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.

eNOS/Hsp70 interaction on rosuvastatin cytoprotective effect in neonatal obstructive nephropathy.

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/BcL(2) 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.

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 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.