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In an attempt to examine the mechanisms of activation of (Na, K)-ATPase when epithelial transport is stimulated, the binding of ouabain to rectal gland tissue was measured before and after stimulation with dibutyryl cAMP and theophylline. Stimulation significantly altered the characteristics of ouabain binding to slices of Squalus acanthias rectal gland and to isolated rectal gland cells, accelerating the rate of binding and increasing the amount of ouabain bound at equilibrium when low concentrations of ouabain (10(-9) to 10(-7) M) were present in the medium. Scatchard plots of ouabain binding were nonlinear, suggesting at least two classes of binding sites, one of higher and one of lower affinity. Stimulation with cAMP and theophylline appeared to increase the affinity of the high-affinity site. Ouabain binding was increased by cAMP and theophylline even in the presence of furosemide (10(-4) M) or bumetanide (10(-5) M), and when Li+ was substituted for Na+, or NO3- for Cl- -maneuvers known to inhibit rectal gland secretion. The changes in ouabain binding induced by cAMP and theophylline do not appear, therefore, to be secondary to secretory activity but may reflect a change in the configuration, environment or location of existing enzyme so as to enhance its activity. Stimulation of ouabain binding cannot be demonstrated in whole homogenates of rectal gland, indicating that intact cells are necessary for the cyclic AMP-induced increase in ouabain binding to become manifest. [ABSTRACT FROM AUTHOR]

The endothelium, as the interface between blood and all tissues, plays a critical role in inflammation. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, highly abundant in plasma, that potently regulates endothelial responses through interaction with its receptors (S1PRs). Here, we studied the role of S1PR2 in the regulation of the proadhesion and proinflammatory phenotype of the endothelium. By using genetic approaches and a S1PR2-specific antagonist (JTE013), we found that S1PR2 plays a key role in the permeability and inflammatory responses of the vascular endothelium during endotoxemia. Experiments with bone marrow chimeras (S1pr2(+/+) → S1pr2(+/+), S1pr2(+/+) → S1pr2(-/-), and S1pr2(-/-) → S1pr2(+/+)) indicate the critical role of S1PR2 in the stromal compartment, in the regulation of vascular permeability and vascular inflammation. In vitro, JTE013 potently inhibited tumor necrosis factor α-induced endothelial inflammation. Finally, we provide detailed mechanisms on the downstream signaling of S1PR2 in vascular inflammation that include the activation of the stress-activated protein kinase pathway that, together with the Rho-kinase nuclear factor kappa B pathway (NF-kB), are required for S1PR2-mediated endothelial inflammatory responses. Taken together, our data indicate that S1PR2 is a key regulator of the proinflammatory phenotype of the endothelium and identify S1PR2 as a novel therapeutic target for vascular disorders.

During murine embryogenesis, the Ets factor Erg is highly expressed in endothelial cells of the developing vasculature and in articular chondrocytes of developing bone. We identified seven isoforms for the mouse Erg gene. Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes. The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells. Homozygous Erg(ΔEx3/ΔEx3) knockout mice are viable, fertile and do not display any overt phenotype. By contrast, homozygous Erg(ΔEx4/ΔEx4) knockout mice are embryonic lethal, which is associated with a marked reduction in endocardial-mesenchymal transformation (EnMT) during cardiac valve morphogenesis. We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

ERG is a member of the ETS transcription factor family that is highly enriched in endothelial cells (ECs). To further define the role of ERG in regulating EC function, we evaluated the effect of ERG knock-down on EC lumen formation in 3D collagen matrices. Blockade of ERG using siRNA completely interferes with EC lumen formation. Quantitative PCR (QPCR) was used to identify potential downstream gene targets of ERG. In particular, we identified RhoJ as the Rho GTPase family member that is closely related to Cdc42 as a target of ERG. Knockdown of ERG expression in ECs led to a 75% reduction in the expression of RhoJ. Chromatin immunoprecipitation and transactivation studies demonstrated that ERG could bind to functional sites in the proximal promoter of the RhoJ gene. Knock-down of RhoJ similarly resulted in a marked reduction in the ability of ECs to form lumens. Suppression of either ERG or RhoJ during EC lumen formation was associated with a marked increase in RhoA activation and a decrease in Rac1 and Cdc42 activation and their downstream effectors. Finally, in contrast to other Rho GTPases, RhoJ exhibits a highly EC-restricted expression pattern in several different tissues, including the brain, heart, lung, and liver.

We recently demonstrated that the 3-kb 5'-flanking region of the human ROBO4 gene directs endothelial cell-specific expression in vitro and in vivo. Moreover, a GA-binding protein (GABP)-binding motif at -119 was necessary for mediating promoter activity in vitro. The goal of the present study was to confirm the functional relevance of the -119 GABP-binding site in vivo. To that end, the Hprt locus of mice was targeted with a Robo4-LacZ transgenic cassette in which the GABP site was mutated. In other studies, the GABP mutation was introduced into the endogenous mouse Robo4 locus in which LacZ was knocked-in. Compared with their respective controls, the mutant promoters displayed a significant reduction in activity in embryoid bodies, embryos, and adult animals. Together, these data provide strong support for the role of the GABP-binding motif in mediating Robo4 expression in the intact endothelium.

Robo4, a member of the roundabout family, is expressed exclusively in endothelial cells and has been implicated in endothelial cell migration and angiogenesis. Here we report the cloning and characterization of the human Robo4 promoter. The 3-kb 5'-flanking region directs endothelial cell-specific expression in vitro. Deletion and mutation analyses revealed the functional importance of two 12-bp palindromic DNA sequences at -2528 and -2941, 2 SP1 consensus motifs at -42 and -153, and an ETS consensus motif at -119. In electrophoretic mobility shift assays using supershifting antibodies, the SP1 motifs bound SP1 protein, whereas the ETS site bound a heterodimeric member of the ETS family, GA binding protein (GABP). These DNA-protein interactions were confirmed by chromatin immunoprecipitation assays. Transfection of primary human endothelial cells with small interfering RNA against GABP and SP1 resulted in a significant (approximately 50%) reduction in endogenous Robo4 mRNA expression. The 3-kb Robo4 promoter was coupled to LacZ, and the resulting cassette was introduced into the Hprt locus of mice by homologous recombination. Reporter gene activity was observed in the vasculature of adult organs (particularly in microvessels), tumor xenografts, and embryos, where it colocalized with the endothelial cell-specific marker CD31. LacZ mRNA levels in adult tissues and tumors correlated with mRNA levels for endogenous Robo4, CD31, and vascular endothelial cadherin. Moreover, the pattern of reporter gene expression was similar to that observed in mice in which LacZ was knocked into the endogenous Robo4 locus. Together, these data suggest that 3-kb upstream promoter of human Robo4 contains information for cell type-specific expression in the intact endothelium.

Susceptibility to schizophrenia is considered familial, but the mechanism for transmission has not been found. Since widespread cognitive deficits have been found in patients with schizophrenia, several of these have been proposed as candidate familial endophenotypes that may or may not be predictive of who develops the illness. The current study examines these candidates in individuals from 32 families with at least 2 members having the diagnosis of chronic schizophrenia and normal comparison subjects using an extensive neuropsychological battery. Consistent with previous literature, family members with schizophrenia were significantly impaired on all measures compared with controls. Well relatives demonstrated significantly worse performance on a measure of verbal learning, delayed visual recall, perceptual-motor, and pure motor speed. Expressive and receptive language, but not other functions, were highly correlated within both concordant for schizophrenia and discordant sibling pairs, suggesting that they are familial vulnerability endophenotypes, but not predictive of whom becomes ill. On the other hand, some measures of perceptual-motor, pure motor speed, and frontal/executive functioning were significantly correlated in concordant, but not discordant pairs. These latter correlations suggest that some cognitive measures may be genetically related to the illness., ((c) 2004 Wiley-Liss, Inc.)

Vascular endothelial growth factor (VEGF) is a potent vascular endothelial cell-specific mitogen that modulates endothelial cell function. In the present study, we show that VEGF induces manganese-superoxide dismutase (MnSOD) mRNA and protein in human coronary artery endothelial cells (HCAEC) and pulmonary artery endothelial cells. VEGF-mediated induction of MnSOD mRNA was inhibited by pretreatment with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI), and 4-(2-aminoethyl)-benzenesulfonyl fluoride, but not with the nitric oxide synthase inhibitor L-NAME (N-monomethyl-L-arginine) or the xanthine oxidase inhibitor allopurinol. VEGF stimulation of MnSOD was also inhibited by adenoviral-mediated overexpression of catalase Cu, Zn-SOD and a dominant-negative form of the small GTPase component of NADPH oxidase Rac1 (Rac1N17). Treatment of HCAEC with VEGF resulted in a transient increase in ROS production at 20 min, as measured by 2,7-dichlorodihydrofluorescein oxidation. This effect was abrogated by expression of Rac1N17. Taken together, these findings suggest that VEGF induces MnSOD by an NADPH oxidase-dependent mechanism and that VEGF signaling in the endothelium is coupled to the redox state of the cell.

Early growth response-1 (Egr-1) is a transcription factor that couples short-term changes in the extracellular milieu to long-term changes in gene expression. Under in vitro conditions, the Egr-1 gene has been shown to respond to many extracellular signals. In most cases, these findings have not been extended to the in vivo setting. The goal of the present study was to explore the role of epidermal growth factor (EGF) in mediating Egr-1 expression in hepatocytes under both in vitro and in vivo conditions. In HepG2 cells, Egr-1 protein and mRNA were upregulated in the presence of EGF. In stable transfections of HepG2 cells, a 1,200-bp Egr-1 promoter contained information for EGF response via a protein kinase C-independent, mitogen-activated protein kinase-dependent signaling pathway. A promoter region containing the two most proximal serum response elements was sufficient to transduce the EGF signal. In transgenic mice that carry the Egr-1 promoter coupled to the LacZ reporter gene, systemic delivery of EGF by intraperitoneal injection resulted in an induction of the endogenous Egr-1 gene and the Egr-1-lacZ transgene in hepatocytes. Together, these results suggest that the 1,200-bp promoter contains information for EGF response in hepatocytes both in vitro and in intact animals.

Cells derived from the inner medullary collecting duct undergo in vitro branching tubulogenesis to both the c-met receptor ligand hepatocyte growth factor (HGF) as well as epidermal growth factor (EGF) receptor ligands. In contrast, many other cultured renal epithelial cells respond in this manner only to HGF, suggesting that these two receptors may use independent signaling pathways during morphogenesis. We have therefore compared the signaling pathways for mIMCD-3 cell morphogenesis in response to EGF and HGF. Inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway with the mitogen-activated protein kinase kinase (MKK1) inhibitor PD98059 (50 microm) markedly inhibits HGF-induced cell migration with only partial inhibition of EGF-induced cell motility. Similarly, HGF-dependent, but not EGF-dependent, branching morphogenesis was more greatly inhibited by the MKK1 inhibitor. Examination of EGF-stimulated cells demonstrated that extracellular-regulated kinase 5 (ERK5) was activated in response to EGF but not HGF, and that activation of ERK5 was only 60% inhibited by 50 microm PD98059. In contrast, the MKK inhibitor U0126 markedly inhibited both ERK1/2 and ERK5 activation and completely prevented HGF- and EGF-dependent migration and branching process formation. Expression of dominant negative ERK5 (dnBMK1) likewise inhibited EGF-dependent branching process formation, but did not affect HGF-dependent branching process formation. Our results indicate that activation of the ERK1/ERK2 signaling pathway is critical for HGF-induced cell motility/morphogenesis in mIMCD-3 cells, whereas ERK5 appears to be required for EGF-dependent morphogenesis.

Renal epithelial tubule formation in cultured cells occurs after the addition of tubulogenic growth factors such as the hepatocyte growth factor (HGF). HGF activates the tyrosine kinase receptor c-met, initiating a series of complex events that regulate cell morphology, cell-cell interactions, and cell-matrix interactions and eventually result in the formation of branching tubular structures. The discovery that disruption of the formin gene locus in mice causes agenesis of the kidneys secondary to failure of ureteric bud outgrowth and branching tubule formation suggested that this family of proteins may be critical to the development of renal epithelial tubules. In this study, we investigated whether formin is involved in the HGF/c-met signaling pathway of in vitro tubulogenesis in renal epithelial cells. mIMCD-3 cells were analyzed by reverse transcription-PCR and found to express formin IV mRNA. With the use of an antibody that recognizes the carboxy terminus of all known formin isoforms, it was observed a formin isoform of approximately 165 kD markedly increased in the detergent soluble cell lysate after 10 min of stimulation with HGF. An antibody that is specific for formin IV was then generated and confirmed that the formin isoform regulated by HGF was formin IV. Cell fractionation and confocal localization of formin IV revealed that formin IV is primarily found in a submembranous band that co-localizes with the actin cytoskeleton and in a perinuclear location in quiescent epithelial cells but undergoes a rapid relocalization after HGF stimulation with translocation into the cell cytosol and into the nucleus. Formin IV was found to be a phosphorylation substrate for activated extracellular signal-regulated kinase in vitro, and pretreatment of cells with the mitogen-activated protein kinase inhibitor U0126 prevented the translocation of formin IV and inhibited HGF-dependent phosphorylation of formin IV in intact cells. In conclusion, activation of the c-met receptor results in cellular relocalization of formin IV in a mitogen-activated protein kinase-dependent manner.

c-Ret, a protein tyrosine kinase receptor, and its ligand glial-derived neurotropic factor (GDNF) are critical for early regulation of ureteric bud development and nephrogenesis. To address whether c-ret directly initiates epithelial cell morphogenesis, the c-ret receptor was expressed in murine inner medullary collecting duct cells (mIMCD-3, a cell line of ureteric bud origin, which has no detectable endogenous c-ret expression). Stable expression of wild-type c-ret was found to yield a constitutively tyrosine-phosphorylated receptor, with no change after the addition of GDNF. Examination of mRNA from these cells demonstrated the message for endogenous GDNF, suggesting that c-ret was potentially being constitutively activated by an autocrine mechanism. When mIMCD-3 cells stably expressing the phosphorylated c-ret receptor were cultured in a type I collagen matrix, they exhibited little GDNF-independent or -dependent branching process formation at early time points compared with the known morphogen hepatocyte growth factor (HGF) (48 h; control, 0.33 +/- 0.33; GDNF, 1.0 +/- 0.58, P = nonsignificant; and HGF, 6.33 +/- 0.33 processes/20 cell clusters, P < 0.001), whereas extended culture (7 days) under serum-free conditions revealed a marked increase in cell survival and the spontaneous development of rudimentary branching process formation. Extended culture (7 days) of c-ret-expressing clones in type I collagen with the epithelial morphogens HGF and/or epidermal growth factor (EGF) resulted in the development of complex three-dimensional spiny cysts, whereas parental mIMCD-3 cells died under these conditions. We conclude that activated c-ret appears to mediate epithelial morphogenesis by prolonging cell survival and, in conjunction with activation of the morphogenic receptors c-met and the EGF receptor, initiates the events required for very early branching morphogenesis.

The isolated perfused rat kidney provides a model of selective hypoxia to the medullary thick ascending limb. To investigate the relationship between immediate early gene expression and the extent of hypoxic damage, we determined expression of the immediate early genes (IEG) c-fos and egr-1 in isolated perfused kidneys during standard perfusion and after various measures shown previously to be protective. mRNA levels of c-fos and egr-1 were markedly increased in kidneys after 90 minutes of standard perfusion with Krebs-Henseleit buffer containing albumin. Gene expression was most prominent in the outer medulla followed by papilla and cortex, a pattern reflected by the immunohistochemical demonstration of a prominent accumulation of both egr-1 and c-fos polypetides mainly in the medullary thick ascending limb (mTAL). Protective measures known to minimize morphological damage to the mTAL, including hyperoncotic perfusion, perfusion with glycine, or perfusion with a mixture of amino acids, decreased mRNA levels of c-fos and egr-1 in the outer medulla (by 50% and 35%, respectively) and the papilla (by 60 and 30%, respectively). Renal cortex showed only minor changes. In contrast, prevention of tubular transport by perfusion with 1 mM ouabain increased mRNA levels of c-fos and egr-1 in the outer medulla by 100% and 60%, respectively. Ouabain also dramatically increased mRNA levels of both IEGs in two lines of cultured renal epithelial cells. Changes in the level and distribution of the protein products of these IEGs were not detectable in perfused kidneys by immunohistochemistry. Hypoxic injury of the kidney stimulates IEG expression even in the absence of reperfusion. Protection against hypoxic injury in the mTAL correlates with suppression of IEG mRNA levels when protection is provided by amino acids or hyperoncotic perfusion, but not when provided by inhibition of Na,K-ATPase, which stimulates IEG expression. We conclude that diminished IEG expression is not a necessary concomitant of protection against hypoxic injury.

Expression of HSP-27, HSP-60 and HSP-70 was estimated in the cortex, outer medulla and inner medulla (papilla) of rats undergoing water diuresis or water restriction for two days. The mRNAs for HSP-27 and HSP-60 in renal papilla were two- to threefold greater in rats during water restriction than in those excreting a dilute urine, but levels of mRNA for HSP-70 were not reduced by water diuresis and Western analysis for HSP-70 protein showed no difference between water-loaded and water-restricted animals.

The possible protective effect of heat-shock proteins (HSPs) on ischemic injury to renal cells was assessed in two different experimental models: ischemia-reflow in intact rats and medullary hypoxic injury as seen in the isolated perfused rat kidney. Heat shock was induced by raising the core temperature of rats to 42 degrees C for 15 minutes. Following this, Northern blots showed enhanced gene expression of HSP70, HSP60 and ubiquitin at one hour and reaching a maximum by six hours after heat shock in all regions of the kidney, but most prominently in medulla and papilla. The HSP70 protein in the kidney, estimated by immunohistochemical means, was detectable 24 hours following heat shock and further increased at 48 hours following heat shock. In the first set of experiments, the animals underwent uninephrectomy followed by cross clamping of the remaining renal artery for 40 minutes prior to reflow. Serum creatinine and urea nitrogen rose to 3.15 +/- 0.98 and 126.4 +/- 62.5 mg/dl at 24 hours. No significant differences were observed at 24, 48 and 72 hours after reflow between these values in control rats and rats pretreated with heat shock 48 hours earlier. Severe morphological damage to proximal tubules of the renal cortex was observed to the same extent in both groups. In a second set of experiments, the right kidney was removed either 24 or 48 hours after heat shock and perfused in isolation for 90 minutes. Functional and morphological parameters were compared with those of isolated perfused kidneys obtained from animals that had not been subjected to heat shock.(ABSTRACT TRUNCATED AT 250 WORDS)

The straight portion (S3) of the proximal tubule lies in close proximity to the thick ascending limbs (TALs) at the cortico-medullary junction. Since a delicate balance exists between oxygen demand and the limited oxygen supply in this region, we hypothesized that reduction of thick limb metabolic activity might augment oxygen availability to S3 segments, which depend heavily upon aerobic metabolism, and prevent hypoxic damage. The degree of functional deterioration and morphological damage to S3 was assessed in isolated rat kidneys perfused with an erythrocyte-free medium. Bumetanide (10(-5) M) and furosemide (10(-4) M) reduced S3 fragmentation from 9.8 +/- 3.9% of tubules in controls to 0 and 1.4 +/- 0.9%, respectively (P < 0.0005). Tubular glucose reabsorption was better preserved in kidneys exposed to loop diuretics than in control kidneys (P < 0.01), and urinary alkaline phosphatase (P < 0.05) and the total amount of LDH released into the perfusate and urine (P < 0.01) were lower in the treatment groups. Morphological damage to S3 was closely correlated with medullary TAL necrosis (r = 0.66, P < 0.001), urinary alkaline phosphatase excretion (r = 0.89, P < 0.001) and glycosuria (r = 0.83, P < 0.001). We conclude that under hypoxic conditions TALs and S3 segments may compete with each other for a limited oxygen supply. Reduction of active transport in the mTAL might augment oxygen availability to S3 segments and improve their survival.

Infusion of radiocontrast agents in vivo results in renal artery constriction and subsequent renal hypoperfusion. To examine the role of endothelin and of prostaglandins in radiocontrast-mediated renal vasoconstriction, rats were treated with an endothelin receptor antagonist, CP170687, and with indomethacin. The dose of CP170687 utilized was sufficient to reverse endothelin1-mediated constriction of isolated aortic rings and of renal blood flow in intact rats. In normal rats there was a transient drop in renal blood flow to 80% of baseline following sodium iothalamate injection, an effect which was not prevented by CP170687. In rats first given indomethacin, the drop in renal blood flow was more pronounced (to 63% of baseline) and was sustained. In this instance, CP170687 fully reversed the sustained decrease of renal perfusion. CP170687 also diminished the rise in systemic blood pressure seen following iothalamate injection. In the absence of indomethacin, iothalamate increased urinary prostaglandin E2 to a maximum of sevenfold above baseline values. In summary, injection of radiocontrast results in an immediate decrease in renal blood flow that is counteracted by an increase in renal prostaglandin formation. When prostaglandin synthesis is inhibited, prolonged endothelin-mediated renal vasoconstriction is observed that is reversed by an endothelin receptor antagonist.

The two major hypotheses for the pathogenesis of amphotericin nephrotoxicity are direct interaction with epithelial cell membranes and vasoconstriction. Studies indicating the special vulnerability of the medullary ray and medulla to hypoxia led to a reexamination of amphotericin nephrotoxicity. Twenty-four rats were divided into four groups: amphotericin injection (5 mg/kg daily for 3 wk), amphotericin plus salt depletion, vehicle, and salt depletion and vehicle. The amphotericin group had polyuria (P < 0.01) but normal serum creatinine. In contrast, amphotericin plus salt depletion rats exhibited renal failure (creatinine of 1.49 +/- 0.05 versus amphotericin alone 0.98 +/- 0.01; P < 0.01). Semiquantitative histologic analysis of cortical and medullary injury correlated with functional impairment. Cortical changes in the amphotericin group were largely restricted to the medullary ray, where focal rupture and calcification of thick ascending limbs were noted. The S2/S3 tubules in the medullary rays showed focally diminished cell complexity with histiocytic/lymphocytic infiltration. However, calcification was also seen in the area of the macula densa. Morphometry revealed that the thick ascending limbs in the medulla were hypertrophied (1,420 +/- 63 versus 1,195 +/- 48 microns 2 for vehicle; P < 0.05). In contrast, in the amphotericin and salt depletion group, the changes in the medullary ray extended to the labyrinth and the thick ascending limbs in the inner stripe showed atrophic changes (772 +/- 23 microns 2; P < 0.01 versus vehicle). Thus, changes as a result of amphotericin toxicity take place both in areas known to be most vulnerable to hypoxia (medullary ray and medulla), and in areas rich in oxygen (adjacent to glomerulus). Salt depletion potentiates the cortical changes and converts medullary hypertrophy to atrophy. These findings support a dual pathogenesis for amphotericin nephropathy (direct toxicity and vasoconstriction).

We evaluated the effect of glycine infusions on the early renal uptake of cisplatin, measured one hour after cisplatin was injected, as well as five days following cisplatin administration. Glycine (1.25 mmol per 100 g body wt) markedly attenuated the early uptake of platinum by the kidney, an effect not observed with control infusions of saline or of L-alanine. The kidney content of platinum at five days, on the other hand, was similar in glycine-treated animals and saline controls. Early inhibition of renal uptake of platinum may be responsible for glycine's protective action in experimental cisplatin nephrotoxicity.

Rationale and Objectives: Certain radiocontrast agents, including iothalamate, iohexol, and ioxaglate, release the renal vasoconstrictor peptide endothelin from vascular endothelium in a way that might contribute to radiocontrast nephropathy. The effects of the nonionic, low osmolar agent, ioversol, on endothelin release and renal function are investigated., Methods: Effects of ioversol were compared with equi-iodine doses of iothalamate when applied to cultured bovine aortic endothelial cells or injected into normal rats and rats preconditioned by uninephrectomy, salt depletion, and indomethacin (USIC) to develop radiocontrast nephropathy., Results: In comparison with iothalamate, ioversol had a greatly reduced propensity to stimulate the release of endothelin, from cultured cells and when injected into anesthetized rats. Ioversol produced less renal vasoconstriction than did iothalamate, in control and in USIC rats, and the development of radiocontrast nephropathy, assessed by creatinine clearance and morphologic damage to the renal medulla, was largely avoided., Conclusions: These results strengthen the hypothesis that endothelin release induced by radiocontrast agents is correlated with their renal toxicity and therefore, may play a role in radiocontrast nephropathy.

Addition of glycine to the recirculating perfusate of isolated perfused rat kidneys protects against hypoxic injury to the medullary thick ascending limb and slows functional deterioration in the course of perfusion. This effect is dependent on dose; the earliest significant protection is seen at 0.25 mM, and the protective effects increase as glycine concentration is increased to 2 mM, the highest level tested. Two specific agonists of the strychnine-insensitive (NMDA) glycine receptor in neural membranes, 1-aminocyclopropane carboxylic acid (ACC) and d-serine, also exerted a cytoprotective effect at a concentration of 2 mM. On the other hand, 1-serine and taurine, ineffective agonists of the NMDA-glycine receptor but effective agonists of the strychnine-sensitive glycine receptor, had no protective effect in this system. Two antagonists to glycine at its binding site on the N-methyl-D-Aspartate (NMDA) receptor, 7-chlorokynurenic acid (2 mM) and indole-2-carboxylic acid (12.5 mM), did not reverse the cytoprotective action of 0.25 mM glycine. The data are consistent with a ligand-acceptor type of interaction to account for cytoprotection. The configuration of the glycine acceptor may resemble, but is not identical with, that of certain glycine receptors in the nervous system.

Glycine preserves tubular cell integrity under hypoxic and toxic conditions in vitro. It also ameliorates cisplatin nephrotoxicity in vivo. We studied the effect of glycine on tubular necrosis from ischemia reflow and on inner stripe injury in an animal model of radiocontrast nephropathy. In all experiments, glycine (75 mg/100 g/h) increased tubular damage in the inner stripe. In the model of radiocontrast nephropathy, the percentage of medullary thick ascending limb (mTAL) necrosis at 24 hours increased from 22% +/- 6% to 41% +/- 9% or 55% +/- 7% with glycine infusion of 75 or 135 minutes, respectively (mean +/- SE, P less than 0.05, analysis of variance [ANOVA]). Renal function was not significantly affected. In rat kidneys subjected to ischemia reflow, mTAL injury following glycine increased from 1% +/- 0% to 12% +/- 6% (P less than 0.05) and from 8% +/- 5% to 49% +/- 8% (P less than 0.01) 24 hours after 30 minutes and 45 minutes ischemia, respectively. Tubular injury in the inner stripe was maximal in the deep interbundle zone, typical of hypoxic, rather than reperfusion, injury. Prior uninephrectomy increased inner stripe damage, but protected the proximal tubules. Both uninephrectomy and glycine infusion were found to contribute to mTAL necrosis. The infusion of glycine for 1 hour in intact rats increased renal blood flow by 44% and tripled urine volume (P less than 0.01). A parallel increase in glomerular filtration rate GFR; by 22% over 90 minutes) fell short of statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)