Renal nitric oxide production during the early phase of experimental diabetes mellitus.

BACKGROUND: Diabetic nephropathy (DN) is characterized by hyperfiltration and hypertrophy in experimental models of diabetes mellitus (DM). Several studies have demonstrated that the pathophysiologic and morphologic changes in DN are mediated by either an increase or decrease in renal nitric oxide (NO) production and/or activity. The goal of the present study was to determine the effects that the early diabetic state has on NO production in the kidney of rats with streptozotocin-induced DM. METHODS: Experimental DM was induced in rats with streptozotocin. Urinary NO production was measured, and levels and activity of the different NOS isoforms were determined by a combination of techniques, including immunoblotting, immunohistochemistry, diaphorase staining, and reverse transcription-polymerase chain reaction. RESULTS: During the first week of DM, urinary NO metabolites (uNO2 + NO3) were reduced as compared with controls, which were unrelated to changes in serum levels of NO. Total NO synthase (NOS) activity was reduced in the renal cortex beginning at 30 hours after the induction of DM. NADPH diaphorase staining of renal cortical slices showed reduced NOS activity in the macula densa in diabetic animals. By immunohistochemical staining with antibodies to the different isoforms of NOS, it was found that protein levels of the neuroneal NOS (nNOS) isoform was diminished in the macula densa. No changes were found in the levels of endothelial NOS (eNOS) activity and protein in the renal cortex in the early diabetic state. CONCLUSIONS: This study provides strong evidence that renal production of NO is reduced in early DM and that this reduction is associated with decreased levels of nNOS activity and protein in the macula densa.

Time course of lipopolysaccharide-induced nitric oxide synthase mRNA expression in rat glomeruli.

The decrease in glomerular filtration rate that is characteristic of sepsis has been shown to result from the local glomerular inhibition of endothelial nitric oxide synthase (NOS) by nitric oxide (NO) generated from the inducible isoform of NOS (iNOS). iNOS activation depends on de novo synthesis of both RNA and protein. Therefore it is assumed that several hours are required for its full activation. Yet the renal hemodynamic response in sepsis has been documented as early as 60 minutes after lipopolysaccharide (LPS) administration. Experiments were designed to determine the time course of LPS-induced glomerular iNOS mRNA expression and activity in rats. Rats were treated with LPS (2 mg/kg body weight IP). Kidneys were removed after 1,2, 4, 6, and 16 hours. Glomeruli were isolated and incubated. Nitric oxide generation was measured with a Griess assay, and iNOS mRNA was studied by reverse transcriptase-polymerase chain reaction. Similar time course experiments were repeated in glomeruli isolated from normal rats and exposed to LPS in vitro. A significant increase in iNOS mRNA expression was evident as early as 60 minutes after both in vivo and in vitro administration of LPS. The quantity of iNOS mRNA reached its peak between 2 to 4 hours after administration and declined to baseline levels after 16 hours. Immunohistochemical studies were remarkable for a significant increase in the staining for iNOS in glomeruli 2 hours after the in vivo administration of LPS. Plasma nitric oxide concentration after the in vivo administration of LPS increased from a baseline level of 11.25 +/- 0.8 micromol/L to a peak level of 62.9 +/- 3.8 micromol/L (P < .05 vs baseline) at 4 hours and then decreased to 17.5 +/-1.9 micromol/L at 16 hours. Similar results were obtained when the glomerular generation of nitric oxide after in vivo administration of LPS was measured (2.6 +/- 0.8 pmol/h/microg tissue, 17.2 +/- 2.1 pmol/h/microg tissue (P < .05 vs baseline), and 0.4 +/- 0.65 pmol/h/microg tissue, respectively). These results provide evidence of the rapid activation of glomerular iNOS after in vivo and ex vivo administration of LPS and thus support the role of nitric oxide in the early renal hemodynamic response to LPS.

Stable and temperature-sensitive transformation of baby rat kidney cells by SV40 suppresses expression of membrane dipeptidase.

Membrane dipeptidase (MDP) is a zinc metalloenzyme located in the lungs and on the brush border membranes of the kidney and intestine. The gene for MDP (also termed DPEP1) is both frequently lost in Wilm's tumours and is located on human chromosome 16q24.3, a region of the genome known to contain a tumour suppressor gene(s). We now report on the regulation of MDP gene expression in normal and transformed cells. MDP enzyme activity and mRNA was detected in primary baby rat kidney (BRK) cells maintained in culture for up to 4 weeks. In contrast all stable transformed cell lines that were tested, derived either by transformation with the DNA tumour viruses SV40 or adenovirus, or in human tumour cell lines, contained very low levels of or no detectable MDP mRNA or enzyme activity. In BRK cells transformed by the temperature-sensitive tsA58 mutant of SV40 T antigen, MDP activity was not detectable, in cell lines grown at the permissive temperature (33 degrees C) but after 5-14 days of incubation at the non-permissive temperature (39.5 degrees C), MDP protein and enzyme activity could be readily detected. Taken together, these results indicate that MDP expression is characteristic of differentiated kidney epithelial cells and is down-regulated in proliferating, transformed cells.

Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase.

Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin-Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

Site-directed mutagenesis of conserved cysteine residues in porcine membrane dipeptidase. Cys 361 alone is involved in disulfide-linked dimerization.

Membrane dipeptidase (EC 3.4.13.19) is a glycosylphosphatidylinositol-anchored glycoprotein of the renal brush border which exists as a disulfide-linked homodimer. Porcine membrane dipeptidase has a subunit M(r) of 47 kDa, and the mature protein contains seven cysteine residues per subunit, six of which are conserved in the human enzyme. Chemical modification established that cysteine residues are not involved in enzyme activity. In order to determine which of the cysteine residues are involved in the interchain disulfide bond, we have used a site-directed mutagenesis approach. Each of the conserved cysteine residues was replaced by glycine or alanine. The single mutants (C71G, C93A, C154G, C226A, C258G, and C361G) were expressed in COS-1 cells and their enzymatic activity and oligomeric structure determined. Only the C361G mutant migrated as a polypeptide of 47 kDa when subjected to denaturing polyacrylamide gel electrophoresis under nonreducing conditions. Thus, cysteine 361 is the only residue involved in disulfide linkage between the subunits. This places the disulfide bond close to the site of GPI anchor addition (Ser 368 in the porcine enzyme) and to the membrane surface. Titration of the human and porcine proteins with 2-nitro-5-thiosulfabenzoate indicates that membrane dipeptidase additionally possesses two intrachain disulfide bonds. On native polyacrylamide gel electrophoresis, the C361G mutant migrates in a manner identical to that of the wild type, indicating that the protein remains associated as a noncovalent homodimer. The expressed C361G mutant, unlike the wild type, is released from COS-1 cell membranes by trypsin and by an endogenous serine protease.

The renal membrane dipeptidase (dehydropeptidase I) inhibitor, cilastatin, inhibits the bacterial metallo-beta-lactamase enzyme CphA.

The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a metallo-beta-lactamase which is highly active against carbapenem antibiotics such as imipenem. Here we show that the cphA gene product shares inhibitory similarities with a mammalian zinc peptidase, membrane dipeptidase (MDP; dehydropeptidase I). Both enzymes are able to hydrolyze imipenem and are inhibited by cilastatin. The active site similarities of these enzymes are not reflected in any significant primary sequence similarity.

Directed mutagenesis of pig renal membrane dipeptidase. His219 is critical but the DHXXH motif is not essential for zinc binding or catalytic activity.

Pig renal membrane dipeptidase cDNA has been expressed in COS-1 cells. Directed mutagenesis was used to investigate the roles of some conserved histidyl and aspartyl residues. Mutation of His219 to Arg, Lys or Leu results in complete abolition of enzyme activity, although the mutants are expressed at the cell-surface. Residues in a proposed motif (DHXDH; residues 269-273) for zinc binding have been mutated individually. Each retained activity comparable to that of the wild-type, excluding an essential role for components of this motif. The zinc-binding ligands in membrane dipeptidase therefore represent a novel domain for a metallopeptidase with His219 being one candidate.

Expression of a cloned gamma-aminobutyric acid transporter in mammalian cells.

The cDNA clone GAT-1, which encodes a Na(+)- and Cl(-)-coupled GABA transporter from rat brain, has been expressed in mammalian cells using three different systems: (1) transient expression upon transfection of mouse Ltk- cells with a eukaryotic expression vector containing GAT-1; (2) stable expression in L-cells transfected with the same vector; (3) transfection of HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase. Similar results both qualitatively and quantitatively were obtained with all systems. The GABA transporter expressed in HeLa and L-cells retains all the properties described previously for GABA transport into synaptosomes and synaptic plasma membrane vesicles. It was fully inhibited by cis-3-aminocyclohexanecarboxylic acid (ACHC) and not by beta-alanine. The KM for GABA transport and the IC50 for ACHC inhibition were similar to the presynaptic transporter. Accumulated [3H]GABA was released from transfected cells by dissipating the transmembrane Na+ gradient with nigericin or by exchange with unlabeled external GABA. Accumulation was stimulated by both Na+ and Cl- in the external medium. However, in the absence of external Cl-, a small amount of GABA transport remained which was dependent on GAT-1 transfection. Functional expression of the GABA transporter was abolished by tunicamycin. An antitransporter antibody specifically immunoprecipitates a polypeptide with an apparent molecular mass of about 70 kDa from GAT-1-transfected cells. When cells were grown in the presence of tunicamycin, only a faint band of apparent mass of about 60 kDa was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of a rat brain GABA transporter.

A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

Structural and functional studies on the sodium- and chloride-coupled gamma-aminobutyric acid transporter: deglycosylation and limited proteolysis.

The sodium- and chloride-coupled gamma-aminobutyric transporter, an 80-kDa glycoprotein, has been subjected to deglycosylation and limited proteolysis. The treatment of the 80-kDa band with endoglycosidase F results in its disappearance and reveals the presence of a polypeptide with an apparent molecular mass of about 60 kDa, which is devoid of 125I-labeled wheat germ agglutinin binding activity but is nevertheless recognized by the antibodies against the 80-kDa band. Upon limited proteolysis with papain or Pronase, the 80-kDa band was degraded to one with an apparent molecular mass of about 60 kDa. This polypeptide still contains the 125I-labeled wheat germ agglutinin binding activity but is not recognized by the antibody. The effect of proteolysis on function was examined. The transporter was purified by use of all steps except that for the lectin chromatography [Radian, R., Bendahan, A., & Kanner, B.I. (1986) J. Biol. Chem. 261, 15437-15441]. After papain treatment and lectin chromatography, gamma-aminobutyric transport activity was eluted with N-acetylglucosamine. The characteristics of transport were the same as those of the pure transporter, but the preparation contained instead of the 80-kDa polypeptide two fragments of about 66 and 60 kDa. The ability of the anti-80-kDa antibody to recognize these fragments was relatively low. The observations indicate that the transporter contains exposed domains which are not important for function.

gamma-Aminobutyric acid transport in reconstituted preparations from rat brain: coupled sodium and chloride fluxes.

Transport of gamma-aminobutyric acid (GABA) is electrogenic and completely depends on the presence of both sodium and chloride ions. These ions appear to be cotransported with gamma-aminobutyric acid through its transporter [reviewed in Kanner, B. I. (1983) Biochim. Biophys. Acta 726, 293-316]. Using proteoliposomes into which a partially purified gamma-aminobutyric acid transporter preparation was reconstituted, we have been able--for the first time--to provide direct evidence for sodium- and chloride-coupled gamma-aminobutyric acid transport. This has been done by measuring the fluxes of 22Na+, 36Cl-, and [3H]GABA. These fluxes have the following characteristics: There are components of the net fluxes of sodium and chloride that are gamma-aminobutyric acid dependent. The sodium flux is chloride dependent; i.e., when Cl- is replaced by inorganic phosphate or by SO4(2-), gamma-aminobutyric acid dependent sodium fluxes are abolished. The chloride flux is sodium dependent; i.e., when Na+ is replaced by Tris+ or by Li+, gamma-aminobutyric acid dependent chloride fluxes are abolished. Thus, the gamma-aminobutyric acid dependent sodium and chloride fluxes appear to be catalyzed by the transporter. Using these fluxes we have attempted to determine the stoichiometry of the process. We measured the initial rate of sodium-dependent gamma-aminobutyric acid fluxes and that of gamma-aminobutyric acid dependent sodium fluxes. This yields the stoichiometry between sodium and gamma-aminobutyric acid (2.58 +/- 0.99). Similarly, we measured the stoichiometry between chloride and gamma-aminobutyric acid, which is found to be 1.27 +/- 0.12.(ABSTRACT TRUNCATED AT 250 WORDS)