RESUMEN
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.
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Proteínas HSP70 de Choque Térmico/metabolismo , Hipertensión Renal/tratamiento farmacológico , Riñón/patología , Losartán/uso terapéutico , Nefritis/tratamiento farmacológico , Sustancias Protectoras/uso terapéutico , Animales , Transición Epitelial-Mesenquimal/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Losartán/farmacología , Sustancias Protectoras/farmacologíaRESUMEN
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.
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Lesión Renal Aguda , Infecciones por Escherichia coli , Síndrome Hemolítico-Urémico , Escherichia coli Shiga-Toxigénica , Lesión Renal Aguda/etiología , Lesión Renal Aguda/terapia , Niño , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/terapia , Síndrome Hemolítico-Urémico/complicaciones , Síndrome Hemolítico-Urémico/terapia , Humanos , Lipopolisacáridos , Estudios Retrospectivos , Toxina ShigaRESUMEN
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.
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Lesión Renal Aguda/tratamiento farmacológico , Antioxidantes/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Hipertensión/tratamiento farmacológico , Losartán/farmacología , Angiotensina II/metabolismo , Animales , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , NADPH Oxidasa 4/metabolismo , Ratas , Ratas Endogámicas SHRRESUMEN
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.
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Angiotensina II/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Losartán/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Animales , Cadherinas/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Adhesiones Focales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/genética , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Vinculina/metabolismoRESUMEN
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 10â¯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 10â¯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.
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Endocitosis , Síndrome Hemolítico-Urémico/metabolismo , Síndrome Hemolítico-Urémico/patología , Toxina Shiga/metabolismo , Receptor Toll-Like 4/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Enfermedad Aguda , Niño , Preescolar , Citocinas/sangre , Estudios de Seguimiento , Síndrome Hemolítico-Urémico/sangre , Humanos , Lactante , Receptores de Lipopolisacáridos/metabolismo , Monocitos/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
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.
Asunto(s)
Antioxidantes/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Túbulos Renales Proximales/citología , Losartán/farmacología , NADPH Oxidasas/metabolismo , Proteolisis/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Caveolina 1/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Masculino , Modelos Biológicos , NADPH Oxidasa 4 , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Especies Reactivas de Oxígeno/metabolismo , Receptor de Angiotensina Tipo 1/metabolismoRESUMEN
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.
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Proteínas de Transporte de Catión/fisiología , Células Epiteliales/fisiología , Túbulos Renales/citología , Túbulos Renales/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Animales , Apoptosis/fisiología , Tamaño de la Célula , Supervivencia Celular/fisiología , Humanos , Transporte Iónico/fisiología , Transducción de Señal/fisiología , Intercambiador 1 de Sodio-HidrógenoRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
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Antihipertensivos/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Losartán/farmacología , Músculo Liso Vascular/efectos de los fármacos , NADPH Oxidasas/metabolismo , Angiotensina II/farmacología , Animales , Caveolina 1/metabolismo , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Proteínas del Choque Térmico HSP72/antagonistas & inhibidores , Proteínas del Choque Térmico HSP72/genética , Proteínas del Choque Térmico HSP72/metabolismo , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , NADPH Oxidasa 4 , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Wistar , Receptor de Angiotensina Tipo 1/química , Receptor de Angiotensina Tipo 1/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
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.
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Síndrome Hemolítico-Urémico/metabolismo , Leucocitos/metabolismo , Receptor Toll-Like 4/genética , Preescolar , Citocinas/sangre , Femenino , Regulación de la Expresión Génica , Humanos , Lactante , Masculino , Monocitos/química , Neutrófilos/química , Receptor Toll-Like 4/sangreRESUMEN
Perturbation of renal tubular antioxidants and overproduction of reactive oxygen species may amplify the proinflammatory state of renal obstruction, culminating in oxidative stress and tubular loss. Here, we analyzed the heat shock protein 70 (Hsp70) response and the function and signal transduction of NF-E2-related protein 2 (Nrf2) transcription factor on oxidative stress modulation in obstruction. Rats were subjected to unilateral ureteral obstruction or sham operation and kidneys harvested at 5, 7, 10, and 14 days after obstruction. Hsp70 expression and Nrf2 activity and its downstream target gene products were assessed. After 10 and 14 days of obstruction, enhanced lipid peroxidation through higher thiobarbituric acid reactive substances levels and increased oxidative stress resulted in reduced total antioxidant activity and enhanced nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity were demonstrated. This was accompanied by decreased inducible Hsp70 expression and a progressive reduction of nuclear Nrf2 and its target gene products glutathione S-transferase A2 (GSTA2) and NADPH/quinone oxidoreductase 1 (NQO1), whereas the Nrf2 repressor Kelch-like ECH-associated protein-1 (Keap1) was upregulated. By contrast, on early obstruction for 7 days, lack of increased oxidative markers associated with higher inducible Hsp70 protein levels and a rapid nuclear accumulation of Nrf2, Keap1 downregulation, and mRNA induction of the identified Nrf2-dependent genes, NQO1 and GSTA2, were shown. For these results, we suggest that the magnitude of cytoprotection in early obstruction depends on the combined contribution of induced activation of Nrf2 upregulating its downstream gene products and Hsp70 response. Impaired ability to mount the biological response to the prevailing oxidative stress leading to renal injury was shown in prolonged obstruction.
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Proteínas HSP70 de Choque Térmico/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Proteínas/metabolismo , Obstrucción Ureteral/metabolismo , Animales , Animales Recién Nacidos , Citoprotección/genética , Femenino , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Proteínas HSP70 de Choque Térmico/fisiología , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/genética , Isoenzimas/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch , Corteza Renal/metabolismo , Corteza Renal/patología , Peroxidación de Lípido , Masculino , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , NADPH Oxidasas/metabolismo , Ratas , Ratas Wistar , Transducción de Señal , Tiobarbitúricos/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
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.
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Fluorobencenos/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Enfermedades Renales/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Pirimidinas/farmacología , Sulfonamidas/farmacología , Obstrucción Ureteral/metabolismo , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Caspasa 3/análisis , Caspasa 3/metabolismo , Citoprotección , Femenino , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/etiología , Enfermedades Renales/patología , Masculino , NADPH Oxidasas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas WKY , Rosuvastatina Cálcica , Superóxido Dismutasa/metabolismo , Obstrucción Ureteral/complicaciones , Proteína X Asociada a bcl-2/metabolismoRESUMEN
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.