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.

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.

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.

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.

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.

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.

Effect of red wine on adipocytokine expression and vascular alterations in fructose-fed rats.

BACKGROUND: Imbalance in adipocytokines secretion is related to the development of metabolic syndrome (MS). In addition, moderate consumption of red wine (RW) decreases the risk of cardiovascular disease. The aim of this study was to evaluate the effects of moderate consumption of RW or ethanol (E) on adiponectin and resistin expression, and vascular alterations in fructose-fed rats (FFRs) as an experimental model of MS. METHODS: Thirty-day-old male Wistar rats were assigned to control (C), F (10% fructose in drinking water), F+E (4.5 ml/kg), and F+RW (35 ml/kg of Malbec RW containing 4.5 ml/kg E). E and RW were administered during the last 4 weeks of a 10-week period. RESULTS: RW administration to F rats was able to significantly decrease insulin resistance, mesenteric adipose tissue weight, and systolic blood pressure (SBP) compared to F group. F+E only reduced the SBP (P < 0.05 vs. F). F+RW also reduced aortic NAD(P)H-oxidase activity, NAD(P)H subunits Nox4 expression in mesenteric tissue, plasma thiobarbituric acid reactive substances (TBARS), and recovered plasma total antioxidant activity (TAA) compared to F and F+E groups (P < 0.05). Adiponectin expression decreased, whereas resistin, vascular cell adhesion molecules-1 (VCAM-1), and nuclear factor-κB (NF-κB) expression and vascular remodeling in mesenteric arteries were higher in F than in C group (P < 0.05). Only RW was able to partially reverse the aforementioned alterations. CONCLUSION: In this study, Malbec RW, but not alcohol alone, improved the balance of adipocytokines and attenuated the oxidative stress and vascular inflammation in a model of MS, suggesting that nonalcohol components of RW are responsible for the beneficial effects.

WT-1 mRNA expression is modulated by nitric oxide availability and Hsp70 interaction after neonatal unilateral ureteral obstruction

Wilms tumor gene 1 (wt-1), a key regulator of mesenchymal-epithelial transformation, is downregulated during congenital obstructive nephropathy, leading to apoptosis. There is a functional interaction between WT-1 and inducible nitric oxide synthase (iNOS). In this regard, we reported that after neonatal unilateral ureteral obstruction, rosuvastatin prevents apoptosis through an increase in nitric oxide bioavailability, which in turn is linked to higher Hsp70 expression. Hence, the goal of this study was to determine whether a nitric oxide/Hsp70 interaction is involved in changes in WT-1 mRNA expression after ureteral obstruction. Neonatal rats submitted to experimental ureteral obstruction were treated with either vehicle or rosuvastatin for 14 days. Decreased nitric oxide and iNOS/Hsp70 expression associated wit h WT-1 low expression was shown in obstructed kidneys. Apoptosis was induced and it was associated with an increased Bax/BcL2 ratio. Conversely, iNOS/Hsp70 upregulation and an increased WT-1 mRNA expression, without an apoptotic response, were observed in the cortex of obstructed kidneys of rosuvastatin-treated rats. Nitric oxide also modulated Hsp70 and WT-1 mRNA expression in MDCK cells. Finally, in vivo experiments with nitric oxide modulators support our hypothesis that WT-1 mRNA expression is associated with nitric oxide level. Results suggest that rosuvastatin may modulate WT-1 mRNA expression through renal nitric oxide bioavailability, preventing neonatal obstruction-induced apoptosis associated with Hsp70 interaction.

WT-1 mRNA expression is modulated by nitric oxide availability and Hsp70 interaction after neonatal unilateral ureteral obstruction

Wilms tumor gene 1 (wt-1), a key regulator of mesenchymal-epithelial transformation, is downregulated during congenital obstructive nephropathy, leading to apoptosis. There is a functional interaction between WT-1 and inducible nitric oxide synthase (iNOS). In this regard, we reported that after neonatal unilateral ureteral obstruction, rosuvastatin prevents apoptosis through an increase in nitric oxide bioavailability, which in turn is linked to higher Hsp70 expression. Hence, the goal of this study was to determine whether a nitric oxide/Hsp70 interaction is involved in changes in WT-1 mRNA expression after ureteral obstruction. Neonatal rats submitted to experimental ureteral obstruction were treated with either vehicle or rosuvastatin for 14 days. Decreased nitric oxide and iNOS/Hsp70 expression associated wit h WT-1 low expression was shown in obstructed kidneys. Apoptosis was induced and it was associated with an increased Bax/BcL2 ratio. Conversely, iNOS/Hsp70 upregulation and an increased WT-1 mRNA expression, without an apoptotic response, were observed in the cortex of obstructed kidneys of rosuvastatin-treated rats. Nitric oxide also modulated Hsp70 and WT-1 mRNA expression in MDCK cells. Finally, in vivo experiments with nitric oxide modulators support our hypothesis that WT-1 mRNA expression is associated with nitric oxide level. Results suggest that rosuvastatin may modulate WT-1 mRNA expression through renal nitric oxide bioavailability, preventing neonatal obstruction-induced apoptosis associated with Hsp70 interaction.(AU)

WT-1 mRNA expression is modulated by nitric oxide availability and Hsp70 interaction after neonatal unilateral ureteral obstruction.

Wilms tumor gene 1 (wt-1), a key regulator of mesenchymal-epithelial transformation, is downregulated during congenital obstructive nephropathy, leading to apoptosis. There is a functional interaction between WT-1 and inducible nitric oxide synthase (iNOS). In this regard, we reported that after neonatal unilateral ureteral obstruction, rosuvastatin prevents apoptosis through an increase in nitric oxide bioavailability, which in turn is linked to higher Hsp70 expression. Hence, the goal of this study was to determine whether a nitric oxide/Hsp70 interaction is involved in changes in WT-1 mRNA expression after ureteral obstruction. Neonatal rats submitted to experimental ureteral obstruction were treated with either vehicle or rosuvastatin for 14 days. Decreased nitric oxide and iNOS/Hsp70 expression associated with WT-1 low expression was shown in obstructed kidneys. Apoptosis was induced and it was associated with an increased Bax/BcL2 ratio. Conversely, iNOS/Hsp70 upregulation and an increased WT-1 mRNA expression, without an apoptotic response, were observed in the cortex of obstructed kidneys of rosuvastatin-treated rats. Nitric oxide also modulated Hsp70 and WT-1 mRNA expression in MDCK cells. Finally, in vivo experiments with nitric oxide modulators support our hypothesis that WT-1 mRNA expression is associated with nitric oxide level. Results suggest that rosuvastatin may modulate WT-1 mRNA expression through renal nitric oxide bioavailability, preventing neonatal obstruction-induced apoptosis associated with Hsp70 interaction.

Caveolin-1 and Hsp70 interaction in microdissected proximal tubules from spontaneously hypertensive rats as an effect of Losartan.

BACKGROUND: Caveolin is required to traffic the AT1 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 AT1 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 (40 mg/kg per day) (SHRLos) or no treatment (SHRH2O) 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. WKYH2O. Hsp70 membrane translocation was demonstrated in SHRLos through out the up-regulation of Hsp70 expression in microdissected proximal tubule membrane fractions when compared with WKYH2O (P < 0.001). Conversely, decreased Hsp70 protein levels were shown in microdissected proximal tubule cytosol fraction from SHRLos (P < 0.01). Interaction between caveolin-1 and Hsp70 was further determined by coimmunoprecipitation and by immunofluorescence co-localization in SHRLos proximal tubule membranes. After membrane translocation of Hsp70, the decreased NADPH oxidase activity (RFU/microprot per min incubation) near controls demonstrated on microdissected proximal tubule membranes from SHRLos vs. SHRH2O (P < 0.01) was reversed by the preincubation with anti-Hsp70 antibody. In addition, interaction between Hsp70 and Nox4 was determined by the coimmunoprecipitation strategy showing that membrane overexpression of Hsp70 was associated with decreased Nox4 after Losartan treatment in SHRs. CONCLUSION: After Losartan administration interaction of caveolin-1 and Hsp70 was shown in SHR proximal tubules. Translocation of Hsp70 to proximal tubule membranes in SHRLos might exert a cytoprotective effect by down-regulation of NADPH subunits Nox4.