RESUMEN

Ischaemia-reperfusion (IR)-associated acute kidney injury (AKI) is a severe clinical condition that lacks effective pharmacological treatments. Our recent research revealed that pretreatment with the angiotensin II type 2 receptor (AT2R) agonist C21 alleviates kidney damage during IR. Primary cilia are organelles crucial for regulation of epithelial cell homeostasis, which are significantly affected by IR injury. This study aimed to evaluate the impact of AT2R activation on cilia integrity during IR and to identify pathways involved in the nephroprotective effect of C21. Rats were subjected to 40 min of unilateral ischaemia followed by 24 h of reperfusion. Immunofluorescence analysis of the kidneys showed that the nephroprotective effect of C21 was associated with preservation of cilia integrity in tubular cells. AT2R agonists increased α-tubulin acetylation in primary cilia in tubular cells in vivo and in a cell model. Analysis of ERK phosphorylation indicated that AT2R activation led to diminished activation of ERK1/2 in tubular cells. Similar to AT2R agonists, inhibitors of α-tubulin deacetylase HDAC6 or inhibitors of ERK activation ameliorated IR-induced cell death and preserved cilia integrity. Immunofluorescence analysis of tubular cells revealed significant ERK localization at primary cilia and demonstrated that ERK inhibition increased cilia levels of acetylated α-tubulin. Overall, our findings demonstrate that C21 elicits a preconditioning effect that enhances cilia stability in renal tubular cells, thereby preserving their integrity when exposed to IR injury. Furthermore, our results indicate that this effect might be mediated by AT2R-induced inhibition of ERK activation. These findings offer potential insights for the development of pharmacological interventions to mitigate IR-associated AKI. KEY POINTS: The AT2R agonist C21 prevents primary cilia shortening and tubular cell deciliation during renal ischaemia-reperfusion. AT2R activation inhibits ERK1/2 in renal tubular cells. Both AT2R agonists and ERK1/2 inhibitors increase alpha-tubulin acetylation at the primary cilium in tubular cells. AT2R activation, ERK1/2 inhibition or inhibition of alpha-tubulin deacetylation elicit protective effects in tubular cells subjected to ischaemia-reperfusion injury.

Asunto(s)

Cilios , Receptor de Angiotensina Tipo 2 , Daño por Reperfusión , Animales , Masculino , Ratas , Acetilación , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Cilios/metabolismo , Cilios/efectos de los fármacos , Imidazoles , Túbulos Renales/metabolismo , Túbulos Renales/patología , Sistema de Señalización de MAP Quinasas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 2/metabolismo , Receptor de Angiotensina Tipo 2/agonistas , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Sulfonamidas , Tiofenos , Tubulina (Proteína)/metabolismo

RESUMEN

Acetaminophen (APAP) is a widely prescribed analgesic and antipyretic drug. In the present work, we studied the effects of glutamine (Gln) in an in vivo model of APAP-induced nephrotoxicity in male Wistar rats. Renal function, histological characteristics, and Na+,K+-ATPase cortical abundance and distribution were analyzed. The appearance of HSP70 and actin in urine was also evaluated. Myeloperoxidase (MPO) activity in cortical tissue was measured as an index of the inflammatory response. Gln administration 30 min before APAP protected from the renal functional and histological damage promoted by APAP. Rats that received the dual treatment Gln and APAP (Gln/APAP) showed the same level of Na+,K+-ATPase cortical induction as APAP-treated animals, but the enzyme maintained its normal basolateral localization. HSP70 abundance was increased up to the same level in the Gln, APAP, and Gln/APAP groups. Urinary HSP70 and actin were detected only in the APAP-treated animals, reinforcing the protection of renal tubular integrity afforded by the Gln pretreatment. Gln pretreatment also protected from the increment in MPO activity promoted by APAP. Our results support the idea that Gln pretreatment could be a therapeutic option to prevent APAP-induced renal injury.

Asunto(s)

Acetaminofén/efectos adversos , Glutamina/uso terapéutico , Enfermedades Renales/inducido químicamente , Enfermedades Renales/tratamiento farmacológico , Sustancias Protectoras/uso terapéutico , Actinas/metabolismo , Animales , Modelos Animales de Enfermedad , Glutamina/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/orina , Riñón/efectos de los fármacos , Riñón/patología , Riñón/fisiopatología , Enfermedades Renales/patología , Enfermedades Renales/fisiopatología , Masculino , Peroxidasa/metabolismo , Sustancias Protectoras/farmacología , Transporte de Proteínas/efectos de los fármacos , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/metabolismo

RESUMEN

It has been proposed that, during metabolic acidosis, the liver downregulates mitochondrial ammonia detoxification via ureagenesis, a bicarbonate-consuming process. Since we previously demonstrated that hepatocyte mitochondrial aquaporin-8 channels (mtAQP8) facilitate the uptake of ammonia and its metabolism into urea, we studied whether mtAQP8 is involved in the liver adaptive response to acidosis. Primary cultured rat hepatocytes were adapted to acidosis by exposing them to culture medium at pH 7.0 for 40 h. Control cells were exposed to pH 7.4. Hepatocytes exposed to acid medium showed a decrease in mtAQP8 protein expression (-30%, p < 0.05). Ureagenesis from ammonia was assessed by incubating the cells with (15)N-labeled ammonia and measuring (15)N-labeled urea synthesis by nuclear magnetic resonance. Reduced ureagenesis was found in acidified hepatocytes (-31%, p < 0.05). In vivo studies in rats subjected to 7 days acidosis also showed decreased protein expression of hepatic mtAQP8 (-50%, p < 0.05) and reduced liver urea content (-35%; p < 0.05). In conclusion, our in vitro and in vivo data suggest that hepatic mtAQP8 expression is downregulated in acidosis, a mechanism that may contribute to decreased ureagenesis from ammonia in response to acidosis.

Asunto(s)

Acidosis/metabolismo , Amoníaco/metabolismo , Acuaporinas/metabolismo , Regulación hacia Abajo , Mitocondrias/metabolismo , Urea/metabolismo , Animales , Células Cultivadas , Masculino , Ratas , Ratas Wistar

RESUMEN

We recently reported that hepatocyte mitochondrial aquaporin-8 (mtAQP8) channels facilitate the uptake of ammonia and its metabolism into urea. Here we studied the effect of bacterial lipopolysaccharides (LPS) on ammonia-derived ureagenesis. In LPS-treated rats, hepatic mtAQP8 protein expression and diffusional ammonia permeability (measured utilizing ammonia analogues) of liver inner mitochondrial membranes were downregulated. NMR studies using 15N-labeled ammonia indicated that basal and glucagon-induced ureagenesis from ammonia were significantly reduced in hepatocytes from LPS-treated rats. Our data suggest that hepatocyte mtAQP8-mediated ammonia removal via ureagenesis is impaired by LPS, a mechanism potentially relevant to the molecular pathogenesis of defective hepatic ammonia detoxification in sepsis.

Asunto(s)

Amoníaco/metabolismo , Acuaporinas/metabolismo , Hepatocitos/metabolismo , Lipopolisacáridos/farmacología , Mitocondrias Hepáticas/metabolismo , Urea/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Glucagón/fisiología , Hepatocitos/inmunología , Masculino , Metilaminas/metabolismo , Mitocondrias Hepáticas/inmunología , Ratas , Ratas Wistar

RESUMEN

Mitochondrial ammonia synthesis in proximal tubules and its urinary excretion are key components of the renal response to maintain acid-base balance during metabolic acidosis. Since aquaporin-8 (AQP8) facilitates transport of ammonia and is localized in inner mitochondrial membrane (IMM) of renal proximal cells, we hypothesized that AQP8-facilitated mitochondrial ammonia transport in these cells plays a role in the response to acidosis. We evaluated whether mitochondrial AQP8 (mtAQP8) knockdown by RNA interference is able to impair ammonia excretion in the human renal proximal tubule cell line, HK-2. By RT-PCR and immunoblotting, we found that AQP8 is expressed in these cells and is localized in IMM. HK-2 cells were transfected with short-interfering RNA targeting human AQP8. After 48 h, the levels of mtAQP8 protein decreased by 53% (P < 0.05). mtAQP8 knockdown decreased the rate of ammonia released into culture medium in cells grown at pH 7.4 (-31%, P < 0.05) as well as in cells exposed to acid (-90%, P < 0.05). We also evaluated mtAQP8 protein expression in HK-2 cells exposed to acidic medium. After 48 h, upregulation of mtAQP8 (+74%, P < 0.05) was observed, together with higher ammonia excretion rate (+73%, P < 0.05). In vivo studies in NH(4)Cl-loaded rats showed that mtAQP8 protein expression was also upregulated after 7 days of acidosis in renal cortex (+51%, P < 0.05). These data suggest that mtAQP8 plays an important role in the adaptive response of proximal tubule to acidosis possibly facilitating mitochondrial ammonia transport.

Asunto(s)

Acidosis/metabolismo , Acuaporinas/metabolismo , Túbulos Renales Proximales/metabolismo , Mitocondrias/metabolismo , Animales , Animales Modificados Genéticamente , Acuaporinas/genética , Análisis de los Gases de la Sangre , Western Blotting , Línea Celular , Supervivencia Celular , Humanos , Corteza Renal/metabolismo , Túbulos Renales Proximales/citología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Microscopía Confocal , Compuestos de Amonio Cuaternario/orina , Interferencia de ARN , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

Cumulative evidence demonstrated effective downstream metabolism of pregnenolone in renal tissue. The aim of this study was to evaluate the expression and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1), which converts cholesterol into pregnenolone, in adult rat kidney. Immunohistochemical labeling for CYP11A1 was observed in renal cortex and medulla, on structures identified as distal convoluted tubule and thick ascending limb of Henle's loop, respectively. Immunoblotting analysis corroborated the renal expression of the protein in inner mitochondrial membrane fractions. The incubation of isolated mitochondria with the membrane-permeant cholesterol analogue 22R-hydroxycholesterol resulted in efficient formation of pregnenolone, the immediate precursor for the synthesis of all the steroid hormones. The low progesterone production rate observed in these experiments suggested a poor activity of 3ß-hydroxysteroid dehydrogenase enzyme in renal mitochondria. The steroidogenic acute regulatory protein (StAR), involved in the mitochondrial import of cholesterol, was detected in renal tissue at both mRNA and protein level. Immunostaining for StAR showed similar distribution to that observed for CYP11A1. The expression of StAR and CYP11A1 was found to be higher in medulla than in cortex. This enhanced expression of steroidogenesis-related proteins correlated with a greater pregnenolone synthesis rate and higher steroid hormones tissular content measured in medulla. In conclusion, we have established the expression and localization of StAR and CYP11A1 protein, the ability of synthesizing pregnenolone and a region-specific content of sex hormones in the adult rat kidney. These data clearly show that the kidney is a steroid hormones synthesizing organ. It is proposed that the existence in the kidney of complete steroidogenic machinery would respond to a physiological significance.

Asunto(s)

Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Riñón/enzimología , Animales , Proteínas Portadoras/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Riñón/anatomía & histología , Masculino , Membranas Mitocondriales/enzimología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Pregnenolona/biosíntesis , Progesterona/biosíntesis , Ratas , Ratas Wistar , Receptores de GABA-A/metabolismo , Testosterona/metabolismo

RESUMEN

Acetaminophen (APAP) is an analgesic-antipyretic drug widely used in children. In the present study, we used an in vivo model of APAP-induced nephrotoxicity in male Wistar rats. We analyzed whether toxic doses of APAP could induce heat shock protein 70 (HSP70) in the kidney and whether HSP70 could be detected in urine. Renal function and histological evaluation of the kidneys were performed at different times after APAP administration (1,000 mg/kg body weight i.p.). Cellular injury was assessed by Triton X-100 solubilization of Na(+)/K(+) ATPase. Renal and hepatic glutathione levels were also measured. Urinary N-acetyl-beta-D glucosaminidase (NAG) excretion increased 4 h after intoxication. At this time, urea and creatinine were at control levels and a slight degree of histological alteration was detected. Kidney microscopic evaluation, Na(+)/K(+) ATPase solubility, creatinine, and urea levels and NAG excretion did not differ from those of controls 48 h after APAP administration. HSP70 was detected in urine obtained from 4 to 24 h after APAP administration. HSP70 abundance in renal cortex was increased at early time points and 48 h after APAP administration. Urinary HSP70 excretion would be a marker of its renal induction combined with the loss of tubule integrity. NAG would be a suitable early biomarker of APAP-induced nephrotoxicity.

Asunto(s)

Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Proteínas HSP70 de Choque Térmico/biosíntesis , Enfermedades Renales/metabolismo , Acetilglucosaminidasa/orina , Animales , Creatinina/sangre , Modelos Animales de Enfermedad , Glutatión/metabolismo , Proteínas HSP70 de Choque Térmico/orina , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/inducido químicamente , Enfermedades Renales/patología , Pruebas de Función Renal , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Urea/sangre

RESUMEN

BACKGROUND/AIMS: Contributions to the understanding of acute renal failure (ARF) pathogenesis have not been translated into an effective clinical therapy. We studied the effects of pretreatment with the angiotensin II type 1 (AT1) receptor blocker, losartan, on renal function, tissue injury, inflammatory response and serum aldosterone levels in a model of ischemic ARF. METHODS: Rats underwent unilateral renal ischemia followed by 24 h of reperfusion (IR), and were pretreated or not with 8 (IRL8) or 80 (IRL80) mg/kg/day of losartan for 3 days. RESULTS: IR kidneys showed marked renal dysfunction, epithelial damage, capillary congestion, increased myeloperoxidase (MPO) activity and increased TNF-alpha, IL1-beta and IL-6 mRNA levels. IRL80 kidneys showed protection against dysfunction and tissue injury, associated with normal MPO activity and cytokine mRNA levels. The lower dose was not able to achieve the same degree of functional renoprotection and could not prevent an increase of MPO or proinflammatory cytokine mRNA levels. The high losartan dose completely prevented an increase of serum aldosterone levels induced by IR. CONCLUSION: Renoprotection of the high losartan dose would be mainly mediated by its anti-inflammatory actions. Our results show a potential pathophysiological role of AT1 activation in promoting renal dysfunction, structural injury, inflammation and aldosterone elevation after IR injury.

Asunto(s)

Lesión Renal Aguda/etiología , Lesión Renal Aguda/prevención & control , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Riñón/irrigación sanguínea , Losartán/uso terapéutico , Receptor de Angiotensina Tipo 1/fisiología , Daño por Reperfusión/complicaciones , Daño por Reperfusión/prevención & control , Enfermedad Aguda , Animales , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Ratas , Ratas Wistar

RESUMEN

The aim of the present work was to study the effects of an unilateral ischaemic-reperfusion injury on Na+, K+-ATPase activity, alpha1 and beta1 subunits protein and mRNA abundance and ATP content in cortical and medullary tissues from postischaemic and contralateral kidneys. Right renal artery was clamped for 40 min followed by 24 and 48 h of reperfusion. Postischaemic and contralateral renal function was studied cannulating the ureter of each kidney. Postischaemic kidneys after 24 (IR24) and 48 (IR48) hours of reperfusion presented a significant dysfunction. Na+, K+-ATPase alpha1 subunit abundance increased in IR24 and IR48 cortical tissue and beta1 subunit decreased in IR48. In IR24 medullary tissue, alpha1 abundance increased and returned to control values in IR48 while beta1 abundance was decreased in both periods. Forty minutes of ischaemia without reperfusion (I40) promoted an increment in alpha1 mRNA in cortex and medulla that normalised after 24 h of reperfusion. beta1 mRNA was decreased in IR24 medullas. No changes were observed in contralateral kidneys. This work provides evidences that after an ischaemic insult alpha1 and beta1 protein subunit abundance and mRNA levels are independently regulated. After ischaemic-reperfusion injury, cortical and medullary tissue showed a different pattern of response. Although ATP and Na+, K+-ATPase activity returned to control values, postischemic kidney showed an abnormal function after 48 h of reflow.

Asunto(s)

Daño por Reperfusión/enzimología , Daño por Reperfusión/fisiopatología , ATPasa Intercambiadora de Sodio-Potasio/genética , Adenosina Trifosfato/análisis , Animales , Regulación de la Expresión Génica , Corteza Renal/irrigación sanguínea , Corteza Renal/metabolismo , Corteza Renal/patología , Médula Renal/irrigación sanguínea , Masculino , Subunidades de Proteína/análisis , Subunidades de Proteína/genética , ARN Mensajero/análisis , Ratas , Ratas Wistar , Arteria Renal/patología , ATPasa Intercambiadora de Sodio-Potasio/análisis

RESUMEN

The aim of this work was to study the effects of chronic administration of aluminum (Al) on the urinary concentrating and diluting mechanisms in the distal tubules and collecting ducts. Male Wistar rats were chronically treated with aluminum lactate for 12 weeks (0.575 mg Al/100g of body weight, i.p., three times per week). After 12 weeks, renal function of control and Al-treated rats was evaluated by clearance techniques. To study urinary concentrating mechanisms, renal function was also measured in control and Al-treated rats deprived of water, after the administration of desmopressin (vasopressin agonist) and after the infusion of hypertonic saline at increasing infusion rates. Sodium and water balance were impaired. We found decreased urinary concentrating ability in situations in which endogenous (thirst or infusion of hypertonic saline) or exogenous plasma antidiuretic hormone was increased. Solute-free water formation, measured during the infusion of hypotonic saline showed normal transport in the thick ascending limb. Aquaporin-2 (AQP2) expression was measured by Western blot to evaluate water permeability in collecting ducts. We found that Al produced downregulation of AQP2 in plasma membranes and intracellular vesicles, that could account for the impaired water handling. Administration of desmopressin increased AQP2 in plasma membranes, suggesting that Al did not impair trafficking of this protein, but could interfere with AQP2 synthesis.

Asunto(s)

Compuestos de Aluminio/toxicidad , Acuaporina 2/metabolismo , Capacidad de Concentración Renal/efectos de los fármacos , Túbulos Renales/efectos de los fármacos , Lactatos/toxicidad , Animales , Desamino Arginina Vasopresina/farmacología , Conducta de Ingestión de Líquido , Túbulos Renales/metabolismo , Túbulos Renales/fisiopatología , Masculino , Transporte de Proteínas , Ratas , Ratas Wistar , Solución Salina Hipertónica , Factores de Tiempo , Urinálisis

RESUMEN

Isopropyl alcohol dehydrogenase (iPDH) is a dimeric mitochondrial alcohol dehydrogenase (ADH), so far detected within the Trypanosomatidae only in the genus Phytomonas. The cloning, sequencing, and heterologous expression of the two gene alleles of the enzyme revealed that it is a zinc-dependent medium-chain ADH. Both polypeptides have 361 amino acids. A mitochondrial targeting sequence was identified. The mature proteins each have 348 amino acids and a calculated molecular mass of 37 kDa. They differ only in one amino acid, which can explain the three isoenzymes and their respective isoelectric points previously found. A phylogenetic analysis locates iPDH within a cluster with fermentative ADHs from bacteria, sharing 74% similarity and 60% identity with Ralstonia eutropha ADH. The characterization of the two bacterially expressed Phytomonas enzymes and the comparison of their kinetic properties with those of the wild-type iPDH and of the R. eutropha ADH strongly support the idea of a horizontal gene transfer event from a bacterium to a trypanosomatid to explain the origin of the iPDH in Phytomonas. Phytomonas iPDH and R. eutropha ADH are able to use a wide range of substrates with similar Km values such as primary and secondary alcohols, diols, and aldehydes, as well as ketones such as acetone, diacetyl, and acetoin. We speculate that, as for R. eutropha ADH, Phytomonas iPDH acts as a safety valve for the release of excess reducing power.

Asunto(s)

Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/genética , Transferencia de Gen Horizontal , Trypanosoma/genética , Alelos , Secuencia de Aminoácidos , Animales , Southern Blotting , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Escherichia coli/genética , Cromatografía de Gases y Espectrometría de Masas , Cinética , Datos de Secuencia Molecular , Filogenia , Isoformas de Proteínas , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Trypanosoma/enzimología , Zinc/química