Caspases are the main executioners of apoptosis as well as interleukin (IL)-1beta and IL-18 conversion to active forms. They are activated after acute kidney injuries. In this study, we evaluated the importance of the caspase family in the pathogenesis and recovery of glycerol-induced acute renal failure in rats (Gly-ARF). Rats were treated with pan-caspase or selective caspase 1 and 3 inhibitors at the moment we injected glycerol. Renal function, renal histology (HE), transferase-mediated deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining for apoptosis, leukocytes infiltration (immunohistochemistry), renal expression of IL-1beta and IL-18 (immunohistochemistry and Western blot), tubular regeneration (5-bromo-2'-deoxyuridine (BrdU) incorporation), and P27(Kip) expression (Western blot) were evaluated at appropriate times. All inhibitors reduced the renal function impairment. Pan-caspase and caspase-3 inhibitors reduced cellular death (necrosis and apoptosis) 24 h after Gly-ARF. All caspases inhibitors reduced macrophages infiltration. The expression of total IL-1beta was enhanced in Gly-ARF, but the active IL-1beta and IL-18 forms were abolished in pan-caspase treated rats. Caspase-1 inhibitor attenuated Gly-ARF but not tubular injury suggesting glomerular hemodynamic improvement. There was striking regenerative response 48 h after Gly-ARF characterized by enhanced BrdU incorporation and reduced expression of p27(Kip). This response was not blunted by caspases inhibition. Our findings demonstrate that caspases participate in important pathogenic mechanisms in Gly-ARF such as inflammation, apoptosis, vasoconstriction, and tubular necrosis. The early inhibition of caspases attenuates these mechanisms and reduces the renal function impairment in Gly-ARF.

Patients that develop rhabdomyolysis of different causes are at high risk of acute renal failure. Efforts to minimize this risk include volume repletion, treatment with mannitol, and urinary alkalinization as soon as possible after muscle injury. This is a retrospective analysis (from January 1, 1992, to December 31, 1995) of therapeutic response to prophylactic treatment in patients with rhabdomyolysis admitted to an intensive care unit (ICU). The diagnosis of rhabdomyolysis was based on creatinine kinase (CK) level (> 500 Ui/L) and the criteria for prophylaxis were: time elapsed between muscle injury to ICU admission < 48 h and serum creatinine < 3 mg/dL. Fifteen patients were treated with the association of saline, mannitol, and sodium bicarbonate (S + M + B group) and 9 patients received only saline (S group). Serum creatinine at admission was similar in both groups: 1.6 +/- 0.6 mg/dL in the S + M + B group and 1.5 +/- 0.6 mg/dL in the S group (p > 0.05). Maximum serum CK measured was 3351 +/- 1693 IU/L in the S + M + B group and 1747 +/- 2345 IU/L in the S group (p < 0.05). However the measurement of CK was earlier in S + M + B patients (1.7 vs 2.7 days after rhabdomyolysis). APACHE II scores were 16.9 +/- 7.4 and 13.4 +/- 4.9 in the S + M + MB and S groups, respectively (p > 0.05). Despite the treatment protocol the serum levels of creatinine had similar behavior and reached normal levels in all patients in 2 or 3 days. The saline infusion during the first 60 h on the ICU was 206 mL/h in the S group and 204 mL/h in S + M + B (p > 0.05). Mannitol dose was 56 g/day, and bicarbonate 225 mEq/day during 4.7 days. Our data show that progression to established renal failure can be totally avoided with prophylactic treatment, and that once appropriate saline expansion is provided, the association of mannitol and bicarbonate seems to be unnecessary.

Background: Hepatocyte growth factor (HGF) is overexpressed after acute kidney injury (AKI). The aim of this study was to evaluate the role of endogenous HGF in the progression of the inflammatory response in glycerol-induced AKI (Gly-AKI) in rats. Methods: Renal and systemic HGF expressions were evaluated during the development of Gly-AKI. Subsequently, the blockade of endogenous HGF was analyzed in rats treated with anti-HGF antibody concomitant to glycerol injection. Apoptosis, cell infiltration and chemokine and cytokine profiles were investigated. Results: We detected an early peak of renal and plasma HGF protein expressions 3 h after glycerol injection. The pharmacological blockade of the endogenous HGF exacerbated the renal impairment, the tubular apoptosis, the renal expression of monocyte chemoattractant protein-1 and the macrophage, CD43+, CD4+ and CD8+ T lymphocytes renal infiltration. The analysis of mRNA expressions of Th1 (t-bet, TNF-α, IL-1β) and Th2 (gata-3, IL-4, IL-10) cytokines showed a Th1-polarized response in Gly-AKI rats that was aggravated with the anti-HGF treatment. Conclusion: Endogenous HGF attenuates the renal inflammatory response, leukocyte infiltration and Th1 polarization after glycerol injection. The control of cellular immune response may partly explain the protective effect of endogenous HGF in the development of Gly-AKI.

Background/aims: Silymarin is an herbal extract with antioxidant properties that can reduce oxidative stressmediated injuries in murine models of liver, heart, and kidney diseases. Silymarin can also increase p53-mediated cellular apoptosis in vitro. We tested the effect of silymarin administration before glycerol-induced acute kidney injury (Gly-AKI) in rats. Methods: Renal function, tubular injury, oxidative stress, leukocytes infiltration, and renal expression of apoptosis regulating proteins (p53, p-p53, Bax, Bcl-2, survivin, and cleaved caspase-3) were evaluated 6 or 24 h after glycerol. Results: Silymarin exacerbated the renal impairment and tubular apoptosis but had no effect on tubular necrosis or renal leukocytes infiltration. Renal lipid and DNA peroxidation was increased after glycerol and silymarin did not reduce oxidative stress. Proteins p53, p-p53, and proapoptotic Bax were upregulated in Gly-AKI rats treated with silymarin, whereas anti-apoptotic Bcl-2 was reduced in this group. Cleaved caspase-3 was overexpressed in Gly-AKI rats, particularly when treated with silymarin. Survivin was less expressed in Gly-AKI than in controls, but this deficit was not aggravated by silymarin. Conclusion: The persistence of oxidative stress, inflammatory reaction, and tubular necrosis, as well as exacerbation of p53-mediated tubular apoptosis, led to a more severe renal impairment in Gly-AKI rats treated with silymarin.

Interleukin-6 stimulates tubular regeneration in rats with glycerol-induced acute renal failure. Background: Interleukin 6 (IL-6) is a pleiotropic cytokine released after endotoxemia, trauma and organ injury. IL-6 may act in cellular proliferation activating transduction signals and Ras/Map cascade or the HGF/c-met axis. We tested the effect of IL-6 in the regeneration of tubular epithelia after acute tubular necrosis (ATN) in rats. Methods: Rats with glycerol-induced acute renal failure (Gly-ARF) were treated with IL-6 200 µg/kg/day. Functional, histological and immunohistochemical tests were done 24 and 72 h after Gly-ARF to localise mitotic cells (BrdU). The renal expression of c-met (Western-Blot) and circulating levels of HGF (ELISA) were also determined. Results: Rats with Gly-ARF had reduced creatinine clearance that was not influenced by IL-6. The histological appearance of ATN was also unaffected by IL-6. The IL-6 treated rats showed a significant increase in tubular cell proliferation in cortex and medulla, as well as in the expression of c-met protein in the renal cortex, compared to untreated Gly-ARF rats. The plasma HGF concentration was equally elevated in treated and untreated Gly-ARF rats. Discussion: IL-6 stimulates tubular regeneration after Gly-ARF and increases the expression of c-met in the renal cortex. Gly-ARF rats have high circulating levels of HGF that is targeted to act in the injured kidneys by the IL-6 overexpressed renal c-met.