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Angiotensin converting enzyme (ACE) inhibition is a common therapeutic modality in the treatment of autosomal recessive polycystic kidney disease (ARPKD). This study was designed to investigate whether chronic inhibition of ACE would have a therapeutic effect in attenuating the progression of renal cystogenesis in an orthologous rat model of ARPKD, the polycystic kidney (PCK) rat. Lisinopril (3 mg/kg per day) was administered orally for a period of 12 weeks, beginning at post-natal week 4. Lisinopril treatment resulted in an ~30% improvement in the collecting duct cystic indices (CT CI) of PCK animals. Activation of extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2), proliferative signaling markers, and proliferating cell nuclear antigen (PCNA), an end-point marker for proliferation, was reduced following chronic treatment with lisinopril compared to that in vehicle-treated PCK rats. To assess whether apoptotic pathways were altered due to chronic ACE inhibition, we examined p38 mitogen activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/ JNK), which are markers of apoptotic signaling cascades. p38 MAPK was significantly reduced (P Keywords Autosomal recessive polycystic kidneydisease (ARPKD) * Western blot * Cyst * Lisinopril * Angiotensin converting enzyme * Apoptosis * Proliferation, Introduction Autosomal recessive polycystic kidney disease (ARPKD) is a monogenetic disorder that has an incidence of ~ 1:20,000 in live births annually, and there are currently ~20,000 affected people in [...]

Oxidative stress and apoptosis are important factors in the etiology of renal ischemia-repeffusion (I/R) injury. The present study tested the hypothesis that the cell-permeant SOD mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) protects the kidney from I/R-mediated oxidative stress and apoptosis in vivo. Male Sprague-Dawley rats (175-220 g) underwent renal I/R by bilateral clamping of the renal arteries for 45 min followed by reperfusion for 24 h. To examine the role of reactive oxygen species (ROS) in renal I/R injury, a subset of animals were treated with either saline vehicle (I/R Veh) or MnTMPyP (I/R Mn) (5 mg/kg ip) 30 min before and 6 h after surgery. MnTMPyP significantly attenuated the I/R-mediated increase in serum creatinine levels and decreased tubular epithelial cell damage following I/R. MnTMPyP also decreased TNF-[alpha] levels, [gp.sup.91phox], and lipid peroxidation after I/R. Furthermore, MnTMPyP inhibited the I/R-mediated increase in apoptosis and caspase-3 activation. Interestingly, although MnTMPyP did not increase expression of the antiapoptotic protein Bcl-2, it decreased the expression of the proapoptotic genes Bax and FasL. These results suggest that MnTMPyP is effective in reducing apoptosis associated with renal I/R injury and that multiple signaling mechanisms are involved in ROS-mediated cell death following renal I/R injury. acute ischemia-reperfusion; manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin; reactive oxygen species

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-[PK.sub.1] cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-[PK.sub.1] cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release (P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 [micro]M) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 [micro]M) also inhibited cytotoxicity significantly (P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase (P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells (P < 0.05). This was abolished in the presence of HET-0016 (P < 0.05) or MnTMPyP (P < 0.01). These results demonstrate that 20-HETE over-expression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals. apoptosis; cytochrome P-450 4A10; cytochrome P-450 4A12; LLC-[PK.sub.1]; lentiviral vectors; kidney; HET-0016; superoxide; [0.sub.2]

We examined the efficacy and mechanism of action of [N.sup.6]-(1-iminoethyl)-L-lysine (L-NIL), a highly selective inhibitor of inducible nitric oxide (NO) synthase (iNOS), on acute cardiac transplant rejection. L-NIL produced a concentration-dependent attenuation of plasma NO by-products and a decrease in nitrosylation of heme protein without altering protein levels of iNOS. At postoperative day 4, L-NIL did not alter the increased binding activities for transcription factors nuclear factor-[kappa]B and activator protein-1. Whereas L-NIL decreased inflammatory cell infiltration, graft survival was only prolonged at the dose of 1.0 [micro]g/ml that incompletely blocked NO production. Higher L-NIL concentrations (30 and 60 [micro]g/ml) ablated the increased NO production but failed to improve graft survival and even potentiated NF-[kappa]B binding activity examined at day 6. Alloimmune activation indicated by increased cytokine gene expression for interferon-[gamma], interleukin-6, and interleukin-10 was inhibited in grafts only by treatment with 1.0 [micro]g/ml L-NIL. These findings suggest a complex role of NO in acute cardiac allograft rejection. Partial inhibition of iNOS is beneficial to graft survival, whereas total ablation may oppose any benefits to graft survival. These studies have important implications in understanding the dual role of NO in acute rejection and help to reconcile discrepancies in the literature. inducible nitric oxide synthase; nuclear factor-KB; activator protein-1; interferon-[gamma]; interleukin-6; interleukin-10; electron paramagnetic resonance spectroscopy

In this study, we examined the actions of diethyldithiocarbamate-iron (DETC-Fe) complex in acute graft rejection heterotopically transplanted rat hearts. Chronic treatment with DETC-Fe inhibited the increase in plasma nitric oxide (NO) metabolites and nitrosylation of myocardial heme protein as determined by electron paramagnetic resonance (EPR) spectroscopy. Pulse injection with DETC-Fe normalized NO metabolites. We verified intragraft trapping of NO in vivo by pulse injection with DETC-Fe by the detection within allografts of an anisotropic triplet EPR signal for DETC-Fe-NO adduct with resonance positions (g tensor factors for perpendicular and parallel components, respectively g[perpendicular to] = 2.038 and g[parallel] = 2.02; hyperfine coupling of 12.5 G). DETC-Fe prolonged graft survival and decreased histological rejection scores. DNA binding activity for nuclear factor (NF)-[kappa]B and activator protein-1 was increased in allografts and prevented by DETC-Fe. Abrogation of the activation of NF-[kappa]B by DETC-Fe was associated with increased I[kappa]B[alpha] inhibitory protein. Western blotting and RT-PCR analysis revealed that DETC-Fe inhibited inducible NO synthase protein and gene expression. Gene expression for the proinfiammatory cytokine interferon-[gamma] Was also decreased by DETC-Fe. Thus DETC-Fe limits NF-[kappa]B-dependent gene expression and possesses significant immunosuppressive properties. nitric oxide synthase; interferon-[gamma]; electron paramagnetic resonance spectroscopy; nuclear factor-[kappa]B; activator protein-1