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BACKGROUND: Anti-thymocyte globulins are polyclonal T-cell-depleting immunoglobulins used in induction of immunosuppression in kidney transplant recipients. Thymoglobulin is purified rabbit immunoglobulin (Ig)G, obtained by immunization of rabbits with fetal human thymus, which depletes T lymphocytes by complement-dependent lysis and apoptosis, reduces production of cytokines, and decreases expression of adhesion molecules in endothelial cells. METHODS: To determine possible direct effects of Thymoglobulin on kidney cells during transplantation, we used the Human Embryonic Kidney cell line (HEK293) in culture. We measured membrane potential of the cells by use of the slow whole patch-clamp technique. We determined effects of Thymoglobulin on cell death and proliferation during hypoxia/re-oxygenation injury, using a hypoxic chamber. RESULTS: Depolarizations of HEK293 cells caused by Thymoglobulin were concentration-dependent and membrane potential-dependent, showing direct effects of Thymoglobulin on the HEK293 cells, whereas rabbit anti-thymocyte globulin produced against Jurkat cells (ATG-F) and normal rabbit IgG had no effects. To determine the effects of Thymoglobulin in hypoxia/re-oxygenation conditions, cells were incubated for 24 hours with Thymoglobulin in an atmosphere with 5% CO2-95% N2 at 37°C followed by 1 hour in atmosphere with 5% CO2-95% air at 37°C. The effects of hypoxia/re-oxygenation were detected by calculating cell death and determining the cell growth, using scratch test. CONCLUSIONS: Thymoglobulin prevented the cell death induced by hypoxia and re-oxygenation conditions. In addition, it accelerated the cell growth (improved scratch wound-healing). This is the first study to show the direct effects of Thymoglobulin on kidney-derived epithelial cells, which may lead to better understanding of its effects in kidney transplantation.

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Almost 4500 patients are being treated with renal replacement therapies in Croatia. Infections are frequent in the population treated with dialysis and kidney transplantation, being one of the most common causes of morbidity and mortality in these patients. In dialysis patients, infections are usually related to dialysis access (usually central venous dialysis catheter or peritoneal catheter). In kidney transplant recipients, infections are most common in the early postoperative period. Prevention, early recognition, as well as appropriate treatment of infection are all crucial for better survival of these patients, with ever more other medical specialties being involved in their management.

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AIM: Mechanisms that initiate compensatory renal growth following unilateral nephrectomy are incompletely understood. An early event following unilateral nephrectomy is the activation of phospholipase C in the apical membrane of the proximal tubule, mediated by an unknown agonist. We tested the hypothesis that endothelin is responsible for the stimulation of phospholipase C in rat proximal tubule following unilateral nephrectomy. METHODS: Compensatory renal growth was induced in adult male rats by unilateral nephrectomy. 1,2-Diacylglycerol, a product of phospholipase C activation, was measured in renal cortical slices and isolated proximal tubules, respectively, 20 min following unilateral nephrectomy, or after incubation of the slices or proximal tubules with plasma from unilaterally nephrectomized or sham-operated rats. RESULTS: Twenty min following unilateral nephrectomy, an increase in 1,2-diacylglycerol concentration occurred in the renal cortex. Bosentan, a nonselective endothelin receptor antagonist, as well as an anti-endothelin-1 antibody administered intravenously, completely inhibited this 1,2-diacylglycerol accumulation in renal cortex. Incubation of renal cortical slices with plasma from unilaterally nephrectomized or bilaterally nephrectomized rats, stimulated 1,2-diacylglycerol production in isolated proximal tubule apical membranes. Again, bosentan prevented the increase evoked by incubation with plasma from unilaterally nephrectomized rats. Finally, concentration of endothelin-1 increased in renal cortex in response to unilateral nephrectomy. CONCLUSION: These results make evident the role of endothelin in stimulation of phospholipase C in proximal tubule following unilateral nephrectomy, suggesting participation of the endothelin system during the initiation of the compensatory renal growth.

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BACKGROUND: Renal failure is a frequent complication of sepsis with a high mortality. Tumor necrosis factor (TNF) has been suggested to be a factor in the acute renal failure in sepsis or endotoxemia. Recent studies also suggest involvement of nitric oxide (NO), generated by inducible NO synthase (iNOS), in the pathogenesis of endotoxin-induced renal failure. The present study tested the hypothesis that the role of TNF in endotoxic renal failure is mediated by iNOS-derived NO. METHODS: Renal function was evaluated in endotoxemic [Escherichia coli lipopolysaccharide (LPS), 5 mg/kg IP] wild-type and iNOS knockout mice. The effect of TNF neutralization on renal function during endotoxemia in mice was assessed by a TNF-soluble receptor (TNFsRp55). RESULTS: An injection of LPS to wild-type mice resulted in a 70% decrease in glomerular filtration rate (GFR) and in a 40% reduction in renal plasma flow (RPF) 16 hours after the injection. The results occurred independent of hypotension, morphological changes, apoptosis, and leukocyte accumulation. In mice pretreated with TNFsRp55, only a 30% decrease in GFR without a significant change in RPF in response to LPS, as compared with vehicle-treated mice, was observed. Also, the serum NO concentration was significantly lower in endotoxemic wild-type mice pretreated with TNFsRp55, as compared with untreated endotoxemic wild-type mice (260 +/- 52 vs. 673 +/- 112 micromol/L, P < 0.01). In LPS-injected iNOS knockout mice and wild-type mice treated with a selective iNOS inhibitor, 1400W, the development of renal failure was similar to that in wild-type mice. As in wild-type mice, TNFsRp55 significantly attenuated the decrease in GFR (a 33% decline, as compared with 75% without TNFsRp55) without a significant change in RPF in iNOS knockout mice given LPS. CONCLUSIONS: These results demonstrate a role of TNF in the early renal dysfunction (16 h) in a septic mouse model independent of iNOS, hypotension, apoptosis, leukocyte accumulation, and morphological alterations, thus suggesting renal hypoperfusion secondary to an imbalance between, as yet to be defined, renal vasoconstrictors and vasodilators.

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The chronic role of nitric oxide (NO), independent of prostaglandin synthesis, in the primary peripheral vasodilation, increased glomerular filtration rate (GFR), and renal plasma flow (RPF) in normal pregnancy remains to be defined. The purpose of the present study was to chronically inhibit NOS to return systemic vascular resistance (SVR), cardiac output (CO), GFR, and RPF to nonpregnant values. Pregnant rats received the nitric oxide synthase (NOS) inhibitor, nitro-L-arginine methyl ester (L-NAME), orally from gestational days 7 through 14. Results were compared with nonpregnant and untreated pregnant rats. At 14 days gestation, CO significantly increased in pregnant vs. nonpregnant rats (187 +/- 17 vs. 125 +/- 10 ml/min, P < 0.05) as SVR decreased (0.64 +/- 0.08 vs. 1.08 +/- 0.08 mmHg. ml(-1). min, P < 0.05) and mean arterial pressure was unchanged (117 +/- 5 vs. 125 +/- 2 mmHg, not significant). Pregnant rats also demonstrated increased GFR (3,015 +/- 33 vs. 2,165 +/- 136 microl/min, P < 0.01) and RPF (7,869 +/- 967 vs. 5,507 +/- 290 microl/min, P < 0.05) vs. nonpregnant rats. L-NAME-treated pregnant rats had values for CO (118 +/- 7 ml/min), SVR (1.09 +/- 0.07 mmHg. ml(-1). min), GFR (2,264 +/- 150 microl/min), and RPF (5,777 +/- 498 microl/min), which were no different than nonpregnant animals. In summary, similar to human pregnancy, primary peripheral vasodilation occurs early in rat pregnancy. Furthermore, the hyperdynamic circulation and glomerular hyperfiltration of normal rat midterm pregnancy can be chronically reversed by NOS inhibition. These findings suggest a role for endothelial damage and decreased NO in the pathogenesis of preeclampsia.

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Acute renal failure (ARF) is a common clinical condition with significant mortality. Although intermittent hemodialysis (IHD) represents a standard method of renal replacement therapy in ARF, novel methods of continuous renal replacement therapy (CRRT) are becoming more important. CRRT allows an excellent control of uremia, volume and acid-base status, avoiding at the same time rapid fluctuations in effective plasma volume, plasma osmolality, or blood pressure. Methods of CRRT differ with respect to the predominant way of solute removal, utilizing convective clearance (continuous ultrafiltration and hemofiltration), diffusive clearance (continuous hemodialysis), or a combination of the both (continuous hemodiafiltration). Based on their properties, CRRT represent method of choice in the treatment of hemodynamically unstable patients with ARF.

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In cirrhosis of the liver, according to the peripheral arterial vasodilation hypothesis, relative underfilling of the arterial tree triggers a neurohumoral response (activation of renin-angiotensin-aldosterone system, sympathetic nervous system, nonosmotic release of vasopressin) aimed at restoring circulatory integrity by promoting renal sodium and water retention. Evidence has accumulated for a major role of increased vascular production of nitric oxide as the primary cause of arterial vasodilation in cirrhosis. Ascites is a common complication in cirrhosis. Treatment of ascites consists of a low salt diet with diuretics, and paracentesis together with plasma volume expanders in diuretic-resistant patients. Progression of cirrhosis may result in hepatorenal syndrome, a state of functional renal failure that carries an ominous prognosis. Orthotopic liver transplantation has remained the only curative treatment for patients with advanced liver disease; other modalities such as transjugular intrahepatic portosystemic shunt or vasopressin analogues may serve as a bridge to transplantation. Another complication of decompensated cirrhosis is spontaneous bacterial peritonitis, the incidence of which can be reduced by primary or secondary antibiotic prophylaxis by using orally active antibiotics.

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Cirrhosis is typically associated with a hyperdynamic circulation consisting of low blood pressure, low systemic vascular resistance (SVR), and high cardiac output. We have recently reported that nonspecific inhibition of nitric oxide synthase (NOS) with nitro-L-arginine methyl ester reverses the hyperdynamic circulation in rats with advanced liver cirrhosis induced by carbon tetrachloride (CCl(4)). Although an important role for endothelial NOS (eNOS) is documented in cirrhosis, the role of neuronal NOS (nNOS) has not been investigated. The present study was carried out to specifically investigate the role of nNOS during liver cirrhosis. Specifically, physiological, biochemical, and molecular approaches were employed to evaluate the contribution of nNOS to the cirrhosis-related hyperdynamic circulation in CCl(4)-induced cirrhotic rats with ascites. Cirrhotic animals had a significant increase in water and sodium retention. In the aorta from cirrhotic animals, both nNOS protein expression and cGMP concentration were significantly elevated compared with control. Treatment of cirrhotic rats for 7 days with the specific nNOS inhibitor 7-nitroindazole (7-NI) normalized the low SVR and mean arterial pressure, elevated cardiac index, and reversed the positive sodium balance. Increased plasma arginine vasopressin concentrations in the cirrhotic animals were also repressed with 7-NI in association with diminished water retention. The circulatory changes were associated with a reduction in aortic nNOS expression and cGMP. However, 7-NI treatment did not restore renal function in cirrhotic rats (creatinine clearance: 0.76 +/- 0.03 ml. min(-1). 100 g body wt(-1) in cirrhotic rats vs. 0.79 +/- 0.05 ml. min(-1). 100 g body wt(-1) in cirrhotic rats+7-NI; P NS. ). Taken together, these results indicate that nNOS-derived NO contributes to the development of the hyperdynamic circulation and fluid retention in cirrhosis.

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A decade after a unifying hypothesis of sodium and water regulation had been proposed, evidence has accumulated in support of common (patho)physiologic mechanisms of sodium and water retention in congestive heart failure, cirrhosis and pregnancy. Either primary arterial vasodilatation (cirrhosis, pregnancy), or a decrease in cardiac output (low-output heart failure) impair arterial circulatory integrity ("effective arterial blood volume"), which unloads ventricular and arterial baroreceptors. Consequently, activation of the sympathetic nervous system, renin-angiotensin-aldosterone system, arginine-vasopressin, as well as other less well explored mechanisms occurs in an attempt to restore the arterial circulatory integrity. Consequences of this neurohumoral activation with arterial uderfilling include renal sodium and water retention and an increase in peripheral vascular resistance. In this review specific pathophysiologic mechanisms underlying the sodium and water retention in congestive heart failure, cirrhosis and pregnancy will be discussed.

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Renal ischemia-reperfusion (I/R) injury was investigated in inducible nitric oxide synthase (iNOS) knockout mice. After a 26-min bilateral renal pedicle clamp, serum creatinine concentrations (in mg/dl) in wild-type mice after a 24-h reperfusion were 0.25 +/- 0.03 in sham-operated controls and 2.3 +/- 0.38 in ischemic mice (P < 0. 01); after 48 h, concentrations (in mg/dl) were 0.25 +/- 0.03 in controls and 2.0 +/- 0.18 in ischemic mice (P < 0.01). iNOS knockout mice demonstrated an attenuation of serum creatinine concentration after renal I/R injury. Serum creatinine concentrations (mg/dl) after a 24-h reperfusion were 2.3 +/- 0.22 in wild-type ischemic and 1.21 +/- 0.25 in iNOS knockout ischemic mice (P < 0.05); after 48 h, concentrations were 2.0 +/- 0.18 in wild-type ischemic and 0.96 +/- 0.25 in iNOS knockout ischemic mice (P < 0.01). Histological scoring of acute tubular necrosis in iNOS knockout mice was decreased compared with that in wild-type controls (0.88 +/- 0.2 vs. 3.3 +/- 0. 3, P < 0.05). iNOS protein in the renal cortex of wild-type mice subjected to renal I/R injury was undetectable up to 48 h. However, a strong upregulation of heat shock protein 72 expression was observed in renal cortex of iNOS knockout mice under basal conditions. In conclusion, kidneys of iNOS knockout mice were protected against ischemic acute renal failure. This protective effect may be related to a compensatory upregulation of heat shock protein 72.

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1. An obligatory step in the biosynthesis of endothelin-1 (ET-1) is the conversion of its inactive precursor, big ET-1, into the mature form by the action of specific, phosphoramidon-sensitive, endothelin converting enzyme(s) (ECE). Disparate effects of big ET-1 and ET-1 on renal tubule function suggest that big ET-1 might directly influence renal tubule function. Therefore, the role of the enzymatic conversion of big ET-1 into ET-1 in eliciting the functional response (generation of 1,2-diacylglycerol) to big ET-1 was studied in the rat proximal tubules. 2. In renal cortical slices incubated with big ET-1, pretreatment with phosphoramidon (an ECE inhibitor) reduced tissue immunoreactive ET-1 to a level similar to that of cortical tissue not exposed to big ET-1. This confirms the presence and effectiveness of ECE inhibition by phosphoramidon. 3. In freshly isolated proximal tubule cells, big ET-1 stimulated the generation of 1,2-diacylglycerol (DAG) in a time- and dose-dependent manner. Neither phosphoramidon nor chymostatin, a chymase inhibitor, influenced the generation of DAG evoked by big ET-1. 4. Big ET-1-dependent synthesis of DAG was found in the brush-border membrane. It was unaffected by BQ123, an ETA receptor antagonist, but was blocked by bosentan, an ETA.B-nonselective endothelin receptor antagonist. 5. These results suggest that the proximal tubule is a site for the direct effect of big ET-1 in the rat kidney. The effect of big ET-1 is confined to the brush-border membrane of the proximal tubule, which may be the site of big ET-1 sensitive receptors.

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Phospholipid signalling mediated by endothelin (ET) receptor subtypes was studied in the rat proximal tubule. In freshly isolated proximal tubule cells, ET-1, ET-2 and sarafotoxin S6c (S6c) evoked an increase in 1,2-diacylglycerol (DAG), inositol 1,4,5-trisphosphate (InsP3) and phosphocholine (PCho), suggesting stimulation of both phosphatidyl-inositol 4,5-bisphosphate- and phosphatidyl-choline-specific phospholipase C (PLC), while ET-3 increased only DAG and PCho, presumably via phosphatidyl-choline-dependent PLC. Renal cortical slices were also stimulated by the above-mentioned agonists, followed by isolation of either brush border (BBM) or basolateral (BLM) membranes for which mass measurements of inositol lipids and DAG were performed. In BBM, DAG increased in response to ET-1, ET-2 and ET-3, and was followed by protein kinase C (PKC) translocation to the BBM, while in BLM, DAG formation and translocation of PKC were observed only in response to ET-3, suggesting spatial segregation of signalling systems between two membane domains of proximal tubule cells. Tyrphostine, pertussis toxin (PTX) or cholera toxin (CTX) did not influence ET-mediated signalling in either of the membranes, suggesting involvement of PTX- and CTX-insensitive G-protein-mediated stimulation of PLCbeta by ET receptors. ET-dependent stimulation of PLC in BBM and BLM was used as a tool to examine the presence of different ET receptor subtypes in these two cell membrane domains. BQ123, an inhibitor of ETA receptors, did not prevent ET-1-mediated signalling in BBM, but an ETA,B antagonist, bosentan, inhibited ET-3-mediated signalling in BBM. In addition, an ETB agonist, S6c, stimulated PLC in BBM. Neither BQ123 nor bosentan inhibited ET-3 signalling in BLM. Therefore, these data strongly suggest the presence of ETB receptors coupled to phosphatidyl-inositol 4,5-bisphosphate- and phosphatidyl-choline-dependent PLC in BBM and ETC receptors linked to phosphatidyl-choline-dependent PLC in BLM.

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Phorbol 12-myristate 13-acetate (PMA)-mediated signalling was investigated in relation to the ability of murine (CBA) bone marrow cells to form colonies in vitro. Treatment of marrow cells with PMA did not influence the 1,2-diacylglycerol or cyclic AMP concentrations, the intracellular Ca2+ concentration or phospholipase D activity. PMA increased particulate phospholipase A2 (PLA2) activity, lysophosphatidylcholine formation and arachidonic acid release from bone marrow cells; these effects were abolished when cells were pretreated with the putative PLA2 inhibitors heparin and mepacrine. While indomethacin and nordihydroguaiaretic acid inhibited either the cyclo-oxygenase or lipoxygenase pathway of arachidonic acid metabolism, as measured by their products prostaglandin E2 and leukotriene B4, they did not influence PMA-mediated PLA2 activation or translocation of protein kinase C (PKC) from the soluble to the particulate fraction. Treatment of cells with PMA increased the amounts of membrane-bound alpha, beta, delta, epsilon and zeta isoforms of PKC in bone marrow cells. Pretreatment of cells with PLA2 inhibitors reduced the amount of membrane-bound PKC-zeta in unstimulated cells and diminished PMA-induced translocation of PKC-zeta to membranes without affecting other PKC isoforms. This effect could be overcome by exogenous addition of arachidonic acid, suggesting that PKC-zeta may operate downstream of the activated PLA2. On the other hand, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, did not influence the amount of PKC-zeta associated with particulate fractions in control cells and could not abolish the PMA-mediated translocation of this isoform. Short-term exposure (45 min) of bone marrow cells to PMA, phorbol 12,13-dibutyrate or arachidonic acid increased the number of colonies formed over 7 days in a methylcellulose-based culture in vitro. The effects of PMA, but not those of arachidonic acid, could be prevented by putative PLA2 inhibitors. This suggests that PMA-mediated activation of conventional PKCs and novel PKCs leads to PLA2 activation which, by releasing arachidonic acid from phospholipids, activates PKC-zeta. This signalling pathway appears to be mitogenic for bone marrow cells.

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Using highly specific mass assays, concentrations of inositol lipids and 1,2-diacylglycerol (DAG) were determined in plasma membranes isolated from rat kidney cortex. Significantly higher concentrations of inositol lipids were determined in brush-border (BBM) than in basal-lateral (BLM) plasma membranes, although DAG concentrations were similar in both. After unilateral nephrectomy, a decrease in PtdIns(4,5)P2 and PtdIns4P, with a concomitant increase in DAG and translocation of protein kinase C (PKC), were observed in BBM but not in BLM isolated from the remaining kidney. On the other hand, stimulation of renal cortical slices with insulin-like growth factor II (IGF-II) or phenylephrine caused similar effects in BLM but not in BBM. Stimulation of phospholipase C activity with translocation of PKC only to BBM in one kidney was also induced by occlusion of blood flow through the contralateral kidney for 15 min. At 30 min after the occlusion was removed and reflow established, DAG concentration and the amount of PKC in BBM returned to control values. These results suggest that an early signal after unilateral nephrectomy is transmitted to cells through BBM and can be switched on and off by blood occlusion and reflow through the contralateral kidney, while hormonal signals caused by IGF-II and phenylephrine are transmitted to cells through BLM.

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