P < 0.05). At 48 h, the renal scan demonstrated functional renal recovery of the I/R mice and was comparable to sham-treated mice. Our study shows that using dynamic imaging, renal dysfunction can be detected and quantified reliably as early as 5 h after I/R insult, allowing for evaluation of early treatment interventions. acute kidney injury; nuclear imaging; mice models of acute renal failure

CCN1 (cysteine-rich protein 61; Cyr61) is an extracellular matrix-associated signaling molecule that functions in cell migration, adhesion, and differentiation. We previously reported that CCN1 is induced at podocytes in rat anti-Thy-1 glomerulonephritis, a well-known model of reversible glomerular injury, but its expression and significance in the human kidney remain totally unknown (Sawai K, Mori K, Mukoyama M, Sugawara A, Suganami T, Koshikawa M, Yahata K, Makino H, Nagae T, Fujinaga Y, Yokoi H, Yoshioka T, Yoshimoto A, Tanaka I, Nakao K. J Am Soc Nephrol 14:1154-1163, 2003). Here we report that, in the human kidney, CCN1 expression was confined to podocytes in normal adult and embryonic glomeruli from the capillary loop stage. Podocyte CCNI expression was decreased in IgA nephropathy, diabetic nephropathy, and membranous nephropathy, whereas it remained unchanged in minimal change disease and focal segmental glomerulosclerosis. Downregulation of CCN1 was significantly greater in diseased kidneys with severe mesangial expansion. CCN1 protein was also localized in the thick ascending limb of Henle's loop, distal and proximal tubules, and collecting ducts, which was not altered in diseased kidneys. In vitro, recombinant CCN1 protein enhanced endothelial cell adhesion, whereas it prominently inhibited mesangial cell adhesion. CCN1 also completely suppressed mesangial cell migration, suggesting its role as a mesangial-repellent factor. In cultured podocytes, CCN1 markedly induced the expression of cyclin-dependent kinase inhibitor [p27.sup.Kip1] as well as synaptopodin in a dose-dependent manner and suppressed podocyte migration. These data indicate that CCN1 is expressed in podocytes, can act on glomerular cells to modulate glomerular remodeling, and is downregulated in diseased kidneys, suggesting that impairment of CCN1 expression in podocytes may contribute to the progression of glomerular disease with mesangial expansion. podocyte; glomerular visceral epithelial cell; cysteine-rich protein 61; mesangial cell; synaptopodin

Acute renal injury induces metabolic acidosis, but its specific effects on the collecting duct, the primary site for urinary ammonia secretion, the primary component of net acid excretion, are incompletely understood. We induced ischemia-reperfusion (I/R) acute renal injury in Sprague-Dawley rats by clamping the renal pedicles bilaterally for 30 min followed by reperfusion for 6 h. Control rats underwent sham surgery without renal pedicle clamping. I/R injury decreased urinary ammonia excretion significantly but did not persistently alter urine volume, [Na.sup.+], [K.sup.+], or bicarbonate excretion. Histological examination demonstrated cellular damage in the outer and inner medullary collecting duct, as well as in the proximal tubule and the thick ascending limb of the loop of Henle. A subset of collecting duct cells were damaged and/or detached from the basement membrane; these cells were present predominantly in the outer medulla and were less frequent in the inner medulla. Immunohistochemistry identified that the damaged/detached cells were A-type intercalated ceils, not principal cells. Both TdT-mediated dUTP nick-end labeling (TUNEL) staining and transmission electron microscopic examination demonstrated apoptosis but not necrosis. However, immunoreactivity for caspase-3 was observed in the proximal tubule, but not in collecting duct intercalated cells, suggesting that mechanism(s) of collecting duct intercalated cell apoptosis differ from those operative in the proximal tubule. We conclude that I/R injury decreases renal ammonia excretion and is associated with intercalated cell-specific detachment and apoptosis in the outer and inner medullary collecting duct. These effects likely contribute to the metabolic acidosis frequently observed in acute renal injury. acid-base; apoptosis; intercalated cell; acute tubular necrosis

The interaction of renal basolateral organic anion transporter 3 (Oat3) with commonly used pharmacotherapeutics (e.g., NSAIDs, [beta]-lactams, and methotrexate) has been studied extensively in vitro. However, the in vivo role of Oat3 in drug disposition, in the context of other transporters, glomerular filtration, and metabolism, has not been established. Moreover, recent investigations have identified inactive human OAT3 polymorphisms. Therefore, this investigation was designed to elucidate the in vivo role of Oat3 in the disposition of penicillin G and prototypical substrates using an Oat3 knockout mouse model. Oat3 deletion resulted in a doubling of penicillin's half-life (P < 0.05) and a reduced volume of distribution (P < 0.01), together yielding a plasma clearance that was one-half (P < 0.05, males) to one-third (P < 0.001, females) of that in wild-type mice. Inhibition of Oat3 abolished the differences in penicillin G elimination between genotypes. Hepatic accumulation of penicillin was 2.3 times higher in male knockouts (P < 0.05) and 3.7 times higher in female knockouts (P < 0.001). Female knockouts also exhibited impaired estrone-3-sulfate clearance. Oat3 deletion did not impact p-aminohippurate elimination, providing correlative evidence to studies in Oat1 knockout mice that suggest Oat1 governs tubular uptake of p-aminohippurate. Collectively, these findings are the first to indicate that functional Oat3 is necessary for proper elimination of xenobiotic and endogenous compounds in vivo. Thus Oat3 plays a distinct role in determining the efficacy and toxicity of drugs. Dysfunctional human OAT3 polymorphisms or instances of polypharmacy involving OAT3 substrates may result in altered systemic accumulation of [beta]-lactams and other clinically relevant compounds. kidney; liver; transport; Oat1; drug disposition

Aging is associated with an increased incidence and severity of acute renal failure. However, the molecular mechanism underlying the increased susceptibility to injury remains undefined. These experiments were designed to investigate the influence of age on the response of the kidney to ischemic injury and to identify candidate genes that may mediate this response. Renal slices prepared from young (5 mo), aged ad libitum (aged-AL; 24 mo), and aged caloric-restricted (aged-CR; 24 mo) male Fischer 344 rats were subjected to ischemic stress (100% [N.sub.2]) for 0-60 min. As assessed by biochemical and histological evaluation, slices from aged-AL rats were more susceptible to injury than young counterparts. Importantly, caloric restriction attenuated the increased susceptibility to injury. In an attempt to identify the molecular pathway(s) underlying this response, microarray analysis was performed on tissue harvested from the same animals used for the viability experiments. RNA was isolated and the corresponding cDNA was hybridized to CodeLink Rat Whole Genome Bioarray slides. Subsequent gene expression analysis was performed using GeneSpring software. Using two-sample t-tests and a twofold cut-off, the expression of 92 genes was changed during aging and attenuated by caloric restriction, including claudin-7, kidney injury molecule-1 (Kim-1), and matrix metalloproteinase-7 (MMP-7). Claudin-7 gene expression peaked at 18 mo; however, increased protein expression in certain tubular epithelial cells was seen at 24 mo. Kim-1 gene expression was not elevated at 8 or 12 mo but was at 18 and 24 mo. However, changes in Kim-1 protein expression were only seen at 24 mo and corresponded to increased urinary levels. Importantly, these changes were attenuated by caloric restriction. MMP-7 gene expression was decreased at 8 mo, but an age-dependent increase was seen at 24 mo. Increased MMP-7 protein expression in tubular epithelial cells at 24 mo was correlated with the gene expression pattern. In summary, we identified genes changed by aging and changes attenuated by caloric restriction. This will facilitate investigation into the molecular mechanism mediating the age-related increase in susceptibility to injury. ischemia; microarray analysis

We used the patch-clamp technique and Western blot analysis to explore the effect of [PGE.sub.2] on ROMK-like small-conductance K (SK) channels and [Ca.sup.2+]-activated big-conductance K channels (BK) in the cortical collecting duct (CCD). Application of 10 [micro]M [PGE.sub.2] inhibited SK and BK channels in the CCD. Moreover, either inhibition of PKC or blocking mitogen-activated protein kinase (MAPK), P38 and ERK, abolished the effect of [PGE.sub.2] on SK channels in the CCD. The effect of [PGE.sub.2] on SK channels was completely blocked in the presence of SC-51089, a specific EP1 receptor antagonist, and mimicked by application of sulprostone, an agonist for EP1 and EP3 receptors. To determine whether [PGE.sub.2] stimulates the phosphorylation of P38 and ERK, we treated mouse CCD cells (M-1) with [PGE.sub.2]. Application of [PGE.sub.2] significantly stimulated the phosphorylation of P38 and ERK within 5 min. The dose-response curve of [PGE.sub.2] effect shows that 1, 5, and 10 [micro]M [PGE.sub.2] increased the phosphorylation of P38 and ERK by 20-21, 50-80, and 80-100%, respectively. The stimulatory effect of [PGE.sub.2] on MAPK phosphorylation was not affected by indomethacin but abolished by inhibition of PKC. This suggests that the effect of [PGE.sub.2] on MAPK phosphorylation is PKC dependent. Also, the expression of cyclooxygenase II and [PGE.sub.2] concentration in renal cortex and outer medulla was significantly higher in rats fed a K-deficient diet than those on a normal-K diet. We conclude that [PGE.sub.2] inhibits SK and BK channels and that there is an effect of [PGE.sub.2] on SK channels in the CCD through activation of EP1 receptor and MAPK pathways. Also, high concentrations of [PGE.sub.2] induced by K restriction may be partially responsible for increasing MAPK activity during K restriction. cyclooxygenase; arachidonic acid; P38; ERK; ROMK; renal K secretion

The profibrotic effect of plasminogen activator inhibitor-1 (PAI-1) in renal fibrosis is widely recognized, but its mechanism remains controversial especially in chronic progressive kidney disease. In the present study, pioglitazone (Pio) and candesartan (CD), which are reported to inhibit PAI-1, were administered to spontaneously hypercholesterolemic (SHC) rats, a model of chronic progressive kidney disease. Therapeutic effects and effects on the intrarenal plasmin cascade were examined. Eight-wk-old SHC rats were used as controls. Oral administration of vehicle alone, Pio, or CD was performed starting at 8 wk of age and was continued for 24 wk. The degree of renal fibrosis was evaluated by sirius red staining of kidney sections and by total collagen assay of renal homogenates. The renal PAI-1 protein level was assessed by Western blotting, and plasmin activity was analyzed by chromogenic assay and casein gel zymography. Urinary protein and blood urea nitrogen were significantly increased in the vehicle-treated group, but the increase was attenuated in the Pio- and CD-treated groups. This correlated well with the degree of fibrosis as assessed by sirius red staining and total collagen assay. The PAI-1 protein level was also increased significantly in the vehicle-treated group, and the increase was attenuated in the Pio- and CD-treated groups. Despite the presumed plasmin-inhibitory function of PAI-1, plasmin activity changed in parallel with PAI-1. These results suggest that Pio and CD inhibit PAI-1 and exert renoprotective effects against chronic progressive renal disease, but its action is independent of the regulatory function on plasmin activity. angiotensin II type 1 receptor blocker; peroxisome proliferator-activated receptor-[gamma] agonist; plasminogen activator inhibitor-1

Furosemide is a loop diuretic agent that has been used to treat hypercalcemia because it increases renal calcium excretion. The effect of furosemide on calcium transport molecules in distal tubules has yet to be investigated. We conducted studies to examine the effects of furosemide on renal calcium excretion and expression of calcium transport molecules in mice. Mice were administered with a single dose of furosemide (15 mg/kg) and examined 4 h later or were given twice-daily furosemide injections for 3 days. To evaluate the effects of volume depletion, drinking water was supplemented with salt. Our results showed that, in acute experiments, furosemide enhanced urinary calcium excretion, which was associated with a significant increase in mRNA levels of TRPV5, TRPV6, and calbindin-D28k but not calbindin-D9k as measured by real-time PCR (TRPV5 and TRPV6 are transient receptor potential vanilloid 5 and 6). Chronic furosemide administration induced three- to fourfold increases in urinary calcium excretion and elevated mRNA levels of TRPV5, TRPV6, calbindin-D28k, and calbindin-D9k without or with salt supplement. Similar upregulation of calcium transport molecules was observed in mice with gentamicin-induced hypercalciuria. Coadministration of chlorothiazide decreased furosemide-induced calciuria, either acutely or chronically, although still accompanied by upregulation of these transport molecules. Immunofluorescent staining studies revealed comparably increased protein abundance in TRPV5 and calbindin-D28k. We conclude that furosemide treatment enhances urinary calcium excretion. Increased abundance of calcium transport molecules in the distal convoluted tubule represents a solute load-dependent effect in response to increased calcium delivery and serves as a compensatory adaptation in the downstream segment. thiazides; TRPV5; TRPV6; calbindin-D28k; calbindin-D9k

Descending vasa recta (DVR) are capillary-sized microvessels that supply blood flow to the renal medulla. They are composed of contractile pericytes and endothelial cells. In this study, we used the whole cell patch-clamp method to determine whether inward rectifier potassium channels (Kin) exist in the endothelia, affect membrane potential, and modulate intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.cyt]). The endothelium was accessed for electrophysiology by removing abluminal pericytes from collagenase-digested vessels. [K.sub.IR] currents were recorded using symmetrical 140 mM [K.sup.+] solutions that served to maximize currents and eliminate cell-to-cell coupling by closing gap junctions. Large, inwardly rectifying currents were observed at membrane potentials below the equilibrium potential for [K.sup.+]. [Ba.sup.2+] potently inhibited those currents in a voltage- dependent manner, with affinity k = 0.18, 0.33, 0.60, and 1.20 [micro]M at -160, -120, -80, and -40 mV, respectively. [Cs.sup.+] also blocked those currents with k = 20, 48, 253, and 1,856 [micro]M at -160, -120, -80, and -40 mV, respectively. In the presence of 1 mM ouabain, increasing extracellular [K.sup.+] concentration from 5 to 10 mM hyperpolarized endothelial membrane potential by 15 mV and raised endothelial [[[Ca.sup.2+]].sub.cyt]. Both the [K.sup.+]-induced membrane hyperpolarization and the [[[Ca.sup.2+]].sub.cyt] elevation were reversed by [Ba.sup.2+]. Immunochemical staining verified that both pericytes and endothelial cells of DVR express [K.sub.IR]2.1, [K.sub.IR]2.2, and [K.sub.IR]2.3 subunits. We conclude that strong, inwardly rectifying [K.sub.IR]2.x isoforms are expressed in DVR and mediate [K.sup.+]-induced hyperpolarization of the endothelium. kidney; medulla; microcirculation; electrophysiology; potassium channel; endothelium

In renal epithelia, vasopressin influences salt and water transport, chiefly via vasopressin [V.sub.2] receptors ([V.sub.2]Rs) linked to adenylyl cyclase. A combination of vasopressin-induced effects along several distinct portions of the nephron and collecting duct system may help balance the net effects of antidiuresis in cortex and medulla. Previous studies of the intrarenal distribution of [V.sub.2]Rs have been inconclusive with respect to segment- and cell-type-related [V.sub.2]R expression. Our study therefore aimed to present a high-resolution analysis of [V.sub.2]R mRNA expression in rat, mouse, and human kidney epithelia, supplemented with immunohistochemical data. Cell types of the renal tubule were identified histochemically using specific markers. Pronounced [V.sub.2]R signal in thick ascending limb (TAL) was corroborated functionally; phosphorylation of [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter type 2 (NKCC2) was established in cultured TAL cells from rabbit and in rats with diabetes insipidus that were treated with the [V.sub.2]R agonist desmopressin. We found solid expression of [V.sub.2]R mRNA in medullary TAL (MTAL), macula densa, connecting tubule, and cortical and medullary collecting duct and weaker expression in cortical TAL and distal convoluted tubule in all three species. Additional [V.sub.2]R immunostaining of kidneys and rabbit TAL cells confirmed our findings. In agreement with strong [V.sub.2]R expression in MTAL, kidneys from rats with diabetes insipidus and cultured TAL cells revealed sharp, selective increases in NKCC2 phosphorylation upon desmopressin treatment. Macula densa cells constitutively showed strong NKCC2 phosphorylation. Results suggest comparably significant effects of vasopressin-induced [V.sub.2]R signaling in MTAL and in connecting tubule/collecting duct principal cells across the three species. Strong [V.sub.2]R expression in macula densa may be related to tubulovascular signal transfer. antidiuretic hormone; thick ascending limb; kidney; sodium-potassium-chloride cotransporter type 2; bumetanide-sensitive cotransporter type 1

The turnover and repair of peritubular capillaries is essential for the maintenance of normal renal tubular structure and function. Following injury, ineffective capillary repair/angiogenesis may result in chronic disease, whereas effective repair attenuates the injury process. Thus the process of healing in the kidney is likely dependent on an intricate balance between angiogenic and anti-angiogenic factors to maintain the renal microvasculature. We investigated the role of cytoprotective heine oxygenase-1 (HO-1) in the regulation of chemokines in human renal proximal tubular epithelial cells (RPTEC). Transfection of RPTEC with a HO-1 over-expression plasmid promoted a marked induction in the mRNA expression of the anti-angiogenic chemokine CXCL-10, along with angiogenic chemokines CXCL-8 and CCL-2. Utilizing a CXCL-10 promoter luciferase construct, we observed that HO-l-induced CXCL-10 expression is regulated at the transcriptional level. However, with increases in concentrations and time intervals of HO-1 induction, there was a marked decrease in CXCL-10 expression. Using pharmacological inhibitors, we found that HO-1-induced early robust CXCL-10 transcription is mediated through the PKC signaling pathway. To evaluate the functional significance of HO-1-induced CXCL-10 release, we cultured human vascular endothelial cells in the absence and presence of culture supernatants of the HO-1 plasmid-transfected RPTEC. We found that early (24 h) supernatants of the HO-1 plasmid-transfected cells (RPTEC) inhibited endothelial cell proliferation, and this effect was blocked by addition of a CXCL-10 neutralizing antibody. Thus HO-1 can regulate the expression of the anti-angiogenic CXCL-10 and may alter a critical balance between angiogenic vs. anti-angiogenic factors that are important to maintain renal microvasculature during injury. renal injury: renal tubule: cytoprotection

Cyclooxygenase-2 (Cox-2) is an inducible enzyme responsible for the formation of inflammatory prostanoids such as prostaglandins and thromboxane. Its role in the pathophysiology of inflammatory states like sepsis is increasingly recognized. Recently, we demonstrated that sepsis up-regulates the endotoxin receptor Toll-like receptor 4 (TLR4) in rat kidney. Because Cox-2 is one of the downstream products of TLR4 activation, we hypothesized that sepsis-induced changes in renal Cox-2 expression are TLR4 dependent. Indeed, we show that in Sprague-Dawley rats, cecal ligation and puncture (a sepsis model) increases Cox-2 expression in cortical and medullary thick ascending loops (cTAL and mTAL, respectively) as well as inner medullary collecting ducts. These are all sites of increased TLR4 expression during sepsis. To determine the actual dependence on TLR4, we measured Cox-2 expression in wild-type and mutant mice which harbor a TLR4 gene deletion (TLR4-/-). In wild-type mice, sepsis increased Cox-2 expression in proximal tubules, cTAL, and mTAL. In contrast, septic TLR4-/- mice showed no significant increase in cTAL or mTAL Cox-2 expression. Furthermore, renin was absent from juxtaglomerular cells of TLR4-/- mice. We conclude that the dependence of sepsis-induced renal Cox-2 expression on TLR4 is tubule specific. The TLR4-dependent Cox-2 expression is mostly restricted to cortical and medullary thick ascending loops of Henle that characteristically express and secrete Tamm-Horsfall protein. Toll-like receptors; renin; Tamm-Horsfall protein; macula densa; cyclooxygenase

To determine the effects of long-term angiotensin-converting enzyme inhibition (ACEI) and blood pressure (BP) lowering on renal sodium transporter abundance and distribution in spontaneously hypertensive rats (SHR), 9-wk SHR were treated with enalapril (30 mg x [kg.sup.-1] x [day.sup.-1]) for 4 wk. BP decreased from 156 [+ or -] 4 to 96 [+ or -] 8 mmHg. [Na.sup.+]/[H.sup.+] exchanger isoform 3 (NHE3) and [Na.sup.+]-[P.sub.i] cotransporter type 2 (NaPi2) localized to the body of the microvilli (MV) in normotensive rat strains. In untreated SHR, NHE3 partially retracted from the body to base of the MV and NaPi2 retracted to subapical vesicles. Alter enalapril treatment of SHR, NHE3 fully retracted to the base of the MV and, by density gradient fractionation, NHE3, NaPi2, dipeptidyl peptidase IV, myosin VI, Na-Cl cotransporter, and cortical Na-K-Cl cotransporter redistributed from low-density (apical enriched) to high-density (endosome enriched) membranes. Enalapril decreased total abundance of myosin VI (to 0.51 [+ or -] 0.18 of untreated), ACE (0.67 [+ or -] 0.22), and cortical NaPi2 (0.83 [+ or -] 0.10). Normalizing SHR BP with HRH (7.5 mg/day hydralazine, 0.15 mg/day reserpine, and 3 mg/day hydrochlorothiazide) did not change [Na.sup.+] transporter density distribution or abundance. We conclude that lowering BP to normal levels in SHR does not normalize [Na.sup.+] transporter distribution, rather, chronic ACEI treatment provokes retraction of [Na.sup.+] transporters and associated proteins from transport-relevant domains of apical membranes and/or reduces their abundance. hypertension; kidney; Na transporter; ACE inhibitor; triple therapy

Sepsis-related acute kidney injury (AKI) is the leading cause of AKI in intensive care units. Endotoxin is a primary initiator of inflammatory and hemodynamic consequences of sepsis and is associated with experimental AKI. The present study was undertaken to further examine the role of the endothelium, specifically prostacyclin ([PGI.sub.2]), in the pathogenesis of endotoxemia-related AKI. A low dose of endotoxin (LPS, 1 mg/kg) in wild-type (WT) mice was associated with stable glomerular filtration rate (GFR) (164.0 [+ or -] 16.7 vs. 173.3 [+ or -] 6.7 [micro]l/min, P = not significant) as urinary excretion of 6-keto-[PGF.sub.1[alpha]], the major metabolite of [PGI.sub.2], increased. When cyclooxygenase inhibition with indomethacin abolished this rise in 6-keto-[PGF.sub.1[varies]], the same low dose of LPS significantly decreased GFR (110.7 [+ or -] 12.1 vs. 173.3 [+ or -]- 6.7 [micro]l/min, P < 0.05). The same dose of indomethacin did not alter GFR in WT mice. To further study the role of [PGI.sub.2] in endotoxemia, renal-specific PGI synthase (PGIs) transgenic (Tg) mice were developed that had increased PGIs expression only in the kidney and increased urinary 6-keto-[PGF.sub.1[alpha]]. These Tg mice, however, demonstrated endotoxemia-related AKI with low-dose LPS (1 mg/kg) (GFR: 12.6 [+ or -] 3.9 vs. 196.5 [+ or -] 21.0 [micro]l/min P < 0.01), which did not alter GFR in WT mice (164.0 [+ or -] 16.7 vs. 173.3 [+ or -] 6.7 [micro]l/min, P = not significant). An elevation in renal cAMP, however, suggested an activation of the [PGI.sub.2]-cAMP-renin system in these Tg mice. Moreover, angiotensin-converting enzyme inhibition afforded protection against endotoxin-related AKI in these Tg mice. Thus endothelial PGIs-mediated [PGI.sub.2], as previously shown with endothelial nitric oxide synthase-mediated nitric oxide, contributes to renal protection against endotoxemia-related AKI. This effect may be overridden by excessive activation of the renin-angiotensin system in renal-specific PGIs Tg mice. endotoxemia

High-potassium diets have been shown to be beneficial in cardiovascular disease partly because of a blood pressure-lowering effect. The effect of potassium on inflammation has not been studied. We investigated the influence of potassium supplementation on the degree of renal inflammation and the intracellular signaling mechanisms that could mediate inflammation in chronic kidney disease (CKD). CKD was created in male Sprague-Dawley rats by subtotal nephrectomy. Two groups of CKD rats were pair fed with diets containing 2.1% potassium (potassium-supplemented diet) or 0.4% potassium (basal diet). Body weight, blood pressure, and blood and urine electrolytes were measured biweekly. The animals were euthanized at week 8, and the remnant kidneys were analyzed by histology, immunohistochemistry, Western blotting, and real-time quantitative PCR. In the CKD pair-fed groups, blood potassium concentration did not differ significantly, but blood pressure was lower in the potassium-supplemented group. Compared with the basal diet, potassium supplementation decreased renal tubulointerstitial injury and suppressed renal inflammation as evidenced by decreased macrophage infiltration, lower expression of inflammatory cytokines, and decreased NF-[kappa]B activation. These renoprotective effects were associated with downregulation of renal transforming growth facto-[beta], upregulation of renal Smad7, and lower blood pressure. Our results show that potassium supplementation can reduce renal inflammation and hence, could modulate the progression of kidney injury in CKD. NF-[kappa]B; potassium; Smad7; TGF-[beta]

An important role for the enzyme ADP-ribosyl cyclase (ADPR cyclase) and its downstream targets, the ryanodine receptors (RyR), is emerging for a variety of vascular systems. We hypothesized that the ADPR cyclase/RyR pathway contributes to regulation of renal vasomotor tone in vivo. To test this, we continuously measured renal blood flow (RBF) in anesthetized Sprague-Dawley rats. Infusion of the ADPR cyclase inhibitor nicotinamide intrarenally at low doses inhibits angiotensin II (ANG II)- and norepinephrine (NE)-induced vasoconstriction by 72 and 67%, respectively (P < 0.001). RBF studies in rats were extended to mice lacking the predominant form of ADPR cyclase (CD38). Acute renal vasoconstrictor responses to ANG II and NE are impaired by 59 and 52%, respectively, in anesthetized CD38-/- mice compared with wild-type controls (P < 0.05). Intrarenal injection of the RyR activator FK506 decreases RBF by 22% (P > 0.03). Furthermore, RyR inhibition with ruthenium red attenuates ANG II and NE responses by 50 and 59%, respectively (P [less than or equal to] 0.01). Given at higher doses, nicotinamide increases basal RBF by 22% (P > 0.001). Non-receptor-mediated renal vasoconstriction by L-type voltage-gated [Ca.sup.2+] channels is also dependent on ADPR cyclase and RyRs. Nicotinamide and ruthenium red inhibit constriction by the L-type channel agonist BAY K 8644 by 59% (P > 0.02) and 63% (P > 0.001). We conclude that 1) ADPR cyclase activity contributes to regulation of renal vasomotor tone under resting conditions, 2) renal vasoconstriction induced by G protein-coupled receptor agonists ANG II and NE is mediated in part by ADPR cyclase and RyRs, and 3) ADPR cyclase and RyRs participate in L-type channel-mediated renal vasoconstriction in vivo. vascular smooth muscle; intracellular calcium; L-type calcium channels; FK506; CD38

Aminopeptidase N/CD13 (Anpep) is a membrane-bound protein that catalyzes the formation of natriuretic hexapeptide angiotensin IV (ANG IV) from ANG III. We previously reported that Anpep is more highly expressed in the kidneys of Dahl salt-resistant (SR/Jr) than salt-sensitive (SS/Jr) rats, Anpep maps to a quantitative trait locus for hypertension, and that the Dahl SR/Jr rat contains a functional polymorphism of the gene. This suggests that renal Anpep may be linked to salt sensitivity; however, its effect on renal Na handling has not been determined. Here, we examined regulation of basolateral [Na.sup.+]-[K.sup.+]-ATPase, a preeminent basolateral Na+ transporter in proximal tubule cells, by Anpep in LLC-PK1 cells. Treatment of the cells with Anpep siRNA increased total cellular [Na.sup.+]-[K.sup.+]-ATPase activity and basolateral [Na.sup.+]-[K.sup.+]-ATPase abundance by approximately twofold. Conversely, Anpep overexpression reduced [Na.sup.+]-[K.sup.+]-ATPase activity and basolateral abundance by ~50%. Similar effects were observed after treatment with ANG IV (10 nM, x30 min and 12 h). ANG IV receptor (AGTRIV) knockdown via specific siRNA relieved the decreases in basolateral [Na.sup.+]-[K.sup.+]-ATPase levels and activity induced by Anpep overexpression. In sum, these results demonstrate that Anpep reduces basolateral [Na.sup.+]-[K.sup.+]-ATPase levels via ANG IV/ AGTRIV signaling. This novel pathway may be important in renal adaptation to high salt. angiotensin IV; kidney

In contrast to other sympathetic outflow tracts, renal sympathetic nerve activity (RSNA) decreases in response to hypotensive hemorrhage. The functional significance of this 'paradox' is not known. We tested the hypothesis that RSNA modulates renal perfusion and thus erythropoietin (EPO) release after transient hypotensive hemorrhage in anesthetized rats. Plasma EPO was measured before and after 30 min of transient hypotensive hemorrhage (i.e., -40 mmHg from mean baseline blood pressure, followed by reinfusion of shed blood) and 120 min thereafter in sham-denervated rats, and after renal denervation (DNX) or bilateral cervical vagotomy (VX) to abolish/blunt the RSNA decrease mediated by a cardiopulmonary reflex. RSNA, renal Doppler flow, renal vascular resistance (RVR), resistance index, and oxygen delivery/uptake ([DO.sub.2]/[[??]O.sub.2]) were measured. RSNA decreased in intact animals (-40 [+ or -] 5% from baseline, P < 0.05). This was blunted by VX. With intact nerves, EPO level did not increase. In DNX rats, EPO was increased at minute 120 (49 [+ or -] 3 vs. 74 [+ or -] 2 mU/ml; P < 0.05), in VX rats this (47 [+ or -] 2 vs. 62 [+ or -] 4 mU/ml; P < 0.05) was less pronounced. [DO.sub.2] in DNX rats was lower compared with intact and VX rats (0.25 [+ or -] 0.04 vs. 0.51 [+ or -] 0.06 and 0.54 [+ or -] 0.05 ml [O.sub.2]/min; P < 0.05) due to lower Doppler flow and increased RVR. RVR and [DO.sub.2] were similar in intact and VX rats, but resistance index differed between all groups (0.70 [+ or -] 0.02 vs. 0.78 [+ or -] 0.02 vs. 0.85 [+ or -] 0.02; P < 0.05, intact vs. VX vs. DNX), indicating differential reactivity of renal vasculature. [[??]O.sub.2] was unaffected by VX and DNX. Renal sympathoinhibition during hypotensive hemorrhage might help to preserve sufficient oxygenation of renal tissue by modulation of hemodynamic mechanisms that act to adapt renal oxygen availability to demand. renal nerve; oxygen delivery

Hyperglycemia-induced oxidative stress is a key mediator of renal tubular hypertrophy in diabetic nephropathy (DN). The molecular mechanisms of antioxidants responsible for inhibition of renal tubular hypertrophy in DN are incompletely characterized. We now aim at verifying the effects of N-acetylcysteine (NAC) and taurine on cellular hypertrophy in renal tubular epithelial cells under high ambient glucose. We found that NAC and taurine treatments significantly attenuated high glucose (HG)-inhibited cellular growth and HG-induced hypertrophy. HG-induced Raf-1, p42/p44 mitogen-activated protein kinase (MAPK), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 1 (STAT1) and STAT3 (but not STAT5) activation was markedly blocked by NAC and taurine. Moreover, NAC and taurine increased cyclin D1/cdk4 activation and suppressed [p21.sup.Waf1/Cip1] and [p27.sub.Kip1] expression in HG-treated cells. It seems that apoptosis was not observed in these treatments. There were no changes in bcl-2 and poly(ADP-ribose) polymerase expression, and mitochondrial cytochrome c release. However, NAC or taurine markedly inhibited the stimulation by HG of fibronectin and type IV collagen protein levels. It is concluded that both NAC and taurine significantly attenuated HG-induced activation of the Raf-1/MAPK and the JAK2-STAT1/ STAT3 signaling pathways and hypertrophic growth in renal tubular epithelial cells. N-acetylcysteine; taurine; high glucose; hypertrophy; renal tubular epithelial cells; antioxidants

Diabetic nephropathy (DN), the leading cause of end-stage renal failure, is clinically manifested by albuminuria and a progressive decline in glomerular filtration rate. The factors and mechanisms that contribute to progression of DN are still undefined. To address the contribution of [B.sub.2-kinin] receptors (B2KR) to the development of DN, we studied B2KR knockout mice ([B2KR.sup.-/-]) and their wild-type littermates ([B2KR.sup.+/+]). Diabetes was induced by daily injections of streptozotocin (50 mg/kg body wt) for 3-5 days. A total of 48 mice divided into 4 groups were used: group 1, wild-type control ([B2KR.sup.+/+] C); group 2, wild-type diabetic ([B2KR.sup.+/+] D); group 3, B2KR knockout control ([B2KR.sup.-/-] C); and group 4, B2KR knockout diabetic ([B2KR.sup.-/-] D). Glucose levels and albumin excretion rate (AER) were measured at predetermined intervals. Half of the mice were killed at 3 mo, and the remaining half, at 6 mo. Plasma glucose levels were markedly elevated in both [B2KR.sup.+/+] D and [B2KR.sup.-/-] D groups of mice compared with their controls. Diabetic [B2KR.sup.-/-] mice displayed reduced AER as well as reduced glomerular and tubular injury compared with diabetic [B2KR.sup.+/+] mice. The renoprotection conferred by deletion of B2KR was associated with increased renal expression of B1-kinin and angiotensin II [AT.sub.2] receptors and decreased expression of connective tissue growth factor. At a cellular level, our findings demonstrate that bradykinin downregulates the expression of [AT.sub.2] receptors in mesangial cells. These findings provide the first evidence that targeted deletion of B2KR protects against the development of DN. kinin receptors; albuminuria

Culture of opossum kidney (OK) cells over 40 passages resulted in marked overexpression of [[alpha].sub.1]-subunit and [[beta].sub.1]-subunit [Na.sup.+]-[K.sup.+]-ATPase, which paralleled with increases in [Na.sup.+]-[K.sup.+]-ATPase activity, and enhanced availability in hydrogen peroxide ([H.sub.2][O.sub.2]) (J Membr Biol 212: 163-175, 2006). The present study evaluated the mechanisms involved in increased [H.sub.2][O.sub.2] production in OK cells cultured over 40 passages (up to passage 80) and whether [H.sub.2][O.sub.2] plays a role in the regulation of [Na.sup.+]-[K.sup.+]-ATPase expression and activity. The accumulation of [H.sub.2][O.sub.2] in the extracellular medium over 24 h (236.5 [+ or -] 2.7 vs. 319.3 [+ or -] 2.9 nM) and the rate of [H.sub.2][O.sub.2] production (5.21 [+ or -] 0.02 vs. 7.37 [+ or -] 0.02 nM/min) in cells with 40 passages was lower (P < 0.05) than in cells with 80 passages. The increase in [H.sub.2][O.sub.2] production in cells with 80 passages was accompanied by overexpression of NOX1, but not of NOX2, and overexpression of SODl, SOD2, and SOD3. Apocynin, a NADPH inhibitor, markedly attenuated the increase in [H.sub.2][O.sub.2] production in cells with 80 passages and significantly decreased [Na.sup.+]-[K.sup.+]-ATPase activity (26% reduction) and [[alpha].sub.1]-subunit [Na.sup.+]-[K.sup.+]-ATPase overexpression. Increases in [Na.sup.+]-[K.sup.+]-ATPase [[alpha].sub.1]-subunit mRNA abundance were also observed in OK cells with increased number of cell passages, but apocynin did not affect transcript abundance. In conclusion, the increased availability of [H.sub.2][O.sub.2] in the intracellular milieu plays an important role in the long-term regulation of [Na.sup.+]-[K.sup.+]-ATPase expression and activity. These cells may constitute an interesting model to study events related to oxidative stress, in which adaptation to increases in [H.sub.2][O.sub.2] had progressively adapted in a more natural manner. NADPH oxidase; superoxide dismutase

Late-gestation lung protein 1 (LGL1) is a glycoprotein secreted by fetal lung mesenchyme that stimulates branching morphogenesis of the developing lung bud. We show that Lgl1 mRNA and protein are also expressed in mesenchymally derived lineages of fetal kidney. Although Lgl1 expression is stimulated by glucocorticoids in kidney cells, cortisol ([10.sup.-7] M) actually suppresses ureteric bud branching of fetal kidneys from HoxB7/GFP mice in explant culture. However, early branching morphogenesis in the lung and kidney is stimulated by retinoic acid, and we identified putative retinoic acid response elements in the Lgl1 promoter. All-trans-retinoic acid ([10.sup.-6] M) stimulated Lgl1 promoter activity and endogenous Lgl1 mRNA expression in vitro. Branching of cultured fetal kidney explants was increased in the presence of all-trans retinoic acid ([10.sup.-6] M). Heterozygous Lgl1 knockout mice were crossed to HoxB7/GFP mice to visualize the extent of ureteric bud branching at fetal stages. At embryonic (E) days E12.5-E13.0, mutant [Lgl1.sup.+/-] embryos showed a 20% reduction in ureteric bud branching compared with wild-type littermates. We propose a model in which retinoic acid stimulates branching morphogenesis by activating Lgl1 early in development. The prominent effects of glucocorticoids on Lgl1 expression in late lung development suggest a second role for LGL1 in alveolar maturation. branching morphogenesis; kidney development; glucocorticoids

Increasing efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve activity (ARNA). To test whether the ERSNA-induced increases in ARNA involved norepinephrine activating [alpha]-adrenoceptors on the renal sensory nerves, we examined the effects of renal pelvic administration of the [[alpha].sub.1]- and [[alpha].sub.2]-adrenoceptor antagonists prazosin and rauwolscine on the ARNA responses to reflex increases in ERSNA (placing the rat's tail in 49[degrees]C water) and renal pelvic perfusion with norepinephrine in anesthetized rats. Hot tail increased ERSNA and ARNA, 6,930 [+ or -] 900 and 4,870 [+ or -] 670%'s (area under the curve ARNA vs. time). Renal pelvic perfusion with norepinephrine increased ARNA 1,870 [+ or -] 210%'s. Immunohistochemical studies showed that the sympathetic and sensory nerves were closely related in the pelvic wall. Renal pelvic perfusion with prazosin blocked and rauwolscine enhanced the ARNA responses to reflex increases in ERSNA and norepinephrine. Studies in a denervated renal pelvic wall preparation showed that norepinephrine increased substance P release, from 8 [+ or -] 1 to 16 [+ or -] 1 pg/min, and [PGE.sub.2] release, from 77 [+ or -] 11 to 161 [+ or -] 23 pg/min, suggesting a role for [PGE.sub.2] in the norepinephrine-induced activation of renal sensory nerves. Prazosin and indomethacin reduced and rauwolscine enhanced the norepinephrine-induced increases in substance P and [PGE.sub.2]. [PGE.sub.2] enhanced the norepinephrine-induced activation of renal sensory nerves by stimulation of EP4 receptors. Interaction between ERSNA and ARNA is modulated by norepinephrine, which increases and decreases the activation of the renal sensory nerves by stimulating [[alpha].sub.1]- and [[alpha].sub.2]-adrenoceptors, respectively, on the renal pelvic sensory nerve fibers. Norepinephrine-induced activation of the sensory nerves is dependent on renal pelvic synthesis/release of [PGE.sub.2]. substance P; EP4 receptor; pelvis; prazosin; rauwolscine

Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in nonmuscle cells. Synthetic integrin-binding peptide triggers [Ca.sup.2+] mobilization and contraction in vascular smooth muscle cells (VSMCs) of rat afferent arteriole, indicating that interactions between the ECM and integrins modulate vascular tone. To examine whether integrins transduce extracellular mechanical stress into intracellular [Ca.sup.2+] signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (natural ligand of [[alpha].sub.5][[beta].sub.1]-integrin in VSMCs). Pulling of fibronectin-coated beads with an electromagnet triggered [Ca.sup.2+] sparks, followed by global [Ca.sup.2+] mobilization. Paramagnetic beads coated with low-density lipoprotein, whose receptors are not linked to cytoskeleton, were minimally effective in triggering [Ca.sup.2+] sparks and global [Ca.sup.2+] mobilization. Preincubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of [Ca.sup.2+] sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of [[alpha].sub.5]and [[beta].sub.1]-integrins triggered [Ca.sup.2+] sparks simulating the effects of fibronectin-coated beads. Preincubation of microperfused afferent arterioles with ryanodine or integrin-specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular [Ca.sup.2+] signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles. myogenic response; paramagnetic bead: fluorescence confocal microscopy

Worsening renal function in the setting of human acute heart failure (AHF) predicts poor outcomes, such as rehospitalization and increased mortality. Understanding potential renoprotective mechanisms is warranted. The guanylate cyclase (GC) enzymes and their second messenger cGMP are the target of two important circulating neurohumoral systems with renoprotective properties. Specifically, natriuretic peptides (NP) released from the heart with AHF target particulate GC in the kidney, while the nitric oxide (NO) system is an activator of renal soluble GC. We hypothesized that both systems are essential to preserve renal excretory and hemodynamic function in AHF but with distinct roles. We investigated these roles in three groups of anesthetized dogs (6 each) with AHF induced by rapid ventricular pacing. After a baseline AHF clearance, each group received intrarenal vehicle (control), [N.sup.G]-monomethyl-L-arginine (L-NMMA), a competitive NO inhibitor (50 [micro]g x [kg.sup.-1] x [min.sup.-1]) or a specific NP receptor antagonist, HS-142-1 (0.5 mg/kg). We observed that intrarenal L-NMMA decreased renal blood flow (RBF) without significant decreases in glomerular filtration rate (GFR), urinary sodium excretion (UNaV), or urinary cGMP. In contrast, HS-142-1 resulted in a decrease in UNaV and cGMP excretion together with a reduction in GFR and an increase in distal fractional tubular sodium reabsorption. We conclude that in AHF, the NP system plays a role in maintaining sodium excretion and GFR, while the function of NO is in the maintenance of RBF. These studies have both physiological and therapeutic implications warranting further research into cardiorenal interactions in this syndrome of AHF.

There is a sex difference in hypertensive renal injury, with men experiencing greater severity and a more rapid progression of renal disease than women; however, the molecular mechanisms protecting against renal injury in women are unknown. The goal of this study was to determine whether sex hormones modulate blood pressure and the progression of albuminuria during the developmental phase of hypertension in male and female spontaneously hypertensive rats (SHR). Studies were also performed to examine how sex and sex hormones influence two major risk factors for albuminuria, overactivation of the renin-angiotensin system and oxidative stress. Blood pressure was measured by telemetry in gonad-intact and gonadectomized male and female SHR. Microalbumin excretion, measured over time, and macrophage infiltration were used to assess renal health. Male SHR had significantly higher blood pressures than female SHR, and gonadectomy decreased blood pressures in males with no effect in females. Male SHR displayed a gonad-sensitive increase in albuminuria over time, and female SHR had a gonad-sensitive suppression in macrophage infiltration. Female SHR had greater plasma ANG II levels and similar levels of renal cortical ANG II vs. levels shown in males but less ATe-receptor protein expression in the renal cortex. Female SHR also had a gonad-sensitive decrease in renal oxidative stress. Therefore, the renal protection afforded to female SHR is associated with lower blood pressure, decreased macrophage infiltration, and decreased levels of oxidative stress. gender; renin-angiotensin system; oxidative stress

We hypothesized that angiotensin subtype-2 receptor ([AT.sub.2]R) inhibits renal renin biosynthesis in young rats via nitric oxide (NO). We monitored changes in renal NO, cGMP, renal renin content (RRC), and ANG II in 4-wk-old rats in response to low sodium ([LNa.sup.+]) intake alone and combined with 8-h direct renal cortical administration of [AT.sub.1] receptor blocker valsartan (VAL), [AT.sub.2]R blocker PD123319 (PD), NO synthase inhibitor [N.sup.G]-nitro-L-arginine methyl ester (L-NAME), NO donor S-nitroso-N-acetyl penicillamine (SNAP), or guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,2-[alpha]] quinoxaline-l-one (ODQ). In addition, we monitored renal endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in response to VAL or PD. [LNa.sup.+], VAL, PD, L-NAME, and ODQ increased RRC, ANG II, and renin mRNA. PD and L-NAME decreased NO and cGMP, while SNAP reduced RRC, ANG II, renin mRNA, and reversed the effects of PD. PD also reduced eNOS and nNOS protein and mRNA. Combined treatment with PD, L-NAME, or ODQ and VAL reversed the effects of VAL and caused further increase in RRC, ANG II, renin mRNA, and protein. ODQ reversed the effects of SNAP. These data demonstrate that the renal [AT.sub.2] receptor decreases renal renin biosynthesis and ANG II production in young rats. Reversal of the PD effects by SNAP and SNAP effects by ODQ confirms that NO and cGMP mediate the [AT.sub.2] receptor inhibition of renal renin production. angiotensin II; [AT.sub.1] receptor; nitric oxide; valsartan