Inflammation and caspase activation in long-term renal ischemia/reperfusion injury and immunosuppression in rats

Inflammation and caspase activation in long-term renal ischemia/reperfusion injury and immunosuppression in rats.BackgroundWe have previously shown the long-term influence of renal ischemia/reperfusion (I/R) injury and immunosuppression on fibrotic genes and apoptosis in a rat model. For the first time, we have now investigated the effects of I/R and immunosuppression on inflammation and caspase activation.MethodsI/R injury was induced in the right kidney and the left was removed. Cyclosporin (CsA) (10 mg/kg), tacrolimus (0.2 mg/kg), rapamycin (1 mg/kg), or mycophenolate mofetil (MMF) (10 mg/kg) was then administered for 16 weeks. The effects of I/R and immunosuppressants on interstitial inflammation, interleukin (IL)-1β expression, caspase-1 and caspase-3 activation, tubulointerstitial damage, and fibrosis were evaluated.ResultsED-1+ (a specific rat monocyte/macrophage marker) cells were mainly localized in the tubulointerstitium and periglomerular areas and increased in I/R group compared to controls (P < 0.01). This was further increased by CsA, but decreased by tacrolimus, rapamycin, or MMF (P < 0.05). The 17 kD active IL-1β remained unchanged, but 35 kD IL-1β precursor was decreased by rapamycin in comparison with I/R group (P < 0.05). The 45 kD or 20 kD caspase-1 was increased by I/R or CsA, respectively, and decreased by rapamycin (P < 0.05). The 24 kD caspase-3, which proved to be an active caspase-3 subunit, was increased in I/R and CsA groups and deceased by tacrolimus, rapamycin, or MMF (P < 0.05), but not 32 kD precursor or 17 kD active caspase-3. The activity data of caspase-1 and caspase-3 exhibited the same trend as Western blotting data. The staining of active caspase-3 was scattered in kidneys, mainly in tubular and interstitial areas, which was consistent with that of ED-1+ cells. There was a strong positive correlation between interstitial inflammation and 24 kD caspase-3 expression or caspase-3 activity (r = 0.814 or 0.484), all of which were also closely related with urinary protein (r = 0.537, 0.529, or 0.517), serum creatinine (r = 0.463, 0.573, or 0.539), tubulointerstitial damage (r = 0.794, 0.618, or 0.712) and fibrosis (r = 0.651, 0.567, or 0.469), all P < 0.01.ConclusionThis study shows that the mechanisms of long-term I/R injury and immunosuppressants treatment include interstitial inflammation and caspase activation, most clearly demonstrated by the 24 kD active caspase-3

Pharmacological enhancement of the kallikrein-kinin system promotes anti-fibrotic responses in human mesangial cells

The aim of the present study was to investigate whether pharmacological enhancement of the renal kallikrein-kinin system using the vasopeptidase inhibitor omapatrilat plays a direct role in modulating the fibrotic responses of human mesangial cells to injury. Treatment with 40µmol/L omapatrilat was able to reduce macrophage-conditioned medium (MPCM)-induced fibronectin levels without affecting mRNA expression. MPCM injury also suppressed kallikrein and low molecular weight kininogen mRNA. Omapatrilat was able to attenuate this suppression. Bradykinin levels in contrast were increased by MPCM and treatment with omapatrilat further augmented levels. Co-incubation with the bradykinin B2 receptor antagonist HOE 140 attenuated the omapatrilat-induced lowering of fibronectin. Moreover, inhibition of cGMP release had a similar effect. Paradoxically, RT-PCR and Southern blotting demonstrated that bradykinin B2 receptor mRNA levels were down regulated in response to omapatrilat. Western blotting supported this data. Supernatant levels of tissue plasminogen activator (tPA), a product of bradykinin stimulation, were decreased by omapatrilat while cell associated tPA levels were increased. Matrix metalloproteinase-9 (MMP-9) mRNA expression was up regulated by omapatrilat treament, although no difference in active zymogen levels was observed. In conclusion enhancement of kallikrein-kinin system appears to play a direct role in promoting anti-fibrotic responses in MPCM-injured human mesangial cells

Kallikrein gene 'knock-down' by small interfering RNA transfection induces a profibriotic phenotype in rat mesangial cells.

Background: Emerging evidence suggests that kallikrein exerts renoprotective effects independent of its haemodynamic actions. The aim of the current investigation was to delineate the role of kallikrein in the regulation of fibrosis, by 'knocking down' its expression using specific small interfering RNAs (siRNA). Methods: Rat mesangial cells were treated with 12, 60, 120 nmol/l kallikrein-specific siRNAs. The consequent cellular genotypes and phenotypes were analysed. Results: Western blotting demonstrated that mesangial cells produced a kallikrein protein, which was of a different molecular weight to urinary kallikrein from rats of the same species. Treatment of cells with siRNA resulted in a dose-dependent decrease in kallikrein mRNA levels, which impacted on other components of the kallikrein-kinin system, dose-dependently reducing bradykinin B2 receptor mRNA expression. Kallikrein suppression resulted in significant increases in fibronectin and transforming growth factor-[beta] protein levels in culture supernatants over control levels. Gelatin zymography demonstrated a siRNA dose-dependent decrease in active MMP-2 enzyme levels. Bradykinin, an effector molecule of the kallikrein system, is known to stimulate tissue plasminogen activator production. Paradoxically, however, tissue plasminogen activator protein levels were augmented with increasing kallikrein mRNA silencing. This was accompanied by a dose-dependent decrease in low-density lipoprotein receptor-related protein mRNA levels, indicating that increased tissue plasminogen activator levels were due to an attenuation of receptor-mediated protease clearance. Conclusion: These data lend strong support to the hypothesis that kallikrein exerts antifibrotic, renoprotective effects that are independent of its classical haemodynamic actions