Cyclooxygenase inhibition improves endothelium-dependent vasodilatation in patients with chronic renal failure.

BACKGROUND: Some studies have demonstrated beneficial effects of L-arginine as a substrate for nitric oxide synthesis, and diclofenac as an inhibitor of cyclooxygenase (COX)-derived vasoconstrictive agents on vascular responses in humans during several pathological conditions. The aim of the present study was to investigate the acute effects of L-arginine and diclofenac on endothelium-dependent vasodilatation (EDV) and endothelium-independent vasodilatation (EIDV) in patients with chronic renal failure (CRF). METHODS: Effects of L-arginine and diclofenac on EDV and EIDV were measured in 15 patients with CRF and in 15 healthy controls by means of forearm blood flow measurements with venous occlusion plethysmography during local intra-arterial infusions of methacholine (2 and 4 micro g/min evaluating EDV) and sodium nitroprusside (5 and 10 micro g/min evaluating EIDV). RESULTS: L-Arginine infusion increased methacholine-induced vasodilatation both in patients with CRF and healthy controls. Diclofenac infusion increased methacholine-induced vasodilatation only in patients with CRF. There was no significant change in nitroprusside-induced vasodilatation after L-arginine and diclofenac infusions both in patients with CRF and healthy controls. CONCLUSIONS: These results suggest that COX inhibition reduces the levels of a prostanoid-derived vasoconstrictive agent contributing to the impaired EDV in patients with CRF, while in this age group L-arginine improves EDV regardless of renal function.     

Homocysteine induced impairment of nitric oxide-dependent vasorelaxation is reversible by the superoxide dismutase mimetic TEMPOL.

BACKGROUND: Elevated plasma homocysteine concentrations in renal patients are associated with accelerated cardiovascular disease. The mechanism(s) by which homocysteine acts remains unclear however, evidence implicates a role involving endothelial dysfunction. METHODS: Rat femoral arteries after acute or 4-h pre-incubation with racemic D,L-homocysteine (100 microM) were mounted on a myograph, pre-constricted with phenylephrine (10 microM) and responses to acetylcholine-dependent vasorelaxation examined. The incubations were repeated in the presence of indomethacin (10 microM), omega-nitro-L-arginine methyl ester (100 microM), L-arginine (100 microM), tetrahydrobiopterin (1 microM), catalase (1200 U/ml), ebselen, a peroxynitrite chelator (20 microM) and TEMPOL, a superoxide dismutase mimetic (1 mM). Results are shown as means+/-standard error, expressed as per cent relaxation to acetylcholine added (nmol/l). RESULTS: Increasing concentrations of homocysteine had no affect when added directly to basally relaxed or pre-constricted freshly isolated vessels. However, 4-h pre-incubation with or without homocysteine significantly shifted the acetylcholine EC(50) (EC(50) was defined as the concentration of acetylcholine that caused relaxation of the phenylephrine contracted tissue by 50%), control((4 h)) = 74.7 nmol/l+/-10.5 vs 100 microM D,L-homocysteine((4 h)) = 159.9 nmol/l+/-20.6; P<0.05) without affecting maximal relaxation. Response to endothelial independent relaxation was unaffected. Indomethacin, indomethacin and omega-nitro-L-arginine methyl ester, l-arginine and tetrahydrobiopterin, catalase and ebselen had no effect on the EC(50) in homocysteine-exposed arteries. However, TEMPOL normalized vasorelaxation in homocysteine-treated arteries (75.2 nmol/l+/-14.6) but had no effect on the 4-h control group. Moreover, washing TEMPOL from the treated vessels restored endothelial dysfunction in D,L-homocysteine-treated vessels (163.9 nmol/l+/-34.1). CONCLUSIONS: We conclude that homocysteine causes endothelial dysfunction by up-regulating a potential superoxide generating system resulting in reduced nitric oxide bio-availability.     

Hypercholesterolaemia and treatment with statins do not alter L-arginine-induced changes of renal haemodynamics.

BACKGROUND: Hypercholesterolaemia has been found to impair endothelial function in the systemic and coronary circulations and lipid-lowering therapy with statins has been shown to improve this abnormality. METHODS: We examined the impact of hypercholesterolaemia on L-arginine-induced renal vascular relaxation by a cross-sectional study, and the effects of lipid-lowering therapy by a double-blind, randomized, placebo-controlled study. Using constant infusion input clearance technique (PAH and inulin respectively), changes of renal plasma flow (RPF) and glomerular filtration rate (GFR) in response to intravenous infusions of L-arginine (100 mg/kg/30 min and 500 mg/kg/30 min) were studied in 21 hypercholesterolaemic humans (age 57+/-9 years, LDL-cholesterol 211+/-35 mg/dl) and in 20 young healthy (age 26+/-2 years, LDL-cholesterol 90+/-27 mg/dl) and 20 older healthy age-matched control individuals (age 50+/-8 years, LDL-cholesterol 106+/-20 mg/dl). In addition, changes of blood pressure, heart rate, urinary excretion of sodium, and cyclic guanosine monophosphate were measured. Patients were analysed before and after 3 months treatment with either fluvastatin (40 mg twice daily, n=11) or placebo (n=10). RESULTS: In hypercholesterolaemic patients, L-arginine increased RPF and GFR (P<0.01) and urinary excretion of sodium (P<0.05) in a dose-dependent manner. Interestingly, changes were similar between the hypercholesterolaemic patients and the young and the age-matched control individuals (DeltaRPF 100 mg/kg/30 min, 40+/-51 ml/min vs 40+/-52 ml/min, P=NS; DeltaRPF 500 mg/kg/30 min, 114+/-85 ml/min vs 130+/-78 ml/min, P=NS). L-arginine significantly lowered systemic arterial pressure and increased heart rate in all groups. Despite significant reductions in LDL-cholesterol levels (291+/-35 mg/dl vs 213+/-30 mg/dl, P<0.001), treatment with fluvastatin did not alter the renal haemodynamic response pattern to L-arginine infusion when compared to baseline values and to those with placebo. CONCLUSION: In contrast to studies performed in the vasculature of the human forearm or the coronary circulation, our results suggest that hypercholesterolaemia is not associated with an impaired L-arginine-induced renal vascular relaxation.     

Net renal extraction of asymmetrical (ADMA) and symmetrical (SDMA) dimethylarginine in fasting humans.

BACKGROUND: Recently, the potential importance of dimethylarginines as endogenously produced inhibitors of nitric oxide synthase has become clearer. Interestingly, elevated levels have been reported in patients with vascular disease, but especially in patients suffering end-stage renal disease. Although the kidney obviously seems to play a key role in the elimination of dimethylarginines, clear insight into the renal handling of these compounds is lacking. Thus, our aim was to investigate the renal extraction of dimethylarginines. METHODS: Plasma concentrations of dimethylarginines were determined in both arterial and renal venous blood in 20 fasting patients with normal renal function. Renal extraction was calculated as the arteriovenous concentration difference divided by the arterial concentration times 100%. RESULTS: A significant renal extraction was found for both dimethylarginines. Renal extraction was significantly higher for asymmetrical dimethylarginine (ADMA) when compared with symmetrical dimethylarginine (SDMA) (16.2 vs 10.5% respectively, P=0.001). In addition, arterial SDMA concentration, but not ADMA concentration, significantly correlated with arterial creatinine concentration. CONCLUSIONS: In healthy humans, the kidney contributes to the regulation of plasma levels of dimethylarginines, since both ADMA and SDMA were significantly extracted from the arterial supply. Interestingly, a higher renal extraction of ADMA was found when compared to SDMA extraction, which strongly suggests the presence of an additional catabolic pathway for ADMA in the kidney.     

Haemodynamic effects of valsartan in acute renal ischaemia/reperfusion injury.

BACKGROUND: Acute deterioration of renal function is an important side-effect of angiotensin-converting enzyme (ACE) inhibitors, especially if accompanied by other nephrotoxic events. Angiotensin II receptor(1) blockers (ARB) are thought to have fewer side-effects on renal perfusion and function. We examined the effects of valsartan (VAL) on kidney function as well as the contribution of the nitric oxide (NO) system in a rat model of ischaemic acute renal failure (ARF). METHODS: ARF was induced by 40 min of clamping of both renal arteries in female Sprague-Dawley rats. Renal haemodynamic and tubular parameters were determined during post-ischaemic infusion of vehicle, VAL, VAL and the NO-synthase substrate L-arginine, and VAL together with inhibition of NO synthases (NOS) by L-NMMA. RESULTS: Clamping induced acute renal failure with marked decreases in glomerular filtration rate (GFR) and renal plasma flow (RPF) accompanied by a rise in renal vascular resistance (RVR) and fractional sodium excretion. Valsartan caused a slight but significant improvement of GFR and RPF without full recovery of renal function and caused a lowering of RVR and tubular sodium loss. L-arginine-co-administration had no additive beneficial effect. Valsartan-induced changes were not significantly depressed by unspecific inhibition of NOS. CONCLUSIONS: Inhibition of the angiotensin II-receptor(1) diminishes the deleterious effects of ischaemia and reperfusion on glomerular function and on the renal microcirculation. An involvement of the NO system could not be demonstrated.     

Soy protein diet improves endothelial dysfunction in renal transplant patients.

BACKGROUND: Since it has been demonstrated that soy diet can improve endothelial function, in the present study we evaluated the effect of dietary substitution of 25 g of animal proteins with soy proteins on endothelial dysfunction in renal transplant patients. METHODS: In 20 renal transplant patients (55 +/- 11 years, serum creatinine 1.7 +/- 0.6 mg/dl), brachial artery flow mediated dilation (FMD) and endothelium-independent vasodilation (sublingual nitroglycerine, 25 microg) were measured at baseline, after 5 weeks of a soy diet and finally after 5 weeks of soy wash-out. Changes in plasma lipids, markers of oxidative stress (lipid peroxides, LOOH) and inflammation (C-reactive protein), isoflavones (genistein and daidzein), asymmetric dimethyl arginine (ADMA) and L-arginine were also evaluated. RESULTS: At baseline, patients showed a significantly lower FMD as compared with age-matched healthy subjects (3.2 +/- 1.8 vs 6.3 +/- 1.9, respectively; P < 0.001), while response to nitroglycerine was similar. After soy diet, actual protein intake was not changed, cholesterol and lipid peroxides were significantly reduced, and isoflavones were detectable in plasma. Soy diet was associated with a significant improvement in FMD (4.4 +/- 2.0; P = 0.003 vs baseline), while response to nitroglycerine was unchanged. Improvement in FMD was related to L-arginine/ADMA ratio changes, but no significant relation was found to changes in cholesterol, lipid peroxides or genistein and daidzein plasma concentrations. After 5 weeks of soy diet discontinuation, FMD (3.3 +/- 1.7%) returned to baseline values and isoflavones were no longer detectable in plasma. CONCLUSIONS: A soy protein diet for 5 weeks improves endothelial function in renal transplant patients. This effect seems to be strictly dependent on soy intake as it disappears after soy withdrawal and is mediated by an increase in the L-arginine/ADMA ratio, independently of change in lipid profile, oxidative stress or isoflavones.     

L-carnitine inhibits a subset of platelet activation responses in chronic uraemia.

BACKGROUND: Activated uraemic platelets expose the aminophospholipid phosphatidylserine (PS) at their outer surface, which generates a cell procoagulant phenotype and seems at least partly due to an increase in cell caspase-3 activity. L-carnitine (LC) may decrease surface-exposed PS in stored apheresis platelets and inhibit the activity of recombinant caspases, but its effects on platelet activation response with PS externalization have not been ascertained in chronic renal failure. In the present study, we investigated in vitro and in vivo the effects of LC on PS exposure in platelets from chronic uraemic patients. METHODS: Platelet PS-exposure was assayed by flow cytometry using annexin V. Caspase activity in platelets was determined by the cleaving activity of the specific substrate DEVD-pNA and by a flow cytometric assay using rhodamine-fluorescence. The effects of LC in vivo were examined in a prospective cross-over trial including 10 patients on maintenance haemodialysis (HD) who were randomly allocated to two different treatment groups: LC (2 g i.v.) for 4 months followed by placebo (2 g i.v.) for another 4 months (group A), or placebo followed by LC (group B). RESULTS: PS-exposing platelets in blood samples obtained from HD patients were significantly higher than in healthy subjects (P<0.001) under both unstimulated and agonist-stimulated conditions. When uraemic platelets were pre-incubated with LC before agonist stimulation, platelet PS exposure proved to be significantly reduced (-13.7% for 0.5 mM LC and -25% for 5 mM LC). Pre-incubation of uraemic platelets with LC again significantly decreased the cells' caspase activity (P<0.05). In HD patients (Group A), LC supplementation was associated with a significant decrease (P<0.05) in platelet PS exposure followed by a progressive increase during treatment with placebo. In the other group of patients, while no change in platelet PS exposure was observed during the first 4 months of treatment with placebo, a significant reduction (P<0.05) in PS-positive platelets occurred after 2 and 4 months of LC therapy. CONCLUSION: Our data show that LC may reduce, possibly via inhibition of caspase activity, the exposure of PS in activated uraemic platelets. These findings may have implications for the thrombophilic tendency of uraemia.     

Effects of L-carnitine infusions on inflammatory and nutritional markers in haemodialysis patients.

BACKGROUND: Carnitine loss through dialysis membranes is shown to be related to the lack of carnitine in long-term haemodialysis patients. It has been previously reported that haemodialysis patients might have benefited from carnitine supplementation. METHODS: A total of 21 chronic haemodialysis patients maintaining carnitine supplementation and 21 controls (haemodialysis patients not receiving carnitine) were included in the study. L-carnitine was used intravenously three times a week after each haemodialysis session, at a 20 mg/kg dose. C-reactive protein (CRP), lipid profile, transferrin, total protein and albumin levels were determined at baseline after 3 and 6 months of treatment, and compared with the control group. RESULTS: CRP levels were significantly decreased in carnitine group in contrast to the increase in the control group. Transferrin, total protein and albumin levels and body mass index (BMI) of the patients rose in the carnitine group. CONCLUSIONS: There was a significant benefit of L-carnitine on CRP, transferrin, total protein and albumin levels of the haemodialysis patients.     

Differential regulation of L-arginine transporters (cationic amino acid transporter-1 and -2) by peroxynitrite in rat mesangial cells.

BACKGROUND: It has become evident that increased nitric oxide (NO) generation may be associated with production of reactive oxygen species, such as peroxynitrite (ONOO-). Peroxynitrite has been postulated to be responsible for several of the cytotoxic effects previously ascribed to NO. Since cellular arginine uptake has been shown to modulate nitric oxide synthase activity, we were intrigued to study the effect of ONOO- on arginine traffic in renal mesangial cells. METHODS: Arginine uptake, CAT-1 and CAT-2 mRNA expression by northern blotting analysis, and CAT-1 protein content using western blotting were determined in mesangial cells pre-treated with peroxynitrite (0.1 and 0.5 mM) for 2 h. RESULTS: Peroxynitrite induced a significant increase in arginine uptake and CAT-2 mRNA expression compared with untreated cells. In contrast, CAT-1 mRNA expression and protein abundance were diminished. CONCLUSIONS: In rat mesangial cells, peroxynitrite augments arginine uptake via augmentation of CAT-2 while decreasing CAT-1 expression.     

Jejunal dopamine and Na,K-ATPase activity in early chronic renal insufficiency

AIM: The uninephrectomised and three-quarter nephrectomised (3/4nx) rats present dopamine-sensitive enhanced natriuresis. This is accompanied in uninephrectomised rats by a reduced jejunal Na(+),K(+)-ATPase activity with recovered sensitivity to inhibition by dopamine. The present study examined the jejunal Na(+),K(+)-ATPase activity and the role of dopamine in 3/4nx animals. METHODS: Fourteen days after surgery, the L-amino acid decarboxylase activity (AADC) activity, the enzyme responsible for the synthesis of dopamine, and the Na(+),K(+)-ATPase activity, were determined in jejunal epithelial cells from 3/4nx and Sham rats. In addition, the effect of dopamine (1 micromol/L) on jejunal Na(+),K(+)-ATPase activity was evaluated in both groups. RESULTS: The 3/4nx rats presented a reduced AADC activity in jejunal epithelial cells (V(max) in nmol/mg prot/15 min, 142 +/- 6 vs 190 +/- 10, P < 0.05). In addition, the jejunal Na(+),K(+)-ATPase activity was increased in 3/4nx rats (Pi release in nmol/mg prot/min, 137 +/- 1 vs 122 +/- 2, P < 0.05). However, dopamine was unable to inhibit the Na(+),K(+)-ATPase activity in jejunal epithelial cells from both 3/4nx and Sham animals. CONCLUSIONS: In contrast to uninephrectomy, the jejunal Na(+),K(+)-ATPase activity is increased in 3/4nx rats and is not sensitive to inhibition by dopamine.     

Handling of asymmetrical dimethylarginine and symmetrical dimethylarginine by the rat kidney under basal conditions and during endotoxaemia.

BACKGROUND: Asymmetrical dimethylarginine (ADMA) is capable of inhibiting nitric oxide synthase enzymes, whereas symmetrical dimethylarginine (SDMA) competes with arginine transport. The potential role of inflammation in the metabolism of ADMA has been elucidated in an in vitro model using tumour necrosis factor-alpha, resulting in a decreased activity of the ADMA-degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH). The kidney probably plays a crucial role in the metabolism of ADMA by both urinary excretion and degradation by DDAH. We aimed to further elucidate the role of the kidney in a rat model under basal conditions and during endotoxaemia. METHODS: Twenty-five male Wistar rats weighing 275-300 g were used for this study. The combination of arteriovenous concentration differences and kidney blood flow allowed calculation of net organ fluxes. Blood flow was measured using radiolabelled microspheres according to the reference sample method. Concentrations of ADMA, SDMA and arginine were measured by high-performance liquid chromatography. RESULTS: The kidney showed net uptake of both ADMA and SDMA and fractional extraction rates were 35% and 31%, respectively. Endotoxaemia resulted in a lower systemic ADMA concentration (P = 0.01), which was not explained by an increased net renal uptake. Systemic SDMA concentrations increased during endotoxaemia (P = 0.007), which was accompanied by increased creatinine concentrations. CONCLUSIONS: The rat kidney plays a crucial role in the regulation of concentrations of dimethylarginines, as both ADMA and SDMA were eliminated from the systemic circulation in substantial amounts. Furthermore, evidence for the role of endotoxaemia in the metabolism of dimethylarginines was obtained as plasma levels of ADMA were significantly lower in endotoxaemic rats.     

L-2-oxothiazolidine-4-carboxylic acid reduces in vitro cytotoxicity of glucose degradation products.

BACKGROUND: Glucose degradation products (GDP) are an important factor that contribute to bioincompatibility of peritoneal dialysis fluids. These substances are generated in the dialysis fluid during heat sterilization. Several approaches have been proposed to reduce the content or toxicity, or both, of GDP present in the dialysis fluid. We examined whether L-2-oxothiazolidine-4-carboxylic acid (OTZ), a precursor for glutathione synthesis, reduces the cytotoxicity of GDP in human peritoneal mesothelial cells. METHODS: Experiments were performed on primary mesothelial cell cultures. Free radical generation in these cells after exposure to acetaldehyde (ACT), glyoxal (GLYO) or methylglyoxal (M-GLYO) was detected with a fluorescent probe. Cell viability measurements were based on release of LDH from cell cytosol, and synthesis of IL-6 and proliferation after exposure to GDP. Effects of individual GDPs and of dialysis fluid free of GDP (GDP-free PDF) or containing GDP (GDP-high PDF) on cell viability were also studied in the presence of OTZ (1 mmol/l). RESULTS: All of the GDPs as well as the autoclaved dialysis fluid caused increased free radical generation. ACT increased LDH release from the cells by 374% (P < 0.001), and this effect was abolished by OTZ. All of the GDPs inhibited cell growth (ACT, 47%, P < 0.01; GLYO, 52%, P < 0.01; M-GLYO, 26%, P < 0.05) and this effect was reversed in presence of OTZ. ACT inhibited Il-6 synthesis in mesothelial cells by 74% P < 0.01 and this effect was prevented by OTZ. GDP-high PDF but not GDP-free PDF reduced synthesis of IL-6 in mesothelial cells by 40% (P < 0.01) an effect that was reversed by OTZ. Mesothelial cell growth was more strongly inhibited by GDP-high PDF (76%, P < 0.01) than by GDP-free PDF (31%, P < 0.05). OTZ improved growth of mesothelial cells in the presence of GDP-high PDF (+150%, P < 0.01) and in presence of GDP-low PDF (+38%, P < 0.05). OTZ prevented the cytotoxic effect of GDP-high PDF on mesothelial cells. CONCLUSIONS: The GDP-induced stimulation of free radicals in mesothelial cells in the present study may provide a possible mechanism of GDP cytotoxicity. Because OTZ reduced the toxic effects of GDP on mesothelial cells, this compound may improve biocompatibility of peritoneal dialysis fluids.     

Effect of L-carnitine on erythroid colony formation in mouse bone marrow cells.

BACKGROUND: l-Carnitine can alleviate uraemic anaemia in haemodialysis patients by improving erythrocyte membrane functions or erythropoiesis, which are depressed under uraemic conditions. l-Carnitine and palmitoyl-l-carnitine were reported to increase the formation of colony-forming unit-erythroid (CFU-E) colonies in cultures of fetal mouse liver cells, an effect that depended on the concentration of palmitoyl-l-carnitine but not of l-carnitine. In this study, we investigated l-carnitine's effect on CFU-E colony formation in cell cultures of mouse bone marrow cells. METHODS: Bone marrow from normal female mice was placed in 35 mm culture dishes containing a medium composed of methylcellulose and various nutrients. The dishes were incubated for 48 h, and the colonies of erythroblasts, which were differentiated from CFU-E, consisting of >/=8 cells, were counted in each dish using an inverted microscope. RESULTS: The numbers of CFU-E colonies correlated well with both the initial numbers of bone marrow cells and concentrations of recombinant human erythropoietin (rhEPO) in the methylcellulose medium. In the presence of 0.5 or 1.0 IU/ml of rhEPO, l-carnitine at concentrations of 200 and 400 micromol/l significantly enhanced CFU-E colony formation (P<0.001). CONCLUSION: l-Carnitine significantly increased the number of CFU-E colonies in mouse bone marrow cell cultures. This finding suggests that l-carnitine stimulates erythropoiesis, partially accounting for its mitigating effect on renal anaemia.     

L-Arginine-enriched preservation solution decreases ischaemia/reperfusion injury in canine kidneys after long-term cold storage.

BACKGROUND: Kidneys stored hypothermically for transplantation show varying degrees of tissue injury, depending on the duration of preservation. The causes of injury are not entirely clear. We investigated the quality of renal functional recovery in canine kidneys after 72 h hypothermic preservation in custodiol solution or custodiol solution plus L-arginine. METHODS: Kidneys obtained from mongrel dogs, weighing 18-25 kg, were subjected to 72-h cold ischaemia after flushing. Animals were divided into two groups (n=18/group). A flush solution of either custodiol solution or custodiol solution plus L-arginine 1 mmol/l was used for each group. After 72-h cold storage all animals had a contralateral nephrectomy, and autotransplantation was performed to external iliac artery and vein. Survivals were evaluated at 3 days. RESULTS: Renal damage was assessed by kidney function tests, serum creatinine (SCr), blood urea nitrogen (BUN) and light histology. Malondialdehyde (MDA) was measured as an index of lipid peroxidation. SCr and BUN (24, 48 and 72 h) were significantly different from the control and L-arginine groups. Histological damage was less in the L-arginine group. MDA levels were significantly different with the lower levels in the L-arginine group. CONCLUSIONS:On the basis of these data, we concluded that exogenous L-arginine (a substrate for NO synthesis) has a beneficial and protective effect on long-term (72 h) hypothermic ischaemical damage in canine kidneys.     

Glyoxylate reductase activity in blood mononuclear cells and the diagnosis of primary hyperoxaluria type 2.

BACKGROUND: Primary hyperoxaluria type 2 (PH2) is a rare monogenic disorder characterized by an elevated urinary excretion of oxalate. Increased oxalate excretion in PH2 patients can cause nephrolithiasis and nephrocalcinosis, and can, in some cases, result in renal failure and systemic oxalate deposition. The disease is due to a deficiency of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) activity. A definitive diagnosis of PH2 is currently made by the analysis of GR activity in a liver biopsy. GRHPR is expressed in virtually every tissue in the body, suggesting that utilization of more readily available cells could be used to determine GRHPR deficiency. In this study, we have evaluated the potential of determining GR and d-glycerate dehydrogenase (DGDH) activity in blood mononuclear cells (BMC) as a diagnostic indicator of PH2. METHODS: Blood samples were obtained from 10 male and 10 female normal subjects, median age 31, range 21-63, at the Wake Forest University Medical Center and from primary hyperoxaluria patients at the Mayo Clinic. The BMC were isolated and GR and DGDH activities measured in cell lysates. RESULTS: An assay of 20 normal individuals indicated that BMC contained a DGDH and GR activity of 0.97+/-0.20 (range 0.62-1.45), and 10.6+/-3.3 (range 8.3-16.6) nmol/min/mg protein, respectively. The intra-assay coefficient of variation for DGDH and GR activity was 8.2 and 11.5%, respectively. The BMC lysates from normal adult subjects and patients with PH1 showed similar GR and DGDH activities. This was confirmed by the presence of immunoreactive GRHPR protein by western blot analysis. In contrast, PH2 BMC lysates did not exhibit DGDH or GR activity, and showed no immunoreactive GRHPR by western blot analysis. CONCLUSION: These results suggest that the assay of DGDH or GR activity in BMC could be used as a minimally invasive diagnostic test for PH2.     

Protective role of L-2-oxothiazolidine-4-carboxylic acid in cisplatin-induced renal injury.

BACKGROUND: Oxidative stress and inflammation are implicated in the pathogenesis of cisplatin-induced nephrotoxicity. l-2-oxothiazolidine-4-carboxylic acid (OTC) is a cysteine prodrug, and increases cellular glutathione (GSH). OTC is converted to cysteine by the intracellular enzyme, oxoprolinase. To date, the protective role of OTC on cisplatin-induced renal injury has not been investigated. The purpose of the present study was to examine the protective effect of OTC on cisplatin-induced renal injury and to examine the mechanism of its protection. METHODS: Mice were treated with cisplatin with or without administration of OTC. The generation of reactive oxygen species (ROS), expression of intercellular adhesion molecule (ICAM)-1 and monocyte chemoattractant protein (MCP)-1 were determined in the kidney using 2',7'-dichlorofluorescein diacetate, immunostaining or western blot analysis. Nuclear factor (NF)-kappaB activity, infiltration of F4/80-positive cells and apoptosis were also investigated in addition to renal function and histology using electrophoretic mobility shift assay, immunostaining, western blot analysis, uridine triphosphate (dUTP) nick-end labelling or periodic acid-Schiff staining. The effect of OTC on superoxide dismutase activity and GSH level in cisplatin-treated normal adult human kidney (HK-2) cells were measured using assay kits. RESULTS: The administration of OTC resulted in a significant reduction of cisplatin-induced ROS production, the p65 subunit of NF-kappaB translocation into nucleus, expression of ICAM-1, caspase 3 activity, expression of MCP-1 and the infiltration of macrophages into renal tissue. OTC markedly ameliorated renal damage induced by cisplatin through antioxidant and anti-inflammatory effect. CONCLUSIONS: These results suggest that OTC can be a potential therapeutic agent in cisplatin-induced renal injury through decreasing the ROS levels and activation of NF-kappaB.