Induction of experimental diabetes and diabetic nephropathy using anomer-equilibrated streptozotocin in male C57Bl/6J mice.

Streptozotocin (STZ) is widely used to induce experimental diabetes in murine models. However, the ability to induce diabetic nephropathy (DN) is more challenging. It has been recommended to inject STZ at multiple low doses within 15 min after dissolution due to its alleged instability. However, some studies suggest that STZ is stable for days due to equilibration of its two anomers (α and ß), 90 min after dissolution, and that this anomer-equilibrated STZ leads to higher survival rates and persistent hyperglycaemia with minimal weight loss. The aim of this study was to determine an optimal dose of anomer-equilibrated STZ to induce kidney tubular damage and compare it with the more commonly used freshly prepared STZ. We hypothesised that anomer-equilibrated STZ provides a better, reproducible experimental model of diabetes-induced kidney damage with improved animal welfare. Body measurements, fasting glycaemia, insulinemia and renal histology were assessed in male C57Bl/6J at two and six months of age treated with fresh (50 mg/kg) or anomer-equilibrated (dose ranging 35-50 mg/kg) STZ or vehicle control. We demonstrated a dose-dependent effect of anomer-equilibrated STZ on the induction of hypo-insulinaemia and hyperglycaemia, as well as body weight in two-month-old mice. Interestingly, in six-month-old mice STZ leads to body weight loss, independently of STZ preparation mode. Anomer-equilibrated STZ provoked moderate to severe kidney tubule structural damage, resulting in significant kidney hypertrophy, whereas freshly prepared STZ only caused mild alterations. In conclusion, our study proposes that anomer-equilibrated STZ provides a robust murine model of diabetes and early-stage diabetic nephropathy, which can be used to test therapeutic approaches to treat and/or prevent renal damage.

Generation of endogenous hydrogen sulfide by cystathionine gamma-lyase limits renal ischemia/reperfusion injury and dysfunction.

The generation of endogenous hydrogen sulfide may either limit or contribute to the degree of tissue injury caused by ischemia/reperfusion. A total of 74 male Wistar rats were used to investigate the effects of endogenous and exogenous hydrogen sulfide in renal ischemia/reperfusion. Administration of the irreversible cystathionine gamma-lyase (CSE) inhibitor, dL-propargylglycine, prevented the recovery of renal function after 45 min ischemia and 72 h reperfusion. The hydrogen sulfide donor sodium hydrosulfide attenuated the (renal, tubular, and glomerular) dysfunction and injury caused by 45 min ischemia and 6 h reperfusion. Western blot analysis of kidneys taken at 30 min reperfusion showed that sodium hydrosulfide significantly attenuated phosphorylation of mitogen-activated protein kinases (p-38, c-JUN N-terminal protein kinase 1/2, and extracellular signal-regulated kinase 1/2) and activation of nuclear factor-kappaB. At 6 h reperfusion, sodium hydrosulfide significantly attenuated the histological score for acute tubular necrosis, the activation of caspase-3 and Bid, the decline in the expression of anti-apoptotic Bcl-2, and the expression of nuclear factor-kappaB-dependent proteins (inducible nitric oxide synthase, cyclo-oxygenase-2, and intercellular adhesion molecule-1). These findings suggest that (1) the synthesis of endogenous hydrogen sulfide by CSE is essential to protect the kidney against ischemia/reperfusion injury and dysfunction and aids in the recovery of renal function following ischemia/reperfusion, (2) hydrogen sulfide generated by sodium hydrosulfide reduces ischemia/reperfusion injury and dysfunction, and morphological changes of the kidney, and (3) the observed protective effects of hydrogen sulfide are due to both anti-apoptotic and anti-inflammatory effects.

Characterization of the Myocardial Inflammatory Response in Acute Stress-Induced (Takotsubo) Cardiomyopathy.

Takotsubo cardiomyopathy is an acute stress-induced heart failure syndrome for which the exact pathogenic mechanisms are unclear, and consequently, no specific treatment exists. In an experimental model of stress-induced takotsubo-like cardiomyopathy, the authors describe the temporal course of a chronic inflammatory response post-induction, with an initial early influx of neutrophils into myocardial tissue followed by macrophages that are typical of a proinflammatory M1 phenotype, and a nonsignificant increase in systemic inflammatory cytokines. Post-mortem myocardium from the more complex clinical takotsubo patients share features of the study's experimental model. These findings suggest modulators of inflammation could be a potential therapeutic option.

Methionine restriction improves renal insulin signalling in aged kidneys.

Dietary methionine restriction (MR) leads to loss of adiposity, improved insulin sensitivity and lifespan extension. The possibility that dietary MR can protect the kidney from age-associated deterioration has not been addressed. Aged (10-month old) male and female mice were placed on a MR (0.172% methionine) or control diet (0.86% methionine) for 8-weeks and blood glucose, renal insulin signalling, and gene expression were assessed. Methionine restriction lead to decreased blood glucose levels compared to control-fed mice, and enhanced insulin-stimulated phosphorylation of PKB/Akt and S6 in kidneys, indicative of improved glucose homeostasis. Increased expression of lipogenic genes and downregulation of PEPCK were observed, suggesting that kidneys from MR-fed animals are more insulin sensitive. Interestingly, renal gene expression of the mitochondrial uncoupling protein UCP1 was upregulated in MR-fed animals, as were the anti-ageing and renoprotective genes Sirt1, FGF21, klotho, and ß-klotho. This was associated with alterations in renal histology trending towards reduced frequency of proximal tubule intersections containing vacuoles in mice that had been on dietary MR for 190days compared to control-fed mice, which exhibited a pre-diabetic status. Our results indicate that dietary MR may offer therapeutic potential in ameliorating the renal functional decline related to ageing and other disorders associated with metabolic dysfunction by enhancing renal insulin sensitivity and renoprotective gene expression.

Inhibitors of calpain activation (PD150606 and E-64) and renal ischemia-reperfusion injury.

Calpain activation has been implicated in the development of ischemia-reperfusion (I-R) injury. Here we investigate the effects of two inhibitors of calpain activity, PD150606 and E-64, on the renal dysfunction and injury caused by I-R of rat kidneys in vivo. Male Wistar rats were administered PD150606 or E-64 (3mg/kg i.p.) or vehicle (10%, v/v, DMSO) 30min prior to I-R. Rats were subjected to bilateral renal ischemia (45min) followed by reperfusion (6h). Serum and urinary biochemical indicators of renal dysfunction and injury were measured; serum creatinine (for glomerular dysfunction), fractional excretion of Na(+) (FE(Na), for tubular dysfunction) and urinary N-acetyl-beta-d-glucosaminidase (NAG, for tubular injury). Additionally, kidney tissues were used for histological analysis of renal injury, immunohistochemical analysis of intercellular adhesion molecule-1 (ICAM-1) expression and nitrotyrosine formation. Renal myeloperoxidase (MPO) activity (for polymorphonuclear leukocyte infiltration) and malondialdehyde (MDA) levels (for tissue lipid peroxidation) were determined. Both PD150606 and E-64 significantly reduced the increases in serum creatinine, FE(Na) and NAG caused by renal I-R, indicating attenuation of renal dysfunction and injury and reduced histological evidence of renal damage caused by I-R. Both PD150606 and E-64 markedly reduced the evidence of oxidative stress (ICAM-1 expression, MPO activity, MDA levels) and nitrosative stress (nitrotyrosine formation) in rat kidneys subjected to I-R. These findings provide the first evidence that calpain inhibitors can reduce the renal dysfunction and injury caused by I-R of the kidney and may be useful in enhancing the tolerance of the kidney against renal injury associated with aortovascular surgery or renal transplantation.

The cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-prostaglandin J2 ameliorates ischemic acute renal failure.

OBJECTIVE: Here we investigate the effects of the endogenous prostaglandin D2 metabolite 15-deoxy-Delta(12,14)-prostaglandin J2, on the renal dysfunction and injury caused by ischemia/reperfusion of the kidney. METHODS: Male Wistar rats, subjected to bilateral renal ischemia for 45 min followed by reperfusion for up to 48 h, were administered 15-deoxy-Delta(12,14)-prostaglandin J2 (1 mg/kg, intravenously) 5 min prior to and again after 3 or 12 h reperfusion. RESULTS: 15-deoxy-Delta(12,14)-prostaglandin J2 significantly reduced (i) renal and tubular dysfunction (serum urea and creatinine levels, creatinine clearance, fractional excretion of Na+ (FENA)), (ii) tubular and reperfusion-injury (urinary N-acetyl-beta-D-glucosaminidase, aspartate aminotransferase (ASP) and gamma-glutamyltransferase (gamma-GT)) and (iii) histological evidence of renal injury. 15-deoxy-Delta(12,14)-prostaglandin J2 also improved renal function (plasma creatinine levels) and reduced the histological signs of renal injury (after 48 h reperfusion). Administration of 15-deoxy-Delta(12,14)-prostaglandin J2 markedly reduced the expression of inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 during reperfusion (determined using immunohistochemistry). Immunohistochemical analysis of p65 translocation and Western blot analysis of IkappaB-alpha degradation revealed that 15-deoxy-Delta(12,14)-prostaglandin J2 inhibited the activation of nuclear factor (NF)-kappaB in renal cells. Subsequently, 15d-PGJ2 was able to significantly reduce nitric oxide production during renal ischemia/reperfusion and by primary cultures of rat proximal tubular (PT) cells incubated with interferon-gamma and bacterial lipopolysaccharide (LPS) in combination. CONCLUSIONS: We demonstrate here, for the first time, that 15-deoxy-Delta(12,14)-prostaglandin J2 significantly reduces renal ischemia/reperfusion-injury via reduction of pro-inflammatory gene expression during reperfusion subsequent to the inhibition of the activation of NF-kappaB.

TEMPONE reduces renal dysfunction and injury mediated by oxidative stress of the rat kidney.

Here we investigate the effects of the stable, water-soluble nitroxyl radical, TEMPONE, on renal dysfunction and injury caused by ischemia/reperfusion (I/R) of the rat kidney in vivo. TEMPONE significantly improved both glomerular and tubular function (serum urea, creatinine, creatinine clearance, and fractional excretion of Na(+)) in a dose-dependent manner and significantly attenuated the reperfusion-injury associated with I/R (urinary N-acetyl-beta-D-glucosaminidase, aspartate aminotransferase, assessment of renal histology). TEMPONE also markedly reduced the immunohistochemical evidence of the formation of nitrotyrosine and poly(ADP-ribose), indicating reduction of nitrosative and oxidative stress, respectively. The latter was reflected in vitro, where TEMPONE significantly reduced cellular injury of primary cultures of rat renal proximal tubular (PT) cells caused by hydrogen peroxide in a dose-dependent manner. Importantly, in contrast to its in vivo metabolite TEMPOL (which also provided protective effects against renal I/R and oxidative stress of PT cells), TEMPONE reduced renal dysfunction and injury without causing a significant reduction in blood pressure upon administration. These results suggest, for the first time, that TEMPONE can reduce the renal dysfunction and injury caused by I/R and the injury caused to PT cells by oxidative stress without producing the adverse cardiovascular effects observed when using other nitroxyl radicals.

Differential effects of caspase inhibitors on the renal dysfunction and injury caused by ischemia-reperfusion of the rat kidney.

Caspase activation has been implicated in the development of ischemia-reperfusion injury. Here, we investigate the effects of different caspase inhibitors on the renal dysfunction and injury caused by ischemia-reperfusion of the rat kidney. Bilateral clamping of renal pedicles (45 min) followed by reperfusion (6 h) caused significant renal dysfunction and marked renal injury. Caspase-1 inhibitor II (N-acetyl-L-tyrosyl-L-valyl-N-[(1S)-1-(carboxymethyl)-3-chloro-2-oxo-propyl]-L-alaninamide, Ac-YVAD-CMK, 3 mg/kg, administered i.p.) significantly reduced biochemical and histological evidence of renal dysfunction and injury. However, although caspase-3 inhibitor I (N-acetyl-L-aspartyl-L-glutamyl-N-(2-carboxyl-1-formylethyl]-L-valinamide, Ac-DEVD-CHO, 3 mg/kg, administered i.p.) produced a significant improvement of renal (glomerular) dysfunction (reduction of serum creatinine levels), it was not able to reduce tubular dysfunction and injury. Furthermore, the pan-caspase inhibitor caspase inhibitor III (N-tert-butoxycarbonyl-aspartyl(OMe)-fluoromethylketone, Boc-D-FMK, 3 mg/kg, administered i.p.) did not reduce renal dysfunction and injury. Both caspase-1 and -3 inhibitors markedly reduced the evidence of oxidative and nitrosative stress in rat kidneys subjected to ischemia-reperfusion. Overall, these results demonstrate that inhibition of caspase-1 reduces renal ischemia-reperfusion injury to a greater extent than caspase-3 inhibition, supporting the notion that the mode of acute cell death in our model of renal ischemia-reperfusion is primarily via necrosis. Furthermore, our finding that a pan-caspase inhibitor did not reduce the renal dysfunction and injury suggests that activation of some caspases during ischemia-reperfusion could provide protection against acute ischemic renal injury. Overall, these results demonstrate that inhibition of caspase-1 activity reduces renal ischemia-reperfusion injury and that this therapeutic strategy may be of benefit against ischemic acute renal failure.

Selective expression of TGF-beta2 and TGF-beta3 isoforms in early mesangioproliferative glomerulonephritis.

BACKGROUND/AIM: Evidence from ex vivo glomerular analysis has implicated overexpression of transforming growth factor (TGF) beta 1 in progressive renal disease. The roles of TGF-beta2 and TGF-beta3 are less clear. The purpose of this study was to define the temporal expression and abundance of TGF-beta isoforms in both acute and progressive Thy-1 glomerulonephritis during the crucial initiation phase of these models. METHODS: Acute Thy-1 glomerulonephritis was induced by a single injection of OX7, while the progressive model was induced by two injections, 7 days apart. RESULTS: Cellular infiltration of glomeruli consisted of transient increases of neutrophils and ED1+ macrophages. The distribution of TGF-beta1, TGF-beta2, and TGF-beta3 revealed distinct differences in normal and nephritic rats. No changes in TGF-beta1 staining were observed within glomeruli of either model. In marked contrast, in the one-shot model, TGF-beta2 and TGF-beta3 stainings increased rapidly, yet transiently, throughout affected glomeruli, followed by more sustained staining in glomerular epithelial cells. Diffuse, transient staining was absent in two-shot glomerulonephritis, but an increase in epithelial cell staining mirrored that seen in the one-shot model. CONCLUSION: Based on these results, we propose that the effects, formerly thought of as solely due to a single entity, TGF-beta1, may be the result of an interplay between individual TGF-beta isoforms.

Mutations in the pre-replication complex cause Meier-Gorlin syndrome.

Meier-Gorlin syndrome (ear, patella and short-stature syndrome) is an autosomal recessive primordial dwarfism syndrome characterized by absent or hypoplastic patellae and markedly small ears¹â»³. Both pre- and post-natal growth are impaired in this disorder, and although microcephaly is often evident, intellect is usually normal in this syndrome. We report here that individuals with this disorder show marked locus heterogeneity, and we identify mutations in five separate genes: ORC1, ORC4, ORC6, CDT1 and CDC6. All of these genes encode components of the pre-replication complex, implicating defects in replication licensing as the cause of a genetic syndrome with distinct developmental abnormalities.

Retransplantation in Alport post-transplant anti-GBM disease.

BACKGROUND: Post transplant anti-glomerular basement membrane (GBM) disease affects up to 5% of patients with Alport's syndrome. Defects in the COL4A5 gene are responsible for most cases, and alpha 5(IV)NC1 is the usual target for alloantibodies. Gene deletions are more commonly associated with this complication than are point mutations. The disease is severe in renal allografts and nearly always results in graft loss. METHODS: Three cases of retransplantation in Alport's syndrome are described here in detail. All cases were started on immunosuppressive therapy early in the course of their disease and one patient (case 2) received pre-emptive anti-T-cell therapy (Campath IH). Anti-GBM antibodies in these cases were investigated by standard anti-GBM enzyme-linked immunosorbent assay (ELISA), by indirect immunofluorescence, and by Western blotting using collagenase-digested human GBM and recombinant type IV collagen NC1 domains made in insect cells. RESULTS: All cases showed early antibody and complement fixation to human GBM. Target alloantibodies were to alpha 5(IV)NC1 domain predominantly. Cases two and three gave negative results on standard ELISA for anti-GBM antibodies. Pathologic examination revealed crescentic glomerulonephritis, which was rapid in onset in case 1, blunted and less aggressive in case 3, and case 2 developed segmental necrosis without crescent formation. Neutrophilic infiltrates were an early feature in all 3 cases. All cases are compared with a review of all retransplanted cases in the literature. CONCLUSION: Alport anti-GBM disease is a severe disease in retransplanted patients. Anti-T-cell therapy seemed to modify the pathologic findings but did not prevent graft loss. Longer term plasma exchange and mycophenolate mofetil may attenuate the illness, but in these cases did not prevent graft loss. Western blotting detected alloantibodies to alpha 5(IV) NC1 domain and is more sensitive and specific for this disease than standard ELISAs.

Bone-marrow-derived macrophages genetically modified to produce IL-10 reduce injury in experimental glomerulonephritis.

Macrophages are intimately involved in the development of immune-mediated inflammation, including glomerulonephritis. We have transduced primary cultures of macrophages to express IL-10 and tested the ability of these cells to control rat nephrotoxic nephritis (NTN), a model of human glomerulonephritis. Ad-IL-10-transduced bone-marrow-derived macrophages (BMDM) produced large amounts of IL-10 in culture, and their TNF-alpha production was decreased in response to interferon-gamma and LPS. Transduced macrophages were injected into the renal artery of rats, 6 h after the induction of NTN, where they localized efficiently to inflamed rat glomeruli. Delivery of IL-10-expressing macrophages to nephritic rats produced a marked reduction in albuminuria compared with unmodified NTN or injection of Ad-null-transduced BMDM. IL-10 treatment decreased the number of glomerular ED1- and ED3-positive cells, MHC class II expression, and the number of fibrinoid lesions. Interestingly, anti-inflammatory changes in the Ad-IL-10-injected kidney were mirrored by changes in the contralateral kidney. These results highlight that Ad-IL-10-transduced macrophages infiltrate inflamed glomeruli and reduce the severity of glomerular inflammation, emphasizing the value of local delivery of genetically modified macrophages in the manipulation of inflammatory disease.

Inhibition of inducible nitric oxide synthase reduces renal ischemia/reperfusion injury.

BACKGROUND: Nitric oxide (NO), produced via inducible nitric oxide synthase (iNOS), is implicated in the pathophysiology of renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of the iNOS inhibitors L-N6-(1-iminoethyl)lysine (L-NIL) and aminoethyl-isothiourea (AE-ITU) on (a) renal dysfunction and injury mediated by bilateral I/R of rat kidneys in vivo and (b) cytokine-stimulated NO production by primary cultures of rat proximal tubule (PT) cells. METHODS: Male Wistar rats subjected to bilateral renal ischemia (45 min) followed by reperfusion (6 h). Rats were administered either L-NIL (3 mg/kg IV bolus 15 min prior to I/R followed by 1 mg/kg/h throughout I/R) or AE-ITU (1 mg/kg IV bolus 15 min prior to I/R followed by 1 mg/kg/h throughout I/R). Serum and urinary biochemical indicators of renal dysfunction and injury were measured; serum creatinine (SCr, glomerular dysfunction), fractional excretion of Na+ (FENa, tubular dysfunction), serum aspartate aminotransferase (sAST, I/R injury) and urinary N-acetyl-beta-d-glucosaminidase (uNAG, tubular injury). Additionally, renal sections were used for histological grading of renal injury and for immunological evidence of nitrotyrosine formation. Nitrate/nitrate levels in plasma were measured using the Griess assay and used as an indicator of NO production. Primary cultures of rat PT cells were incubated with interferon-gamma(IFN-gamma, 100 IU/mL) and lipopolysaccharide (LPS, 10 microg/mL) for 24 h, either in the absence or presence of increasing concentrations of L-NIL or AE-ITU (0.001 to 1 mmol/L) after which nitrite/nitrate levels were measured using the Griess assay. RESULTS: L-NIL and AE-ITU significantly reduced the I/R-mediated increases in SCr, FENa, sAST and uNAG, indicating attenuation of I/R-mediated renal dysfunction and injury. Specifically, L-NIL and AE-ITU reduced the I/R-mediated glomerular and tubular dysfunction and biochemical and histological evidence of tubular injury. Both L-NIL and AE-ITU attenuated the plasma levels of nitrate (indicating reduced NO production) and the immunohistochemical evidence of the formation of nitrotyrosine. In vitro, L-NIL and AE-ITU both significantly reduced cytokine-stimulated NO production by primary cultures of rat PT cells in a dose-dependent manner. CONCLUSIONS: These results suggest that L-NIL and AE-ITU reduce the renal dysfunction and injury associated with I/R of the kidney, via inhibition of iNOS activity and subsequent reduction of NO (and peroxynitrite) generation. We propose that selective and specific inhibitors of iNOS activity may be useful against the NO-mediated renal dysfunction and injury associated with I/R of the kidney.

Lipoteichoic acid from Staphylococcus aureus reduces renal ischemia/reperfusion injury.

BACKGROUND: The aim of this study was to investigate whether in vivo administration of a low, sub-lethal dose of lipoteichoic acid (LTA), a bacterial wall-fragment derived from the Gram-positive bacterium Staphylococcus aureus, protects the kidney against the renal dysfunction and injury caused by ischemia/reperfusion (I/R). METHODS: Male Wistar rats were administered LTA from S. aureus (1 mg/kg, IP). After 24 hours, rats were subjected to bilateral renal ischemia (45 min) followed by reperfusion (6 h). Serum and urinary markers were measured for the assessment of renal function, tubular and reperfusion-injury. Renal sections were used for histological grading of renal injury and for immunohistochemical localization of P-selectin, inducible nitric oxide synthase (iNOS) and nitrotyrosine (indicative of peroxynitrite formation). Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of polymorphonuclear (PMN) cell infiltration and lipid peroxidation, respectively. Nitric oxide (NO) production was determined by measurement of plasma nitrite/nitrate levels. RESULTS: LTA pretreatment significantly reduced renal dysfunction, tubular and reperfusion-injury caused by I/R of the kidney as well as histological evidence of renal injury. LTA also reduced the expression of P-selectin and kidney MPO activity associated with renal I/R. MDA levels were significantly reduced by LTA pretreatment suggesting a reduction in the lipid peroxidation and formation of reactive oxygen species (ROS). LTA pretreatment also markedly reduced both the expression of iNOS and the formation of nitrotyrosine associated with renal I/R. Although LTA significantly reduced plasma nitrite/nitrate levels associated with I/R, nitrite/nitrate levels remained at levels significantly higher than that measured from the plasma obtained from Sham-operated animals. CONCLUSIONS: These data suggest, to our knowledge for the first time, that LTA pretreatment for 24 hours significantly reduces renal I/R injury. We propose that the mechanism of the protective effect involves reduction of the production of NO, ROS and peroxynitrite subsequent to reduced P-selectin and iNOS expression and PMN recruitment. However, although LTA pretreatment resulted in a reduction of iNOS expression and NO production, we hypothesize that the remaining significant levels of NO contribute to the beneficial actions provided by LTA.

GW274150, a potent and highly selective inhibitor of iNOS, reduces experimental renal ischemia/reperfusion injury.

BACKGROUND: Generation of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of GW274150, a novel, highly selective, potent and long-acting inhibitor of iNOS activity in rat and mouse models of renal I/R. METHODS: Rats were administered GW274150 (5 mg/kg intravenous bolus administered 30 minutes prior to I/R) and subjected to bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Serum and urinary indicators of renal dysfunction, tubular and reperfusion injury were measured, specifically, serum urea, creatinine, aspartate aminotransferase (AST) and N-acetyl-beta-d-glucosaminidase (NAG) enzymuria. In addition, renal sections were used for histologic scoring of renal injury and for immunologic evidence of nitrotyrosine formation and poly [adenosine diphosphate (ADP)-ribose] (PAR). Nitrate levels were measured in rat plasma using the Griess assay. Mice (wild-type, administered 5 mg/kg GW274150, and iNOS-/-) were subjected to bilateral renal ischemia (30 minutes) followed by reperfusion (24 hours) after which renal dysfunction (serum urea, creatinine), renal myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured. RESULTS: GW274150, administered prior to I/R, significantly reduced serum urea, serum creatinine, AST, and NAG indicating reduction of renal dysfunction and injury caused by I/R. GW274150 reduced histologic evidence of tubular injury and markedly reduced immunohistochemical evidence of nitrotyrosine and PAR formation, indicating reduced peroxynitrite formation and poly (ADP-ribose) polymerase (PARP) activation, respectively. GW274150 abolished the rise in the plasma levels of nitrate (indicating reduced NO production). GW274150 also reduced the renal dysfunction in wild-type mice to levels similar to that observed in iNOS-/- mice subjected to I/R. Renal MPO activity and MDA levels were significantly reduced in wild-type mice administered GW274150 and iNOS-/- mice subjected to renal I/R, indicating reduced polymorphonuclear leukocyte (PMN) infiltration and lipid peroxidation. CONCLUSIONS: These results suggest that (1). an enhanced formation of NO by iNOS contributes to the pathophysiology of renal I/R injury and (2). GW274150 reduces I/R injury of the kidney. We propose that selective inhibitors of iNOS activity may be useful against renal dysfunction and injury associated with I/R of the kidney.

The tyrosine kinase inhibitor tyrphostin AG126 reduces renal ischemia/reperfusion injury in the rat.

BACKGROUND: We investigate the effects of tyrphostin AG126, an inhibitor of tyrosine kinase activity, on the renal dysfunction and injury caused by ischemia/reperfusion (I/R) of the kidney. METHODS: Tyrphostin AG126 (5 mg/kg intraperitoneally) was administered to male Wistar rats 30 minutes prior to bilateral renal ischemia for 45 minutes followed by reperfusion for up to 48 hours. Biochemical markers of renal dysfunction and injury were measured and renal sections assessed for renal injury. Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and formation of nitrotyrosine and poly (ADP) ribose (PAR) were assessed using immunohistochemistry. Rat proximal tubular cells (PTCs) were incubated with interferon-gamma (100 IU/mL), bacterial lipopolysaccharide (10 microg/mL), and with increasing concentrations of tyrphostin AG126 (0.0001-1 mmol/L) for 24 hours. Nitric oxide production was measured in both plasma from rats subjected to I/R and in incubation medium from PTCs. RESULTS: After 6 hours of reperfusion, tyrphostin AG126 significantly reduced the increase in serum and urinary indicators of renal dysfunction and injury caused by I/R and reduced histologic evidence of renal injury. Tyrphostin AG126 also improved renal function (after 24 and 48 hours of reperfusion) and reduced the histologic signs of renal injury (after 48 hours of reperfusion). Tyrphostin AG126 reduced the expression of iNOS and nitric oxide levels in both rat plasma and in PTC cultures, as well as expression of COX-2. Tyrphostin AG126 also reduced nitrotyrosine and PAR formation, suggesting reduction of nitrosative stress and poly (ADP-ribose) polymerase (PARP) activation, respectively. CONCLUSION: Taken together, these results show that tyrphostin AG126 significantly reduces the renal dysfunction and injury caused by I/R of the kidney. We propose that inhibition of tyrosine kinase activity may be useful against renal I/R injury.

High density lipoprotein (HDL) reduces renal ischemia/reperfusion injury.

High-density lipoproteins (HDL) have been shown to reduce organ injury and mortality in animal models of shock via modulation of the expression of adhesion molecules and pro-inflammatory enzymes. As renal inflammation plays an important role in the development of ischemia/reperfusion (I/R) injury of the kidney, the aim of this study was to investigate the ability of HDL to alleviate renal dysfunction and injury in a rat model of renal I/R. HDL (80 mg/kg, intravenous) was administered to male Wistar rats 30 min before bilateral renal ischemia for 45 min followed by reperfusion for up to 48 h. After 6-h reperfusion, HDL significantly reduced (1) renal and tubular dysfunction, (2) tubular and reperfusion-injury, and (3) histologic evidence of renal injury. HDL also improved renal function (after 24-h and 48-h reperfusion) and reduced histologic signs of renal injury (after 48-h reperfusion). Administration of HDL significantly reduced the numbers of polymorphonuclear leukocytes (PMN) infiltrating into renal tissues during reperfusion, which was reflected by an attenuation of the increase in renal myeloperoxidase activity caused by I/R. Furthermore, HDL markedly reduced expression of the adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), and P-selectin during reperfusion. The increase in renal malondialdehyde levels caused by renal I/R was also significantly reduced by HDL, suggesting attenuation of lipid peroxidation subsequent to oxidative stress. These results demonstrate that HDL significantly reduces renal I/R injury and severity of ischemic acute renal failure. It is proposed that the mechanism of protection involves reduction of the expression of adhesion molecules, resulting in reduction of PMN infiltration and oxidative stress.

EUK-134 reduces renal dysfunction and injury caused by oxidative and nitrosative stress of the kidney.

BACKGROUND/AIMS: Oxidative and nitrosative stress plays important roles in the pathogenesis of renal ischemia/reperfusion (I/R) injury. Here we investigate the effect of EUK-134, a synthetic superoxide dismutase and catalase mimetic, (i) on renal dysfunction and injury caused by I/R in vivo and (ii) on proximal tubular cell (PTC) injury and death caused by oxidative and nitrosative stress. METHODS: Rats, subjected to bilateral renal ischemia (45 min) followed by reperfusion (6 h), were administered EUK-134 (0.3 and 3 mg/kg, i.v.) prior to and during reperfusion, after which biochemical and histological indicators of renal dysfunction and injury were measured. The expression of poly(ADP-ribose) (PAR) and inducible nitric oxide (NO) synthase (iNOS) and nitrotyrosine formation were determined immunohistochemically and used as indicators of oxidative and nitrosative stress. Primary cultures of rat PTCs, isolated and cultured from the kidney cortex, were incubated with hydrogen peroxide (H2O2; 1 mM for 2 h) in the presence of increasing concentrations of EUK-134 (1-100 microM) after which PTC injury and death were measured. The effects of EUK-134 on serum levels of NO in rats subjected to renal I/R or on NO production by PTCs incubated with interferon-gamma (IFN-gamma, 100 IU/ml) and bacterial lipopolysaccharide (LPS, 10 microg/ml) in combination for 24 h were also measured. RESULTS: EUK-134 produced a significant reduction in renal dysfunction and injury caused by I/R. Specifically, serum creatinine levels, an indicator of renal dysfunction, were reduced from 227 +/- 11 (n = 12, I/R only) to 146 +/- 9 microM (n = 12, I/R +3 mg/kg EUK-134). Urinary N-acetyl-beta-D-glucosaminidase activity, an indicator of tubular damage, was reduced from 42 +/- 5 (n = 12, I/R only) to 22 +/- 3 IU/l (n = 12, I/R +3 mg/kg EUK-134). EUK-134 significantly reduced renal injury caused by oxidative stress in vivo (reduction in PAR formation), and in vitro EUK-134 reduced PTC injury and death caused by H2O2. However, EUK-134 also reduced nitrosative stress caused by I/R in vivo (reduction of iNOS expression and nitrotyrosine formation), which was reflected by a significant reduction in serum NO levels in rats subjected to renal I/R. Specifically, serum NO levels were reduced from 57 +/- 12 (n = 12, I/R only) to 23 +/- 3 mM (n = 12, I/R +3 mg/kg EUK-134). In vitro, EUK-134 significantly reduced NO production by PTCs incubated with IFN-gamma/LPS. CONCLUSION: We propose that EUK-134 reduces renal I/R injury not only via reduction of oxidative stress, but also by reducing nitrosative stress caused by renal I/R.

5-Aminoisoquinolinone reduces renal injury and dysfunction caused by experimental ischemia/reperfusion.

BACKGROUND: Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the development of ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of a water-soluble and potent PARP inhibitor, 5-aminoisoquinolinone (5-AIQ), on the renal injury and dysfunction caused by oxidative stress of the rat kidney in vitro and in vivo. METHODS: Primary cultures of rat renal proximal tubular cells, subjected to oxidative stress caused by hydrogen peroxide (H2O2), were incubated with increasing concentrations of 5-AIQ (0.01 to 1 mmol/L) after which PARP activation, cellular injury, and cell death were measured. In in vivo experiments, anesthetized male Wistar rats were subjected to renal bilateral ischemia (45 minutes) followed by reperfusion (6 hours) in the absence or presence of 5-AIQ (0.3 mg/kg) after which renal dysfunction, injury and PARP activation were assessed. RESULTS: Incubation of proximal tubular cells with H2O2 caused a substantial increase in PARP activity, cellular injury, and cell death, which were all significantly reduced in a concentration-dependent by 5-AIQ [inhibitory concentration 50 (IC50) approximately 0.03 mmol/L]. In vivo, renal I/R resulted in renal dysfunction, injury, and PARP activation, primarily in the proximal tubules of the kidney. Administration of 5-AIQ significantly reduced the biochemical and histologic signs of renal dysfunction and injury and markedly reduced PARP activation caused by I/R. CONCLUSION: This study demonstrates that 5-AIQ is a potent, water soluble inhibitor of PARP activity, which can significantly reduce (1) cellular injury and death caused to primary cultures of rat proximal tubular cells by oxidative stress in vitro, and (2) renal injury and dysfunction caused by I/R of the kidney of the rat in vivo.

Agonists of peroxisome-proliferator activated receptor-gamma reduce renal ischemia/reperfusion injury.

BACKGROUND/AIMS: Recent evidence indicates that peroxisome-proliferator activated receptor (PPAR) agonists protect against ischemia/reperfusion (I/R) injury. Here we investigate the effects of the PPAR-gamma agonists, rosiglitazone and ciglitazone, on the renal dysfunction and injury caused by I/R of the rat kidney in vivo. METHODS: Rosiglitazone or ciglitazone were administered to male Wistar rats prior to and during reperfusion. Biochemical indicators of renal dysfunction and injury were measured and histological scoring of kidney sections was used to assess renal injury. Expression of PPAR isoforms and intercellular adhesion molecule-1 during renal I/R were assessed using RT-PCR and Northern blot, respectively. Myeloperoxidase activity and activation of poly(ADP-ribose) polymerase (PARP) were used as indicators of polymorphonuclear (PMN) cell infiltration and oxidative stress, respectively. RESULTS: Expression of PPAR-alpha, PPAR-beta and PPAR-gamma 1 (but not PPAR-gamma 2) was observed in kidneys with down-regulation of PPAR-alpha expression during renal I/R. Rosiglitazone and ciglitazone significantly reduced biochemical and histological signs of renal dysfunction and injury. Renal expression of ICAM-1 caused by I/R was reduced by rosiglitazone and ciglitazone which was reflected by decreased PMN infiltration into reperfused renal tissues. Both rosiglitazone and ciglitazone reduced PARP activation indicating a reduction of oxidative stress. CONCLUSION: These results suggest that the PPAR-gamma agonists rosiglitazone and ciglitazone reduce the renal dysfunction and injury associated with I/R of the kidney. We propose that one mechanism underlying the protective effects involves inhibition of the expression of ICAM-1, a reduction of PMN infiltration into renal tissues and subsequent reduction of oxidative stress.