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.

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.

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.

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.

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.

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.

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 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.

Acute kidney injury: what part do toll-like receptors play?

The innate immune system plays an important role as a first response to tissue injury. This first response is carried out via germline-encoded receptors. Toll-like receptors (TLRs) are the first identified and best studied family of pattern recognition receptors. TLRs are expressed on a variety of cell types, including epithelial cells, endothelia, dendritic cells, monocytes/macrophages, and B- and T-cells. TLRs initiate innate immune responses and concurrently shape the subsequent adaptive immune response. They are sensors of both pathogens, through the exogenous pathogen-associated molecular patterns (PAMPs), and tissue injury, through the endogenous danger-associated molecular patterns (DAMPs). TLR signaling is critical in defending against invading microorganisms; however, sustained receptor activation is also implicated in the pathogenesis of inflammatory diseases. Ischemic kidney injury involves early TLR-driven immunopathology, and the resolution of inflammation is needed for rapid regeneration of injured tubule cells. Notably, the activation of TLRs also has been implicated in epithelial repair. This review focuses on the role of TLRs and their endogenous ligands within the inflammatory response of acute kidney injury.

Granulomatosis with polyangiitis: rapidly progressive necrotizing glomerulonephritis in a pediatric patient.

Granulomatosis with polyangiitis (GPA) is associated with a broad range of clinical manifestations including renal disease. It is a systemic vasculitis that is rarely encountered in children. We present a 14-year-old girl who suffered from pharyngitis 1 week before admittance to hospital. She was admitted for macroscopic hematuria and oliguria, under the possibility of nephritic syndrome. Renal failure with rapidly progressive glomerulonephritis occurred within 24 hours. Immunologic tests showed the presence of type-C anti-neutrophil cytoplasmic antibodies (c-ANCA with antiproteinase 3 specificity) and renal biopsy revealed pauci-immune crescentic focal necrotizing glomerulonephritis. Treatment including methylprednisolone and cyclophosphamide intravenous pulses allowed renal recovery after 3 weeks. The clinical, hematological, and biochemical parameters improved substantially, achieving remission. Granulomatosis with polyangiitis, although rare in children, should be considered in the above clinical scenario. This case underlines that knowledge of renal histology diagnosis and early aggressive immunosuppressive therapy are essential for the management of these patients.

Hsp70 regulation on Nox4/p22phox and cytoskeletal integrity as an effect of losartan in vascular smooth muscle cells.

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.

Comorbidities in chronic pediatric peritoneal dialysis patients: a report of the International Pediatric Peritoneal Dialysis Network.

UNLABELLED: BACKGROUND, OBJECTIVES, AND METHODS: Hospitalization and mortality rates in pediatric dialysis patients remain unacceptably high. Although studies have associated the presence of comorbidities with an increased risk for death in a relatively small number of pediatric dialysis patients, no large-scale study had set out to describe the comorbidities seen in pediatric dialysis patients or to evaluate the impact of those comorbidities on outcomes beyond the newborn period. In the present study, we evaluated the prevalence of comorbidities in a large international cohort of pediatric chronic peritoneal dialysis (CPD) patients from the International Pediatric Peritoneal Dialysis Network registry and began to assess potential associations between those comorbidities and hospitalization rates and mortality. RESULTS: Information on comorbidities was available for 1830 patients 0 - 19 years of age at dialysis initiation. Median age at dialysis initiation was 9.1 years [interquartile range (IQR): 10.9], median follow-up for calculation of hospitalization rates was 15.2 months (range: 0.2 - 80.9 months), and total follow-up time in the registry was 2095 patient-years. At least 1 comorbidity had been reported for 602 of the patients (32.9%), with 283 (15.5%) having cognitive impairment; 230 (12.6%), motor impairment; 167 (9.1%), cardiac abnormality; 76 (4.2%), pulmonary abnormality; 212 (11.6%), ocular abnormality; and 101 (5.5%), hearing impairment. Of the 150 patients (8.2%) that had a defined syndrome, 85% had at least 1 nonrenal comorbidity, and 64% had multiple comorbidities. The presence of at least 1 comorbidity was associated with a higher hospitalization rate [hospital days per 100 observation days: 1.7 (IQR: 5.8) vs 1.2 (IQR: 3.9), p = 0.001] and decreased patient survival (4-year survival rate: 73% vs 90%, p < 0.0001). CONCLUSIONS: Nearly one third of pediatric CPD patients in a large international cohort had at least 1 comorbidity, and multiple comorbidities were frequently reported among patients with a defined syndrome. Preliminary analysis suggests an association between comorbidity and poor outcome in those patients. As this powerful international registry matures, further multivariate analyses will be important to more clearly define the impact of comorbidities on hospitalization rates and mortality in pediatric CPD patients.

Angiotensin AT(1) receptor inhibition-induced apoptosis by RhoA GTPase activation and pERK1/2 signaling pathways in neonatal obstructive nephropathy.

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.

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.

Apoptosis modulated by oxidative stress and inflammation during obstructive nephropathy.

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.

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.