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Angiotensin II exerts a cardinal role in the pathogenesis of hypertension and renal injury via action of angiotensin II type 1 (AT(1)) receptors. Local renin-angiotensin system (RAS) activity is essential for the mechanisms mediating pathophysiological functions. Proximal tubular angiotensinogen and tubular AT(1) receptors are augmented by intrarenal angiotensin II. Caveolin 1 plays an important role as a regulatory molecule for the compartmentalization of redox signaling events through angiotensin II–induced NADPH oxidase activation in the kidney. A role for the renin-angiotensin system in the development and/or maintenance of hypertension has been demonstrated in spontaneously hypertensive rats (SHRs). Many effects of angiotensin II are dependent on the AT(1) stimulation of reactive oxygen species (ROS) production by NADPH oxidase. Angiotensin II upregulation stimulates oxidative stress in proximal tubules from SHR. The NADPH oxidase 4 (Nox4) is abundantly expressed in kidney proximal tubule cells. Induction of the stress response includes synthesis of heat shock protein 70, a molecular chaperone that has a critical role in the recovery of cells from stress and in cytoprotection, guarding cells from subsequent insults. HSP70 chaperones function in part by driving the molecular triage decision, which determines whether proteins enter the productive folding pathway or result in client substrate ubiquitination and proteasomal degradation. This review examines regulation of losartan-mediated antioxidative stress responses by the chaperone HSP70 in proximal tubule cells of spontaneously hypertensive rats.

Hypertensive nephrosclerosis is the second most common cause of end-stage renal disease after diabetes. For years, hypertensive kidney disease has been focused on the afferent arterioles and glomeruli damage and the involvement of the renin angiotensin system (RAS). Nonetheless, in recent years, novel evidence has demonstrated that persistent high blood pressure injures tubular cells, leading to epithelial–mesenchymal transition (EMT) and tubulointerstitial fibrosis. Injury primarily determined at the glomerular level by hypertension causes changes in post-glomerular peritubular capillaries that in turn induce endothelial damage and hypoxia. Microvasculature dysfunction, by inducing hypoxic environment, triggers inflammation, EMT with epithelial cells dedifferentiation and fibrosis. Hypertensive kidney disease also includes podocyte effacement and loss, leading to disruption of the filtration barrier. This review highlights the molecular mechanisms and histologic aspects involved in the pathophysiology of hypertensive kidney disease incorporating knowledge about EMT and tubulointerstitial fibrosis. The role of the Hsp70 chaperone on the angiotensin II–induced EMT after angiotensin II type 1 receptor (AT1R) blockage, as a possible molecular target for therapeutic strategy against hypertensive renal damage is discussed.

Renal injury related to hypertension is characterized by glomerular and tubulointerstitial damage. The overactivation of the renin-angiotensin system mainly by angiotensin II (AII) seems to be a main contributor to progressive renal fibrosis. Epithelial to mesenchymal transition (EMT) is a mechanism that promotes renal fibrosis. Owing to heat shock protein 70 (Hsp70) cytoprotective properties, the chaperone exhibits an important potential as a therapeutic target. We investigate the role of Hsp70 on Angiotensin II induced epithelial mesenchymal transition within the Losartan effect in proximal tubule cells (PTCs) from a genetic model of hypertension in rats (SHR). Methods: Primary cell culture of PTCs from SHR and Wistar Kyoto (WKY) rats were stimulated with AII, treated with Losartan (L), (L+AII) or untreated (Cc ). The functional Hsp70 role in Losartan effect, after silencing its expression by cell transfection, was determined by Immunofluorescence; Western blotting; Gelatin Zymography assays; Scratch wound assays; flow cytometry; and Live Cell Time-lapse microscopy. Results: (L) and (L+AII) treatments induced highly organized actin filaments and increased cortical actin in SHR PTCs. However, SHR PTCs (Cc ) and (AII) treated cells showed disorganized actin. After Hsp72 knockdown in SHR PTCs, (L) was unable to stabilize the actin cytoskeleton. We demonstrated that (L) and (L+AII) increased E-cadherin levels and decreased vinculin, α-SMA, vimentin, pERK, p38 and Smad2-3 activation compared to (AII) and (Cc ) SHR PTCs. Moreover, (L) inhibited MMP-2 and MMP-9 secretion, reduced migration and cellular displacement, stabilizing intercellular junctions. Notably, (L) treatment in shHsp72 knockdown SHR PTCs showed results similar to SHR PTCs (Cc ). Conclusion: Our results demonstrate that Losartan through Hsp70 inhibits the EMT induced by AII in proximal tubule cells derived from SHR. Fil: Costantino, Valeria Victoria. Universidad Nacional de Cuyo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Bocanegra, María Victoria. Universidad Nacional de Cuyo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Cacciamani, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Gil Lorenzo, Andrea Fernanda. Universidad Nacional de Cuyo; Argentina Fil: Benardon, María Eugenia. Universidad Nacional de Cuyo; Argentina Fil: Vallés, Patricia G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo; Argentina

In patients with diabetic kidney disease (DKD), plasma renin activity is usually decreased, but there is limited information on urinary renin and its origin. Urinary renin was evaluated in samples from patients with longstanding type I diabetes mellitus and mice with streptozotocin-induced diabetes mellitus. Renin-reporter mouse model (Ren1d-Cre;mT/ mG) was made diabetic with streptozotocin to examine whether the distribution of cells of the renin lineage was altered in a chronic diabetic environment. Active renin was increased in urine samples from patients with DKD (n=36), compared with those without DKD (n=38; 3.2 versus 1.3 pg/mg creatinine; P

The sodium-hydrogen exchanger isoform-1 [NHE1] is a ubiquitously expressed plasma membrane protein that plays a central role in intracellular pH and cell volume homeostasis by catalyzing an electroneutral exchange of extracellular sodium and intracellular hydrogen. Outside of this important physiological function, the NHE1 cytosolic tail domain acts as a molecular scaffold regulating cell survival and actin cytoskeleton organization through NHE1-dependent signaling proteins. NHE1 plays main roles in response to physiological stress conditions which in addition to cell shrinkage and acidification, include hypoxia and mechanical stimuli, such as cell stretch. NHE1-mediated modulation of programmed cell death results from the exchanger-mediated changes in pHi, cell volume, and/or [Na+]I; and, it has recently become known that regulation of cellular signaling pathways are involved as well. This review focuses on NHE1 functions and regulations. We describe evidence showing how these structural actions integrate with ion translocation in regulating renal tubule epithelial cell survival.

Background: Angiotensin II/Angiotensin II type 1 receptor (AT1R) effects are dependent on ROS production stimulated by NADPH oxidase activation. Hsp70 regulates a diverse set of signaling pathways through their interactions with proteins. CHIP is a E3 ubiquitin ligase that targets proteins for polyubiquitination and degradation. Aim: We study whether Hsp70/CHIP contribute to the negative regulation of Nox4 after AT1R blockage. Methods/Results: Primary culture of proximal tubule epithelial cells (PTCs) from SHR and WKY were stimulated with Angiotensin II (AII) or treated with Losartan (L) or Losartan plus Angiotensin II (L+AII). Losartan decreased AT1R and Nox4 while enhancing caveolin-1 and Hsp70 protein expression in SHR PTCs. Immunoprecipitation and immunofluorescence proved interaction and colocalization of increased Hsp70/CHIP with decreased Nox4 in SHR PTCs (L) vs (All). Hsp72 knockdown resulted in enhanced Nox4 protein levels, NADPH oxidase activity and ROS generation in (L+AII) revealing that Losartan was unable to abrogate AII effects on Nox4 expression and oxidative activity. Moreover, MG132 exposed PTCs (L) demostrated blocked ubiquitinated Nox4 degradation and increased colocalization of Nox4/Ubiquitin by inmunofluorescence. Conversely, Hsp72 depletion reduced Nox4/Ubiquitin colocalization causing Nox4 upregulation due to proteosomal degradation inhibition, although Losartan treatment. Conclusion: Our study demonstrates that Hsp70 and CHIP mediates the ubiquitination and proteasomal degradation of Nox4 as part ofthe antioxidative effect of Losartan in SHR. Fil: Costantino, Valeria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Gil Lorenzo, Andrea Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Lopez Appiolaza, Carlos Martìn. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Cacciamani, Valeria. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Cátedra de Fisiología Patológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Benardon, María Eugenia. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Cátedra de Fisiología Patológica; Argentina Fil: Bocanegra, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Garramuño, Patricia. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Cátedra de Fisiología Patológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina

A series of signaling cascades are activated after angiotensin II binds to angiotensin II type I receptor (AT1R), a peptide that is an important mediator of oxidative stress. Hsp70 regulates a diverse set of signaling pathways through interactions with proteins. Here, we tested the hypothesis of angiotensin II AT1R inhibition effect on Hsp70 interaction with Nox4/p22phox complex and Hsp70 leading to actin cytoskeleton modulation in spontaneously hypertensive rats (SHR) vascular smooth muscle cells (VSMCs). SHR and Wistar-Kyotto rats (VSMCs from 8 to 10 weeks) were stimulated with angiotensin II (100 nmol/L) for 15 min (AII), treated with losartan (100 nmol/L) for 90 min (L), and with losartan for 90 min plus angiotensin in the last 15 min (L + AII). Whereas SHR VSMCs exposure to angiotensin II overexpressed AT1R and Nox4 nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase and slightly downregulated caveolin-1 expression, losartan decreased AT1R protein levels and increased caveolin-1 and Hsp70 expression in SHR VSMC membranes. Immunoprecipitation and immunofluorescence confocal microscopy proved interaction and colocalization of membrane translocated Hsp70 and Nox4/p22phox. Increased levels of Hsp70 contrast with the decreased immunoprecipitation of Nox4/p22phox and RhoA in membranes from SHR VSMCs (L) vs SHR VSMCs (AII). Hsp72 depletion resulted in higher Nox4 expression and increased NADPH oxidase activity in VSMCs (L + AII) from SHR when contrasted with nontransfected VSMCs (L + AII). After Hsp72 knockdown in SHR VSMCs, losartan could not impair angiotensin II-enhanced stress fiber formation and focal adhesion assembly. In conclusion, our data showing a negative regulation of Hsp70 on Nox4/p22phox demonstrates a possible mechanism in explaining the antioxidative function joined to cytoskeletal integrity modulation within the effects of losartan in VSMCs from SHR. Fil: Gil Lorenzo, Andrea Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Bocanegra, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Benardon, María Eugenia. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Departamento de Patología; Argentina Fil: Cacciamani, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Departamento de Patología; Argentina Fil: Vallés, Patricia G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Departamento de Patología; Argentina

Kidney apoptosis and fibrosis are an inevitable outcome of progressive chronic kidney diseases where congenital obstructive nephropathy is the primary cause of the end-stage renal disease in children, and is also a major cause of renal failure in adults. The injured tubular cells linked to interstitial macrophages, and myofibroblasts produce cytokines and growth factors that promote an inflammatory state in the kidney, induce tubular cell apoptosis, and facilitate the accumulation of extracellular matrix. Angiotensin II plays a central role in the renal fibrogenesis at a very early stage leading to a rapid progression in chronic kidney disease. The increasing levels of angiotensin II induce pro-inflammatory cytokines, NF-κB activation, adhesion molecules, chemokines, growth factors, and oxidative stress. Furthermore, growing evidence reports that angiotensin II (a pro-inflammatory hormone) increases the mitochondrial oxidative stress regulating apoptosis induction. This review summarizes our understanding about possible mechanisms that contribute to apoptosis modulated by inflammation and/or oxidative stress during obstructive nephropathy. The new concept of antiinflammatory tools regulating mitochondrial oxidative stress will directly affect the inflammatory process and apoptosis. This idea could have attractive consequences in the treatment of renal and other inflammatory pathologies.

Background Caveolin is required to traffic the AT 1 receptor through the exocytic pathway. The chaperone Hsp70 regulates a diverse set of signaling pathways via their interactions with proteins. Method Here we examined the AT 1 receptor antagonist Losartan effect on caveolin-1 and Hsp70 protein association in spontaneously hypertensive rat (SHR) proximal tubules. Hsp70 involvement in Losartan oxidative stress regulation was also studied. Five-week-old SHRs were randomized for receiving Losartan (40mg/kg per day) (SHRLos) or no treatment (SHRH 2 O) during 6 weeks. Wistar-Kyoto rats (WKY) were normotensive controls. Results By western blotting, the relative abundance of caveolin-1 was two-fold higher in microdissected proximal tubule membrane fractions from treated SHRs vs. WKYH 2 O. Hsp70 membrane translocation was demonstrated in SHRLos through out the up-regulation of Hsp70 expression in microdissected proximal tubule membrane fractions when compared with WKYH 2 O (P

Metabolic alkalosis is a common feature of hypokalemic hypertensive syndromes associated with angiotensin II excess. The alkalosis-generating effect of angiotensin II is usually ascribed to its stimulatory effect on aldosterone secretion, a hormone that upregulates collecting duct hydrogen ion secretion. We studied the effect of angiotensin II infusions on the expression of B1 and a4 protein, subunits of the renal H + -ATPase in adrenalectomized rats. Adrenalectomized rats were given either angiotensin II or vehicle for 7 days via osmotic mini-pumps. H + -ATPase B1 protein expression was evaluated by Western blot analysis in isolated medulla and cortex plasma membrane preparations from one kidney, whereas the contralateral kidney was used for immunostaining. By Western blotting, the relative abundance of B1 protein was 2-fold higher in renal medulla membranes from rats with intact adrenal glands (sham surgery) than from adrenalectomized rats (219±47%, n=12; P P + -ATPase B1 subunit protein immunoreactivity was found in medullary collecting duct segments of rats infused with angiotensin II. In contrast to B1, expression of a4, another subunit of the H + -ATPase was not altered by adrenalectomy or angiotensin II. We conclude that adrenalectomy decreases whereas angiotensin II increases H + -ATPase B1 subunit expression in medullary, but not in cortical collecting ducts. By increasing the relative abundance of the B1 subunit of H + -ATPase in the collecting duct, angiotensin II excess may lead to increased hydrogen ion secretion and thus metabolic alkalosis—a common feature of hypertensive syndromes associated with angiotensin II overactivity.

Angiotensin II, a potent vasoconstrictor, also participates in the regulation of renal sodium and water excretion, not only via a myriad of effects on renal hemodynamics, glomerular filtration rate, and regulation of aldosterone secretion, but also via direct effects on renal tubule transport. In addition, angiotensin II stimulates H+secretion and HCO3–reabsorption in both proximal and distal tubules and regulates H+-ATPase activity in intercalated cells of the collecting tubule. Different results regarding the effect of angiotensin II on bicarbonate reabsorption and proton secretion have been reported at the functional level, depending on the angiotensin II concentration and tubule segment studied. It is likely that interstitial angiotensin II is more important in regulating hemodynamic and transport functions than circulating angiotensin II. In proximal tubules, stimulation of bicarbonate reabsorption, Na+/H+-exchange, and Na+/HCO3–cotransport has been found using low concentrations (–9M), while inhibition of bicarbonate reabsorption has been documented using concentrations higher than 10–8M. Evidence for the regulation of H+-ATPase activityin vivoandin vitroby trafficking/exocytosis has been provided. An additional level of H+-ATPase regulation via protein synthesis may be important as well. Recently, we have shown that both aldosterone and angiotensin II provide such a mechanism of regulationin vivoat the level of the medullary collecting tubule. Interestingly, in this part of the nephron, the effects of aldosterone and angiotensin II are not sodium dependent, whereas in the cortical collecting duct, both aldosterone and angiotensin II, by contrast, affect H+secretion by sodium-dependent mechanisms.

H + -ATPase activity on unilateral ureteral obstruction: Interaction of endogenous nitric oxide and angiotensin II. Background A number of cytokines, vasoactive compounds, chemoattractant molecules, and growth factors are up-regulated in obstruction. Following the onset of ureteral obstruction, angiotensin II production is rapidly stimulated. Cytokine-induced expression of inducible nitric oxide synthase (iNOS) has been reported in primary cultures of inner medullary collecting duct (IMCD) cells. We found that the defective urinary acidification in unilateral ureteral obstruction (UUO) includes an intensive decrease in bafilomycin-sensitive H + -ATPase activity in microdissected IMCD segments. Methods To investigate the interaction between endogenous nitric oxide and angiotensin II on H + -ATPase activity, we used microdissected IMCD segments of unilaterally obstructed, contralateral, and control kidneys to measure the bafilomycin-sensitive ATPase activity and nitric oxide synthase (NOS) activity. The generated NO was also evaluated. Results Preincubation of obstructed IMCD segments in the presence of a competitive inhibitor of NOS, N G -nitro-L-arginine methyl ester (L-NAME) 1 mmol/L, and in the presence of a specific inhibitor of calcium/calmodulin-independent NOS (iNOS), aminoguanidine 1 mmol/L, each for 60 minutes, significantly increased bafilomycin-sensitive H + -ATPase. A greater increase on iNOS activity (fmol [ 3 H] citrulline/min/μg protein) and a lesser increase in calcium/calmodulin-dependent NOS activity (cNOS) were observed in the obstructed renal medulla. This inhibitory effect of obstruction was abolished when IMCDs were incubated with 10 -5 to 10 -8 mol/L losartan. Decreasing doses of the angiotensin II type 1 (AT 1 ) receptor inhibitor caused an increase in bafilomycin-sensitive H + -ATPase, with a maximum increase at 10 -8 mol/L losartan. A decrease on iNOS activity was demonstrated in the obstructed renal medulla incubated with losartan in concentrations of 10 -5 to 10 -8 mol/L, the same losartan concentrations that showed recovery of vacuolar H + -ATPase activity. Similarly, a decrease on the generation of NO after incubation with losartan 10 -5 to 10 -8 mol/L was shown. Conclusion From these results, we suggest that endogenous NO increased by iNOS is involved in the inhibition of H + -ATPase activity in obstructed IMCD segments. The recovery of H + -ATPase activity in IMCD of obstructed kidneys induced by losartan may be related to a decrease of inducible NOS activity.

Renal kallikrein is localized in the connecting tubule cells and secreted into the tubular fluid at late distal nephron segments. The present experiments were performed to further test the hypothesis that renal kallikrein reduces bicarbonate secretion of cortical collecting duct (CCD). The effect of orthograde injections of pig pancreatic kallikrein (1 or 3 μg/ml) into the renal tubular system was investigated. Urine fractions (Fr) were collected after a 2-min stop flow. Changes in the urine fraction with respect to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/Fr:Ff polyfructosan) increased significantly in the first two urine fractions collected after glandular kallikrein administration (kallikrein, 1 μg/ml, P < 0.05; kallikrein, 3 μg/ml, P < 0.01).[Formula: see text] secretion of collecting ducts was significantly reduced dose dependently by orthograde and also reduced by retrograde pig pancreatic kallikrein administration. Release of kinins into the fractions was not affected by the retrograde kallikrein injection, even though the kallikrein activity increased considerably (2.26 ± 0.2 vs. 1.55 ± 0.2, P < 0.05). Adequacy of retrograde injections for delivering substances to the CCD was demonstrated by injecting colloidal mercury and detecting the appearance of this mercury in the renal cortex by transmission electron microscopy. The integrity of the renal tissue after a retrograde ureteral injection was confirmed by scanning electron microscopy. These results confirm and extend previous data (M. Marin-Grez and P. Vallés. Renal Physiol. Biochem. 17: 301–306, 1994; and M. Marin-Grez, P. Vallés, and P. Odigie. J. Physiol. 488: 163–170, 1995) showing that renal kallikrein reduces bicarbonate secretion at the CCD, probably by inhibiting [Formula: see text]transported by a mechanism unrelated to its kininogenase activity. Support for this assessment was obtained in experiments testing the effect of kallikrein on the luminal bicarbonate secretion of a subpopulation of Madin-Darby canine kidney cells capable of extruding the anion. Kallikrein inhibited[Formula: see text]/Cl−exchange, and the degree of inhibition was dose dependent. This inhibition occurred in the absence of kininogen in the bathing solution.

Intrarenal renin-Angiotensin system (RAS) activity is increased during early development and is further enhanced by unilateral ureteral obstruction (UUO). We studied the involvement of mitogen-activated protein (MAP) kinase members and the RhoA GTPase signaling pathways on the regulation of renal cell response after AT1 Angiotensin II receptor inhibition in obstruction. Neonatal rats subjected to sham operation or complete UUO within the first 48 hours of life received saline vehicle, Losartan (AT1 inhibitor), or PD-123319 (AT2 inhibitor) during the first 14 days of life. Cortex tubular epithelial cell apoptotic response was shown by TUNEL and confirmed by electron microscopy associated with mitochondrial signaling pathway through the increased proapoptotic ratio Bax/BcL-2, and consequently increased caspase 3 expression and activity in obstructed kidney before and after Type 1 (AT1) receptor blockade. Non injury of contralateral kidney was shown. The convergence of two independent signal pathways, the RhoA GTPase and pERK and concurrent inhibition of JNK MAP kinase, were required for the apoptotic response in 14 day kidney obstructed tubular cells either with or without Losartan treatment. Absence of increased AT2 protein expression after AT1 receptor inhibition on day 14 of obstruction was shown. Selective AngiotensinAT2-receptor inhibition with PD-123319 had no protective effect on the renal response to complete 14 day UUO. We suggest a role of both RhoA GTPase activation and the opposing actions of the ERK and JNK-MAP kinase signaling pathways as events involved in tubular cell apoptosis regulation in neonatal UUO. The selective AT1-receptor inhibition had no effect on the renal cellular response in the kidney subjected to UUO for 14 days.

Perturbation of renal tubular antioxidants and overproduction of reactive oxygen species may amplify the proinflammatory state of renal obstruction, culminating in oxidative stress and tubular loss. Here, we analyzed the heat shock protein 70 (Hsp70) response and the function and signal transduction of NF-E2-related protein 2 (Nrf2) transcription factor on oxidative stress modulation in obstruction. Rats were subjected to unilateral ureteral obstruction or sham operation and kidneys harvested at 5, 7, 10, and 14 days after obstruction. Hsp70 expression and Nrf2 activity and its downstream target gene products were assessed. After 10 and 14 days of obstruction, enhanced lipid peroxidation through higher thiobarbituric acid reactive substances levels and increased oxidative stress resulted in reduced total antioxidant activity and enhanced nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity were demonstrated. This was accompanied by decreased inducible Hsp70 expression and a progressive reduction of nuclear Nrf2 and its target gene products glutathione S-transferase A2 (GSTA2) and NADPH/quinone oxidoreductase 1 (NQO1), whereas the Nrf2 repressor Kelch-like ECH-associated protein-1 (Keap1) was upregulated. By contrast, on early obstruction for 7 days, lack of increased oxidative markers associated with higher inducible Hsp70 protein levels and a rapid nuclear accumulation of Nrf2, Keap1 downregulation, and mRNA induction of the identified Nrf2-dependent genes, NQO1 and GSTA2, were shown. For these results, we suggest that the magnitude of cytoprotection in early obstruction depends on the combined contribution of induced activation of Nrf2 upregulating its downstream gene products and Hsp70 response. Impaired ability to mount the biological response to the prevailing oxidative stress leading to renal injury was shown in prolonged obstruction.

There is growing evidence that statins may exert renoprotective effects beyond cholesterol reduction. The cholesterol-independent or “pleiotropic” effects of statins include the upregulation of endothelial nitric oxide synthase (eNOS). Here we determined whether eNOS associated with Hsp70 expression is involved in rosuvastatin resistance to obstruction-induced oxidative stress and cell death. Neonatal rats subjected to unilateral ureteral obstruction (UUO) within two days of birth and controls were treated daily with vehicle or rosuvastatin (10 mg/kg/day) for 14 days. Decreased endogenous nitric oxide (NO) and lower mRNA and protein eNOS expression associated with downregulation of heat shock factor 1 (Hsf1) mRNA and Hsp70 protein levels were observed in the obstructed kidney cortex. Increased nicotinamide adenine dinucleotide phosphate (NADHP) oxidase activity and apoptosis induction, regulated by mitochondrial signal pathway through an increased pro-apoptotic Bax/BcL2 ratio and caspase 3 activity, were demonstrated. Conversely, in cortex membrane fractions from rosuvastatin-treated UUO rats, marked upregulation of eNOS expression at transcriptional and posttranscriptional levels linked to increased Hsf1 mRNA expression and enhanced mRNA and protein Hsp70 expression, were observed. Consequently, there was an absence of apoptotic response and transiently decreased NADPH oxidase activity. In addition, interaction between eNOS and Hsp70 was determined by communoprecipitation in cortex membrane fractions, showing an increased ratio of both proteins, after rosuvastatin treatment in obstructed kidney. In summary, our data demonstrate that the effect of rosuvastatin on eNOS interacting with Hsp70, results in the capacity of both to prevent mitochondrial apoptotic pathway and oxidative stress in neonatal early kidney obstruction.

Potassium depletion has been associated with altered sodium reabsorption in tubule segments. We studied if the altered abundance of Na(+) transporters and ROMK are associated with distal potassium secretion that contributes to the development of hypokalemia in protein-deprived rats. After weaning, Wistar rats were fed with a low-protein diet (8%, LP) for 14 days and then recovered with a normal-protein (NP) diet (24%, RP). An age-matched control group was fed with an NP diet (24%, NP). We showed hypokalemia, lower glomerular filtration rate and higher FEK(+) in the LP group. Immunoblotting revealed that the type 3 Na(+)/H(+) exchanger in the cortex was decreased in the LP group. However, the type 2 Na(+)-K(+)-2Cl(-) cotransporter was increased in the outer stripe of the outer medulla in the LP group. The abundance of the aldosterone-regulated Na(+)-Cl(-) cotransporter (NCC) and epithelial Na(+) channel (ENaC) was higher in the LP group and was associated with higher plasma aldosterone level. ROMK protein levels were increased. Na(+)/K(+)-ATPase protein levels were the same in both groups. After the recovery period, the expression of Na(+) transporters and ROMK returned to control values. We conclude that increased expression of NCC, ENaC subunits, and ROMK contributed to distal potassium secretion leading to enhanced potassium excretion, which may explain the hypokalemia resulting from LP feeding. A role of aldosterone may be suggested.

A low-protein (LP) diet induces injury from energy depletion in renal epithelial cells. Overexpression of heat-shock proteins has been implicated in the restoration of the cytoskeletal anchorage of Na(+)/K(+)-ATPase. We tested if Hsp70 stabilizes renal Na(+)/K(+)-ATPase attachment to the cytoskeleton from the cortex and the outer stripe of the outer medulla (OSOM) in rats during recovery from a LP diet. Rats were fed with a LP diet (8% protein) for 14 days, and then the rats were recovered with a 24% protein (RP) diet. The control group received a 24% protein (NP) diet. Increased Na(+)/K(+)-ATPase dissociation was demonstrated in soluble fraction from OSOM with lower ATP content as a result of LP diet vs NP. Meanwhile, decreased Hsp70 levels in the same fraction were shown. Translocation of Hsp70 to the cytoskeletal injured fraction associated with stabilization of Na(+)/K(+)-ATPase was shown in OSOM from LP after in vitro co-incubation of the cytoskeletal fraction of LP and non-cytoskeletal fraction of RP. These effects were abolished by the addition of the anti-Hsp70 antibody. Absence of Na(+)/K(+)-ATPase detachment from its cytoskeletal anchorage was demonstrated in proximal duct segments from cortex in LP. Co-immunoprecipitation showed that the amount of Na(+)/K(+)-ATPase co-precipitating with Hsp70 increased in the OSOM as a result of the LP diet. In the cortex tissues from rats fed the LP and the RP diet, the interaction of both proteins were similar to the control groups. Our results indicate that Hsp70 has a critical role in protecting the integrity of the cytoskeletal anchorage of Na(+)/K(+)-ATPase during recovery from ATP-depleted injury resulting from LP in OSOM.

OBJECTIVE To examine the participation of NHE1 in the regulation of the apoptotic response in neonatal obstruction, as the ubiquitously expressed NHE1 isoform is an important component of regulatory volume increase. MATERIALS AND METHODS Rats had a unilateral ureteric obstruction (UUO) or a sham operation, and the kidneys were harvested 5 and 14 days afterward. Cellular apoptosis in proximal tubules (PT) and collecting ducts (CD) was assessed using a standard assay, and NHE1 expression in the renal cortex assessed using reverse transcription-polymerase chain reaction and Western blots. Mitochondrial apoptosis was evaluated by Bax/BcL2 expression, and caspase-3 expression and activity. In addition, we evaluated the in vivo administration of increasing doses of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on NHE1 inhibition associated with the induction of apoptosis. RESULTS After 14 days there were consistently more apoptotic cells in CD than in PT, associated with a lower expression of NHE1 at the mRNA and protein levels. There was increased expression of the Bax/BcL2 ratio, linked to decreased pro-caspase-3 protein levels and with increased caspase-3 activation. NHE1 inhibition by increasing doses of EIPA induced epithelial cell apoptosis and increased caspase-3 activity in a dose-dependent manner. After in vitro incubation with amiloride (100 mm) there was less NHE1 expression associated with reduced 32 kDa pro-caspase-3 protein levels. Kidneys obstructed for 5 days showed no changes in NHE1 expression or induction of apoptosis. CONCLUSION In neonatal obstruction, we suggest that the decreased NHE1 expression could be a signal-transduction event participating in the induction of epithelial tubular cell apoptosis, through the regulation of the BcL-2 gene family and activation of caspase-3.

The vacuolar H + -ATPase is a multisubunit protein consisting of a peripheral catalytic domain (V 1 ) that binds and hydrolyzes adenosine triphosphate (ATP) and provides energy to pump H + through the transmembrane domain (V 0 ) against a large gradient. This proton-translocating vacuolar H + -ATPase is present in both intracellular compartments and the plasma membrane of eukaryotic cells. Mutations in genes encoding kidney intercalated cell–specific V 0 a4 and V 1 B1 subunits of the vacuolar H + -ATPase cause the syndrome of distal tubular renal acidosis. This review focuses on the function, regulation, and the role of vacuolar H + -ATPases in renal physiology. The localization of vacuolar H + -ATPases in the kidney, and their role in intracellular pH (pHi) regulation, transepithelial proton transport, and acid-base homeostasis are discussed.

Enhanced expression of the genes that encode for components of the renin-angiotensin system (RAS) has been exhibited in low-protein-fed (LP) rats. We examined distal proton secretion in LP rats through the activity of H(+)-ATPase on microdissected CCD, OMCD and IMCD segments. The effect of angiotensin II AT(1) receptor inhibition and protein recovery (24% protein) on H(+)-ATPase activity was studied.Bafilomycin-sensitive H(+)-ATPase activity on CCD, OMCD and IMCD segments of LP (protein 8%) and control rats CP (protein 24%) was evaluated. We examined the levels of mRNA expression of RAS components: angiotensin-converting enzyme (ACE), angiotensinogen and angiotensin II AT(1) expression; AT(1 )receptor binding and distribution were determined by quantitative autoradiography.Increased ACE and AT(1) mRNA expression was found in cortex and medulla of LP compared to NP rats. AT(1) receptor binding density was significantly reduced in renal cortex and inner stripe of the outer medulla of LP compared to NP rats. Minimal radioligand binding was shown in inner medulla of LP. Whole kidney expression of angiotensinogen was unaltered in LP. H(+)-ATPase activity significantly decreased in IMCDs and OMCDs of LP. The inhibitory effect of LP was abolished when OMCD segments were incubated for 60 min in the presence of losartan 10(-6) to 10(-8)M. There was no effect of losartan concentrations from 10(-6) to 10(-8) M on IMCDs. Similar results were observed on H(+)-ATPase activity in OMCD and IMCD segments after readministration of 24% protein in the diet.Both the recovery of H(+)-ATPase activity in OMCD segments induced by losartan and the increased expression of AT(1 )receptor suggest angiotensin II modulation of proton ATPase activity on this duct segments in LP rats. Intense compromise of proton secretion through the continued H(+)-ATPase inhibition in IMCDs from LP was shown.

The effect of altering the acid-base status on urinary kallikrein excretion of barbiturate-anaesthetized rats was investigated. Alkalosis was induced in a group of rats by intravenous (i.v.) infusion of NaOH at 0.45 mmol x h(-1) for 30 min. Acidosis was induced in two groups of rats by i.v. infusion of HCl at 1.5 mmol x h(-1) for 30 min (uncompensated acidosis) or 0.15 mmol x h(-1) for 3 h (compensated acidosis), respectively. Time controls received 0.45 mmol x h(-1) NaCl. Rats with alkalosis excreted less kallikrein than their controls (P0.05). Rats with uncompensated acidosis excreted more active kallikrein (P0.05), whereas rats with compensated acidosis excreted similar amounts when compared with their respective controls. In rats with uncompensated acid-base derangements, the urinary kallikrein excreted per millilitre of glomerular filtrate was correlated with blood H+ activity (r = 0.99, P0.01). Arterial blood pressure, haematocrit, glomerular filtration rate, urine flow rate and Na+ and K+ excretions of experimental and control animals did not differ. Thus, renal kallikrein secretion into the tubular fluid appears to be regulated by blood proton activity. This, along with our previous demonstration that kallikrein inhibits HCO3- secretion into the tubular lumen (Renal Physiol 17:301-306, 1994; J Physiol (Lond) 488:163-170, 1995), indicates that this enzyme is part of a feedback loop regulating acid-base balance.

The luminal membrane of collecting duct cells, especially the intercalated cells, is normally exposed to active kallikrein. This is the consequence of the specific localization of this renal enzyme in the connecting tubule cells and its principal route of secretion being into the tubular lumen. It is conceivable that kallikrein acts downstream on a transporter involved in distal bicarbonate handling. To investigate this possibility, we estimated bicarbonate concentration and measured kallikrein amidolytic activity in urine fractions collected after a classical stop-flow experiment in rabbits. A highly significant inverse correlation was found between these parameters (r = -0.94, p < 0.001) in the peak kallikrein fractions. Neither sodium nor potassium concentration were correlated to kallikrein. This suggests that the physiological role of renal kallikrein may be to regulate extracellular fluid pH by inhibiting collecting duct bicarbonate secretion. To test the hypothesis that tubular fluid kallikrein activity and bicarbonate secretion are causally related, we developed a novel in vivo stop-flow injection model (‘orthograde stop-flow’). A hog-kallikrein containing solution (0.5 µg/ml) was injected through the abdominal aorta into the renal tubular system of one kidney of barbiturate-anesthetized rats, while the renal blood supply was interrupted. The ureter was then occluded and renal blood perfusion reinitiated. After a 2-min contact time five 125-µl urine fractions were collected. Bicarbonate secretion was clearly detected in the second and third fractions (i.e. those coming from the collecting ducts) of the control animals, which had received only the vehicle. There was no bicarbonate secretion peak in the corresponding urine fractions collected from kallikrein-injected animals. We conclude that intraluminal kallikrein inhibits collecting duct bicarbonate secretion, probably by inhibiting the chloride/bicarbonate exchanger of β-intercalated cells.

The cellular stress response can mediate cellular protection through expression of heat shock protein (Hsp70), which can interfere with the process of apoptotic cell death. Factors regulating renal epithelial cell apoptosis include angiotensin II. In the present study, we have examined the relationship between the Hsp70 expression and the apoptotic pathway in the kidneys from low-protein-fed rats (8% protein). The possible cytoprotective role of Hsp70 has been evaluated during low-protein feeding and after reincorporation of 24% protein in the diet. The effect of angiotensin II AT1 receptor inhibition has also been studied. Rats were fed with a low-protein (LP) diet (8% protein) for 14 days, and then the animals were recovered by means of a normal protein diet (24% protein) (RP) for 14, 21, and 30 days, and control rats received 24% protein (NP) in the diet. LP and NP rats treated with Losartan (10 mg/kg) were also evaluated. The following methods were performed on the kidneys: terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay for apoptosis, reverse transcriptase-polymerase chain reaction assay for AT1, Bax, and Bcl-2 messenger ribonucleic acid (mRNA) expression, and immunohistochemical and Western blot for Hsp70 and caspase 3 protein expression and activity. In the LP group, the cells of the medullary ducts (MDs) showed increased apoptosis associated with weak immunoreaction for Hsp70 and decreased Hsp70 protein levels. In these animals, enhanced proapoptotic ratio Bax/Bcl-2 linked to decreased procaspase 3 protein levels with increased caspase 3 activation were demonstrated. A cytoprotection attributed to Hsp70 could be noted in the RP rats after 21 days of reincorporation of the normal diet, and in the LP-fed group treated with Losartan. In these cases, the MD cells displayed decreased apoptosis and increased Hsp70 expression in colocalization staining, and high Hsp70 levels in cytosolic fraction. A decreased proapoptotic ratio Bax/Bcl-2, associated with increased Bcl-2 mRNA, was also observed. Our results provide evidence for an antiapoptotic, cytoprotective effect of Hsp70 in kidney MD cells of rats with LP intake, when the animals were recovered with 24% protein in diet and after angiotensin II AT1 receptor inhibition. Angiotensin II seems to play a role in the pathogenesis of tubule epithelial cell apoptosis during LP feeding.

Losartan modulation on NOS isoforms and COX-2 expression in early renal fibrogenesis in unilateral obstruction. Background Angiotensin II plays a central role in the initiation of renal fibrogenesis at a very early stage leading to a rapid progression in unilateral ureteral obstruction (UUO). We examined the effect of an angiotensin II receptor inhibitor (AT 1 ) losartan, independent from its effects on blood pressure, on nitric oxide synthase (NOS) isoforms and cyclooxygenase-2 (COX-2) expression and the significance of this interaction on interstitial fibrosis in UUO. Methods Rats underwent UUO for 24hours or control sham operation after been treated with losartan in the drinking water at 10mg/kg/day for 15days. AT 1 receptor binding and distribution was determined by in situ autoradiographic study. Renal fibrosis was evaluated through the relative volume of the tubulointerstitium (Vv) measured by an image analyzer, and transforming growth factor-β (TGF-β) at mRNA levels. NOS activity, expression of NOS isoforms by reverse transcription-polymerase chain reaction (RT-PCR) assay and COX-2 protein expression, were determined. Results After administration of a nonhypotensive dose of losartan prevention of renal fibrogenesis was demonstrated in obstructed kidneys by means of Vv values and TGF-β mRNA expression near controls. Decreased AT 1 receptor binding density was observed in cortex and inner stripe of the outer medulla of nontreated obstructed kidney compared to control, whereas no differences were observed in ipsilateral UUO related to obstructed kidney–treated group. The increased inducible NOS (iNOS) activity and expression of obstructed kidney medulla, increased neuronal NOS (nNOS), and endothelial NOS (eNOS) isoforms expression and COX-2 protein expression in obstructed kidney cortex showed down-regulation of iNOS, nNOS, and COX-2 with persistent levels of eNOS after losartan administration. Conclusion These results allowed us to infer an interstitial fibrogenesis prevention independent action of losartan, involving NOS isoforms and COX-2, in unilateral obstructive nephropathy.