Toll-like Receptor 4 in Acute Kidney Injury.

Acute kidney injury (AKI) is a common and devastating pathologic condition, associated with considerable high morbidity and mortality. Although significant breakthroughs have been made in recent years, to this day no effective pharmacological therapies for its treatment exist. AKI is known to be connected with intrarenal and systemic inflammation. The innate immune system plays an important role as the first defense response mechanism to tissue injury. Toll-like receptor 4 (TLR4) is a well-characterized pattern recognition receptor, and increasing evidence has shown that TLR4 mediated inflammatory response, plays a pivotal role in the pathogenesis of acute kidney injury. Pathogen-associated molecular patterns (PAMPS), which are the conserved microbial motifs, are sensed by these receptors. Endogenous molecules generated during tissue injury, and labeled as damage-associated molecular pattern molecules (DAMPs), also activate pattern recognition receptors, thereby offering an understanding of sterile types of inflammation. Excessive, uncontrolled and/or sustained activation of TLR4, may lead to a chronic inflammatory state. In this review we describe the role of TLR4, its endogenous ligands and activation in the inflammatory response to ischemic/reperfusion-induced AKI and sepsis-associated AKI. The potential regeneration signaling patterns of TLR4 in acute kidney injury, are also discussed.

Severely ill pediatric patients with Shiga toxin-associated hemolytic uremic syndrome (STEC-HUS) who suffered from multiple organ involvement in the early stage.

BACKGROUND: Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is the main cause of pediatric acute kidney injury (AKI) in Argentina. Endothelial injury is the trigger event in the microangiopathic process. The host inflammatory response to toxin and E. coli lipopolysaccharide (LPS) is involved in disease pathophysiology. METHODS: This retrospective study describes pediatric STEC-HUS patients with multiorgan involvement at the initial phase of disease. A retrospective study of critically ill HUS patients with evidence of E. coli infection was conducted through a period of 15 years. RESULTS: Forty-four patients 35.4 ± 4.1 months were admitted to the intensive care unit for 21 ± 2 days. Mechanical ventilation was required in 41 patients, early inotropic support in 37, and 28 developed septic shock. Forty-one patients required kidney replacement therapy for 12 ± 1 days. Forty-one patients showed neurological dysfunction. Dilated cardiomyopathy was demonstrated in 3 patients, left ventricular systolic dysfunction in 4, and hypertension in 17. Four patients had pulmonary hemorrhage, and acute respiratory distress syndrome in 2. Colectomy for transmural colonic necrosis was performed in 3 patients. Thirty-seven patients were treated with therapeutic plasma exchange, and 28 patients received methylprednisolone (10 mg/kg for 3 days). Of the surviving 32 patients, neurological sequelae were seen in 11 and chronic kidney failure in 5. CONCLUSIONS: Severe clinical outcome at onset suggests an amplified inflammatory response after exposure to Shiga toxin and/or E. coli LPS. STEC-HUS associated with severe neurological involvement, hemodynamic instability, and AKI requires intensive care and focused therapy.

Losartan through Hsp70 Avoids Angiotensin II Induced Mesenchymal Epithelial Transition in Proximal Tubule Cells from Spontaneously Hypertensive Rats.

BACKGROUND/AIMS: 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.

Rab7b participation on the TLR4 (Toll-like receptor) endocytic pathway in Shiga toxin-associated Hemolytic Uremic Syndrome (HUS).

BACKGROUND: The inflammatory response of the host to Shiga toxin and/or lipopolysaccharide (LPS) of Escherichia coli (E. coli) is included in (HUS). The TLR4-LPS complex is internalized and TLR4 induced inflammatory signaling is stopped by targeting the complex for degradation. Rab7b, a small guanosine triphosphatase (GTPase) expressed in monocytes, regulates the later stages of the endocytic pathway. OBJECTIVE: we studied the Rab7b participation on the TLR4 endocytic pathway and its effect on monocyte cytokine production along the acute course of pediatric Shiga toxin-associated HUS. METHODS AND RESULTS: Monocytes were identified according to their positivity in CD14 expression. Surface TLR4 expression in monocytes from 18 HUS patients significantly increased by day 1 to 6, showing the highest increase on day 4 compared to monocytes of 10 healthy children. Significant higher surface TLR4 expression was accompanied by increased proinflammatory intracellular cytokines, tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). In contrast, after these time points, surface TLR4 expression and intracellular TNF-α levels, returned to near control levels after 10 days. Furthermore, confocal immunofluorescence microscopy proved colocalization of increased intracellular TLR4/Rab7b determined by Pearson's coefficient in monocytes from HUS patients from day 1 on the highest colocalization of both proteins by day 4. Decreased TLR4/Rab7b colocalization was shown 10 days after HUS onset. CONCLUSION: The colocalization of TLR4 and Rab7b allows us to suggest Rab7b participation in the control of the TLR4 endocytic pathway in HUS patient monocytes. A consequential fall in cytokine production throughout the early follow up of HUS is demonstrated.

Heat Shock Protein 70 and CHIP Promote Nox4 Ubiquitination and Degradation within the Losartan Antioxidative Effect in Proximal Tubule Cells.

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 of the antioxidative effect of Losartan in SHR.

Physiological Functions and Regulation of the Na+/H+ Exchanger [NHE1] in Renal Tubule Epithelial Cells.

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.

RhoA and MAPK signal transduction pathways regulate NHE1-dependent proximal tubule cell apoptosis after mechanical stretch.

Mechanical deformation after congenital ureteral obstruction is traduced into biochemical signals leading to tubular atrophy due to epithelial cell apoptosis. We investigated whether Na(+)/H(+) exchanger 1 (NHE1) could be responsible for HK-2 cell apoptosis induction in response to mechanical stretch through its ability to function as a control point of RhoA and MAPK signaling pathways. When mechanical stretch was applied to HK-2 cells, cell apoptosis was associated with diminished NHE1 expression and RhoA activation. The RhoA signaling pathway was confirmed to be upstream from the MAPK cascade when HK-2 cells were transfected with the active RhoA-V14 mutant, showing higher ERK1/2 expression and decreased p38 activation associated with NHE1 downregulation. NHE1 participation in apoptosis induction was confirmed by specific small interfering RNA NHE1 showing caspase-3 activation and decreased Bcl-2 expression. The decreased NHE1 expression was correlated with abnormal NHE1 activity addressed by intracellular pH measurements. These results demonstrate that mitochondrial proximal tubule cell apoptosis in response to mechanical stretch is orchestrated by signaling pathways initiated by the small GTPase RhoA and followed by the opposing effects of ERK1/2 and p38 MAPK phosphorylation, regulating NHE1 decreased expression and activity.

Caveolin-1-eNOS/Hsp70 interactions mediate rosuvastatin antifibrotic effects in neonatal obstructive nephropathy.

Evidence suggesting that statins may contribute to renoprotection has been provided in experimental and clinical studies. Statins restore endothelial nitric oxide (NO) levels by mechanisms including up-regulation of endothelial NO synthase (eNOS) expression. Caveolin-1/eNOS interaction is essential preventing inadequate NO levels. Here, we evaluated whether caveolin-1 associated with eNOS/Hsp70 expression may be involved in the mechanism by which rosuvastatin exerts tubulointerstitial fibrosis protection in neonatal unilateral ureteral obstruction (UUO). Neonatal rats subjected to UUO within 2 days of birth and controls were treated daily with vehicle or rosuvastatin (10 mg/kg/day) by oral gavage for 14 days. After UUO, morphometric evaluation of interstitial fibrosis showed increased interstitial volume (Vv) associated with reduced NO availability, increased mRNA and protein caveolin-1 expression as well as downregulation eNOS and heat shock protein 70 (Hsp70) expression. Conversely, rosuvastatin treatment attenuated the fibrotic response linked to high NO availability, decreased mRNA and protein caveolin-1 expression, and marked upregulation of eNOS and Hsp70 expression at transcriptional and posttranscriptional levels. Moreover, protein-protein interactions determined by immunoprecipitation and by immunofluorescence co-localization have shown decreased caveolin-1/eNOS as well as increased Hsp70/eNOS interaction, after rosuvastatin treatment. A dose dependent effect of rosuvastatin on decreased caveolin-1 expression was shown in control cortex. In conclusion, our data suggest that statins contribute to the protection against tubulointerstitial fibrosis injury in neonatal early kidney obstruction by increased NO availability, involving interaction of up-regulated eNOS/Hsp70 and down-regulated caveolin-1.

Toll-like receptor 4 expression on circulating leucocytes in hemolytic uremic syndrome.

Lipopolysaccharide stimulation of toll-like receptor 4 (TLR4) activates signal transduction pathways leading to proinflammatory cytokine secretion. We investigated TLR4 surface receptor expression on peripheral blood neutrophils and monocytes and their ability to modulate inflammatory cytokine release in 15 patients 1, 3, and 10 days after hemolytic uremic syndrome (HUS) onset. Seven patients with Escherichia coli (EHEC)-associated diarrhea and seven healthy controls were also studied. Isolated leucocytes from HUS-onset patients exhibited significantly higher messenger RNA (mRNA) TLR4 expression than controls. Moreover, TLR4 protein expression on neutrophils, determined by flow cytometry, was upregulated, driving dependent proinflammatory cytokine, tumor necrosis factor alpha (TNF-α), and interleukin 8 (IL-8) increase, and decreased anti-inflammatory IL-10 release at HUS onset compared with patients with EHEC diarrhea and controls. TLR4 expression on neutrophils was positively correlated with serum TNF-α levels. Conversely, significant reduction of neutrophil TLR4 receptor expression and lack of cytokine-responsive element activation was shown in patients 3 and 10 days after HUS onset. No differences were demonstrated in TLR4 receptor expression on monocytes among the studied groups. Our results suggest TLR4 expression may be differently regulated on neutrophils and monocytes. They could be dynamically modulated across the early development of HUS on neutrophils, resulting in negative regulation preceded by TLR4 overactivation.

Molecular Mechanisms of Hypertensive Nephropathy: Renoprotective Effect of Losartan through Hsp70.

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.

The bone and mineral disorder of children undergoing chronic peritoneal dialysis.

The mineral and bone disorder of chronic kidney disease remains a challenging complication in pediatric end-stage renal disease. Here, we assessed symptoms, risk factors and management of this disorder in 890 children and adolescents from 24 countries reported to the International Pediatric Peritoneal Dialysis Network Registry. Signs of this disease were most common in North American patients. The prevalence of hyperphosphatemia increased with age from 6% in young infants to 81% in adolescents. Serum parathyroid hormone (PTH) was outside the guideline targets in the majority of patients and associated with low calcium, high phosphorus, acidosis, dialysis vintage and female gender. Serum calcium was associated with dialytic calcium exposure, serum phosphorus with low residual renal function and pubertal status. PTH levels were highest in Latin America and lowest in Europe. Vitamin D and its active analogs were most frequently administered in Europe; calcium-free phosphate binders and cinacalcet in North America. Clinical and radiological symptoms markedly increased when PTH exceeded 300 pg/ml, the risk of hypercalcemia increased with levels below 100 pg/ml, and time-averaged PTH concentrations above 500 pg/ml were associated with impaired longitudinal growth. Hence, the symptoms and management of the mineral and bone disorder of chronic kidney disease in children on peritoneal dialysis showed substantial regional variation. Our findings support a PTH target range of 100-300 pg/ml in the pediatric age group.

The renal antioxidative effect of losartan involves heat shock protein 70 in proximal tubule cells.

Angiotensin II exerts a cardinal role in the pathogenesis of hypertension and renal injury via action of angiotensin II type 1 (AT1) receptors. Local renin-angiotensin system (RAS) activity is essential for the mechanisms mediating pathophysiological functions. Proximal tubular angiotensinogen and tubular AT1 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 AT1 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.

Adiponectin in children on peritoneal dialysis: relationship to insulin resistance and nutritional status.

AIM: To study whether adiponectin and resistin serum concentrations in children on peritoneal dialysis (PD) were related to insulin resistance (IR) and anthropometric parameters of nutritional status, 11 PD patients, 9 chronic kidney disease (CKD) patients and 10 healthy children were studied. METHODS: Glucose and insulin were measured during the oral glucose tolerance test. Levels of adiponectin and resistin were evaluated by ELISA, insulin by RIA. RESULTS: In CKD patients, higher homeostasis model assessment-insulin resistance (HOMA-IR), fasting and 2-hour serum insulin levels were shown compared to control and to PD patients. Body mass index (BMI) and body fat content were severely decreased while serum adiponectin levels were significantly higher in PD patients relative to controls. No differences among groups were shown in resistin levels. On regression modeling, inverse independent associations were observed between adiponectin with percentile BMI, weight and height z-score, and with body fat content. In contrast, no relationship was found between adiponectin and IR parameters. In multiple regression analysis, adiponectin was negatively correlated with BMI. A negative association of adiponectin and resistin with glomerular filtration rate was also shown. CONCLUSION: A role for adiponectin in terms of its association with clinical wasting parameters in PD pediatric patients might be suggested.

Altered renal expression of Na(+) transporters and ROMK in protein-deprived rats.

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.

Urinary Renin in Patients and Mice With Diabetic Kidney Disease.

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<0.001). In mice with streptozotocin-induced diabetes mellitus, urine renin was also increased compared with nondiabetic controls. By immunohistochemistry, in mice with streptozotocin-induced diabetes mellitus, juxtaglomerular apparatus and proximal tubular renin staining were reduced, whereas collecting tubule staining, by contrast, was increased. To examine the role of filtration and tubular reabsorption on urinary renin, mice were either infused with either mouse or human recombinant renin and lysine (a blocker of proximal tubular protein reabsorption). Infusion of either form of renin together with lysine markedly increased urinary renin such that it was no longer different between nondiabetic and diabetic mice. Megalin mRNA was reduced in the kidney cortex of streptozotocin-treated mice (0.70±0.09 versus 1.01±0.04 in controls, P=0.01) consistent with impaired tubular reabsorption. In Ren1d-Cre;mT/mG with streptozotocin-induced diabetes mellitus, the distribution of renin lineage cells within the kidney was similar to nondiabetic renin-reporter mice. No evidence for migration of cells of renin linage to the collecting duct in diabetic mice could be found. Renin mRNA in microdissected collecting ducts from streptozotocin-treated mice, moreover, was not significantly different than in controls, whereas in kidney cortex, largely reflecting juxtaglomerular apparatus renin, it was significantly reduced. In conclusion, in urine from patients with type 1 diabetes mellitus and DKD and from mice with streptozotocin-induced diabetes mellitus, renin is elevated. This cannot be attributed to production from cells of the renin lineage migrating to the collecting duct in a chronic hyperglycemic environment. Rather, the elevated levels of urinary renin found in DKD are best attributed to altered glomerular filteration and impaired proximal tubular reabsorption.

Cytoprotective role of nitric oxide associated with Hsp70 expression in neonatal obstructive nephropathy.

Nitric oxide (NO) has emerged as an important endogenous inhibitor of apoptosis. In this study, we postulated that the mechanism of apoptosis inhibition by NO would include stimulation of heat shock protein 70 (Hsp70) expression. Rats were subjected to unilateral ureteral obstruction (UUO) or sham operation, and kidneys were harvested 5 and 14 days after obstruction. After 14 days of obstruction, decreased endogenous NO and lower inducible nitric oxide synthase (iNOS) expression at mRNA and protein levels associated with downregulation of Hsp70 protein expression were shown in apoptosis induction, regulated by mitochondrial signal pathway, through the increased pro-apoptotic ratio Bax/BcL(2) and consequently caspase 3 activity. Conversely, 5 days after kidney obstruction, increased Hsp70 expression linked to increase NO and iNOS expression at transcriptional and post-transcriptional levels with absence of apoptotic response, were demonstrated. In obstructed neonatal rats, in vivo administration of l-Arginine induced heat shock protein 70 (Hsp70) expression, which was associated with cytoprotection from apoptosis and transiently decreased nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activity. Opposite effects were obtained after nitro L-Arginine methyl ester (L-NAME) treatment. The interaction between B-cell lymphoma 2 anti-apoptotic members (BcL(2)) and Hsp70 in the presence of L-Arginine and L-NAME, was determined by coimmunoprecipitation. Binding of BcL(2) and Hsp70 increased after L-Arginine administration. These findings suggest that NO can produce resistance to obstruction-induced cell death by mitochondrial apoptotic pathway, through the induction of Hsp70 expression, in neonatal unilateral ureteral obstruction.

Hypokalemic Distal Renal Tubular Acidosis.

Distal renal tubular acidosis (DRTA) is defined as hyperchloremic, non-anion gap metabolic acidosis with impaired urinary acid excretion in the presence of a normal or moderately reduced glomerular filtration rate. Failure in urinary acid excretion results from reduced H+ secretion by intercalated cells in the distal nephron. This results in decreased excretion of NH4+ and other acids collectively referred as titratable acids while urine pH is typically above 5.5 in the face of systemic acidosis. The clinical phenotype in patients with DRTA is characterized by stunted growth with bone abnormalities in children as well as nephrocalcinosis and nephrolithiasis that develop as the consequence of hypercalciuria, hypocitraturia, and relatively alkaline urine. Hypokalemia is a striking finding that accounts for muscle weakness and requires continued treatment together with alkali-based therapies. This review will focus on the mechanisms responsible for impaired acid excretion and urinary potassium wastage, the clinical features, and diagnostic approaches of hypokalemic DRTA, both inherited and acquired.

Heat shock protein 70 expression is associated with inhibition of renal tubule epithelial cell apoptosis during recovery from low-protein feeding.

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.

H-ATPase activity in collecting duct segments in protein-deprived rats - role of angiotensin II on its regulation.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.