Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis

The human 15-lipoxygenase (15-LO) gene was transfected into rat kidneys in vivo via intra-renal arterial injection. Three days later, acute (passive) or accelerated forms of antiglomerular basement membrane antibody-mediated glomerulonephritis were induced in transfected and nontransfected or sham-transfected controls. Studies of glomerular functions (filtration and protein excretion) and ex vivo glomerular leukotriene B4 biosynthesis at 3 hr, and up to 4 days, after induction of nephritis revealed preservation or normalization of these parameters in transfected kidneys that expressed human 15-LO mRNA and mature protein, but not in contralateral control kidneys or sham-transfected animals. The results provide in vivo-derived data supporting a direct anti-inflammatory role for 15-LO during immune-mediated tissue injury.

Human munc13 Is a Diacylglycerol Receptor that Induces Apoptosis and May Contribute to Renal Cell Injury in Hyperglycemia

We have previously shown that human munc13 (hmunc13) is up-regulated by hyperglycemia under in vitro conditions in human mesangial cell cultures. The purpose of the present study was to determine the cellular function of hmunc13. To do this, we have investigated the subcellular localization of hmunc13 in a transiently transfected renal cell line, opossum kidney cells. We have found that hmunc13 is a cytoplasmic protein and is translocated to the Golgi apparatus after phorbol ester stimulation. In addition, cells transfected with hmunc13 demonstrate apoptosis after treatment with phorbol ester, but cells transfected with an hmunc13 deletion mutant in which the diacylglycerol (C1) binding domain is absent exhibit no change in intracellular distribution and no induction of apoptosis in the presence of phorbol ester stimulation. We conclude that both the diacylglycerol-induced translocation and the apoptosis represent functional activity of hmunc13. We have also demonstrated that munc13-1 and munc13-2 are localized mainly to cortical epithelial cells in rat kidney and both are overexpressed under conditions of hyperglycemia in a streptozotocin-treated diabetic rat model. Taken together, our data suggest that hmunc13 serves as a diacylglycerol-activated, PKC-independent signaling pathway capable of inducing apoptosis and that this pathway may contribute to the renal cell complications of hyperglycemia.

Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury.

Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide (.O2-) at lower L-Arg concentrations. By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and .O2- formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the Ca2+ ionophore A23187 triggered NO generation but no .O2- was seen. With cells in L-Arg-free medium, we observed .O2- formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined. .O2- formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells. Activation by A23187 was cytotoxic to L-Arg-depleted, but not to control cells, with marked lactate dehydrogenase release. The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating .O2- and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving .O2-/NO-mediated cellular injury.

Sphingosine: a mediator of acute renal tubular injury and subsequent cytoresistance.

The goal of this study was to determine whether sphingosine and ceramide, second messengers derived from sphingolipid breakdown, alter kidney proximal tubular cell viability and their adaptive responses to further damage. Adult human kidney proximal tubular (HK-2) cells were cultured for 0-20 hr in the presence or absence of sphingosine, sphingosine metabolites (sphingosine 1-phosphate, dimethylsphingosine), or C2, C8, or C16 ceramide. Acute cell injury was assessed by vital dye exclusion and tetrazolium dye transport. Their subsequent impact on superimposed ATP depletion/Ca2+ ionophore-induced damage was also assessed. Sphingosine (> or = 10 microM), sphingosine 1-phosphate, dimethylsphingosine, and selected ceramides (C2 and C8, but not C16) each induced rapid, dose-dependent cytotoxicity. This occurred in the absence of DNA laddering or morphologic changes of apoptosis, suggesting a necrotic form of cell death. Prolonged exposure (20 hr) to subtoxic sphingosine doses (< or = 7.5 microM) induced substantial cytoresistance to superimposed ATP depletion/Ca2+ ionophore-mediated damage. Conversely, neither short-term sphingosine treatment (< or = 8.5 hr) nor 20-hr exposures to any of the above sphingosine/ceramide derivatives/metabolites or various free fatty acids reproduced this effect. Sphingosine-induced cytoresistance was dissociated from the extent of cytosolic Ca2+ loading (indo-1 fluorescence), indicating a direct increase in cell resistance to attack. We conclude that sphingosine can exert dual effects on proximal renal tubular viability: in high concentrations it induces cell necrosis, whereas in low doses it initiates a cytoresistant state. These results could be reproduced in human foreskin fibroblasts, suggesting broad-based relevance to the area of acute cell injury and repair.

Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury.

Disruption of the renal proximal tubule (PT) brush border is a prominent early event during ischemic injury to the kidney. The molecular basis for this event is unknown. Within the brush border, ezrin may normally link the cytoskeleton to the cell plasma membrane. Anoxia causes ezrin to dissociate from the cytoskeleton and also causes many cell proteins to become dephosphorylated in renal PTs. This study examines the hypothesis that ezrin dephosphorylation accompanies and may mediate the anoxic disruption of the rabbit renal PT. During normoxia, 73 +/- 3% of the cytoskeleton-associated (Triton-insoluble) ezrin was phosphorylated, but 88 +/- 6% of dissociated (Triton-soluble) ezrin was dephosphorylated. Phosphorylation was on serine/threonine resides, since ezrin was not detectable by an antibody against phosphotyrosine. After 60 min of anoxia, phosphorylation of total intracellular ezrin significantly decreased from 72 +/- 2% to 21 +/- 9%, and ezrin associated with the cytoskeleton decreased from 91 +/- 2% to 58 +/- 2%. Calyculin A (1 microM), the serine/threonine phosphatase inhibitor, inhibited the dephosphorylation of ezrin during anoxia by 57% and also blocked the dissociation of ezrin from the cytoskeleton by 53%. Our results demonstrate that (i) the association of ezrin with the renal microvillar cytoskeleton is correlated with phosphorylation of ezrin serine/threonine residues and (ii) anoxia may cause disruption of the renal brush border by dephosphorylating ezrin and thereby dissociating the brush border membrane from the cytoskeleton.