Differential induction of PPAR-gamma by luminal glutamine and iNOS by luminal arginine in the rodent postischemic small bowel.

Using a rodent model of gut ischemia-reperfusion (I/R), we have previously shown that the induction of inducible nitric oxide synthase (iNOS) is harmful, whereas the induction of heme oxygenase 1 (HO-1) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is protective. In the present study, we hypothesized that the luminal nutrients arginine and glutamine differentially modulate these molecular events in the postischemic gut. Jejunal sacs were created in rats at laparotomy, filled with either 60 mM glutamine, arginine, or magnesium sulfate (osmotic control) followed by 60 min of superior mesenteric artery occlusion and 6 h of reperfusion, and compared with shams. The jejunum was harvested for histology or myeloperoxidase (MPO) activity (inflammation). Heat shock proteins and iNOS were quantitated by Western blot analysis and PPAR-gamma by DNA binding activity. In some experiments, rats were pretreated with the PPAR-gamma inhibitor G9662 or with the iNOS inhibitor N-[3(aminomethyl)benzyl]acetamidine (1400W). iNOS was significantly increased by arginine but not by glutamine following gut I/R and was associated with increased MPO activity and mucosal injury. On the other hand, PPAR-gamma was significantly increased by glutamine but decreased by arginine, whereas heat shock proteins were similarly increased in all experimental groups. The PPAR-gamma inhibitor G9662 abrogated the protective effects of glutamine, whereas the iNOS inhibitor 1400W attenuated the injurious effects of arginine. We concluded that luminal arginine and glutamine differentially modulate the molecular events that regulate injurious I/R-mediated gut inflammation and injury. The induction of PPAR-gamma by luminal glutamine is a novel protective mechanism, whereas luminal arginine appears harmful to the postischemic gut due to enhanced expression of iNOS.

Protein-protein interactions involving inducible nitric oxide synthase.

AIM: Nitric oxide (NO) is a signaling and effector molecule that contributes to multiple physiological and pathophysiological processes in the kidney, vasculature, and other tissues. High output NO generation by inducible NO synthase (iNOS) participates in host defense against pathogens and contributes to tissue injury during inflammatory states. Because of its potent reactivity and diffusibility, NO generation by iNOS is subject to multiple levels of regulation, including transcriptional, translational, and post-translational controls, including protein-protein interactions. This review examines the experimental basis for these protein-protein interactions and their known and potential importance for kidney and vascular physiology. METHODS: Analysis of the biomedical literature in the area. RESULTS: iNOS interacts with the inhibitory molecules Kalirin and NOS-associated protein 1.10 kd (NAP110), which inhibit iNOS homodimerization, as well as activator proteins, the Rac-GTPases. Interactions with caveolin-1 control the intracellular locale and degradation of iNOS in tumor cells. In polarized epithelial cells, associations of iNOS with the scaffolding protein EBP50 position iNOS in the apical membrane near key ion transport proteins that also interact with EPB50. In addition, protein-protein interactions of proteins governing iNOS transcription function to specify activation or suppression of iNOS induction by cytokines. CONCLUSION: Interactions of iNOS with a diverse group of heterologous proteins provides a selective mechanism to control the activity, spatial distribution, and proximity of iNOS to intended targets, while potentially limiting autotoxicity to the iNOS-expressing cell.

Specific association of nitric oxide synthase-2 with Rac isoforms in activated murine macrophages.

Nitric oxide synthase-2 (NOS2) is responsible for high-output nitric oxide production important in renal inflammation and injury. Using a yeast two-hybrid assay, we identified Rac2, a Rho GTPase member, as a NOS2-interacting protein. NOS2 and Rac2 proteins coimmunoprecipitated from activated RAW 264.7 macrophages. The two proteins colocalized in an intracellular compartment of these cells. Glutathione-S-transferase (GST) pull-down assays revealed that both Rac1 and Rac2 associated with GST-NOS2 and that the NOS2 oxygenase domain was necessary and sufficient for the interaction. [(35)S]methionine-labeled NOS2 interacted directly with GST-Rac2 in the absence of GTP, calmodulin, or NOS2 substrates or cofactors. Stable overexpression of Rac2 in RAW 264.7 cells augmented LPS-induced nitrite generation (~60%) and NOS2 activity (~45%) without measurably affecting NOS2 protein abundance and led to a redistribution of NOS2 to a high-speed Triton X-100-insoluble fraction. We conclude that Rac1 and Rac2 physically interact with NOS2 in activated macrophages and that the interaction with Rac2 correlates with a posttranslational stimulation of NOS2 activity and likely its spatial redistribution within the cell.

Molecular biology of natriuretic peptides and nitric oxide synthases.

Natriuretic peptides and nitric oxide play important roles in cardiovascular and renal physiology and disease. The natriuretic peptides - atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide - comprise a family of proteins that participate in the integrated control of intravascular volume and arterial blood pressure. The natriuretic peptides differentially bind distinct classes of receptors that signal through different mechanisms. Membrane-bound, guanylyl cyclase-coupled natriuretic peptide receptors (A- and B-types) mediate natriuretic peptide effects through the production of 3',5'-cyclic guanosine monophosphate (cGMP). C-Type natriuretic peptide receptors, which lack the guanylyl cyclase domain, alter target cell function through G(i) protein-coupled inhibition of membrane adenylyl cyclase activity, and also serve to clear circulating natriuretic peptides. The expression of the natriuretic peptides and their receptors are subject to complex controls. Similar structural and regulatory diversity exists for the nitric oxide synthases. The three nitric oxide synthase genes are regulated by a variety of mechanisms ranging from alternative splicing and alternative promoter usage to complex post-translational controls. This review highlights the molecular diversity of the natriuretic peptides and nitric oxide synthases and explores recent insights into their regulation.

Post-injury multiple organ failure: the role of the gut.

Despite intensive investigation, the pathogenesis of post-injury multiple organ failure (MOF) remains elusive. Laboratory and clinical research strongly suggests that the gastrointestinal tract (i.e., the gut) plays a pivotal pathogenic role. Since its inception in 1988, the Trauma Research Center (TRC) at the University of Texas-Houston Medical School (UTHMS) has focused its efforts on elucidating the role of the gut in post-injury MOF. On the basis of our observations and those of others, we believe that 1) shock with resulting gut hypoperfusion is an important inciting event, 2) the reperfused gut is a source of proinflammatory mediators that can amplify the early systemic inflammatory response syndrome (SIRS) and thus contribute to early MOF, 3) early gut hypoperfusion causes an ileus in both the stomach and small bowel that sets the stage for progressive gut dysfunction so that the proximal gut becomes a reservoir for pathogens and toxins that contribute to late sepsis-associated MOF, and 4) late infections cause further worsening of this gut dysfunction. Thus, the gut can be both an instigator and a victim of MOF. The purpose of this article is to provide the rationale behind these beliefs and to provide a brief overview of the ongoing research projects in the TRC at UTHMS.

Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells.

Two H(+)-K(+)-ATPase isoforms are present in kidney: the gastric, highly sensitive to Sch-28080, and the colonic, partially sensitive to ouabain. Upregulation of Sch-28080-sensitive H(+)-K(+)-ATPase, or "gastric" H(+)-K(+)-ATPase, has been demonstrated in hypokalemic rat inner medullary collecting duct cells (IMCDs). Nevertheless, only colonic H(+)-K(+)-ATPase mRNA and protein abundance increase in this condition. This study was designed to determine whether Sch-28080 inhibits transporters other than the gastric H(+)-K(+)-ATPase. In the presence of bumetanide, Sch-28080 (200 microM) and ouabain (2 mM) inhibited (86)Rb(+) uptake (>90%). That (86)Rb(+) uptake was almost completely abolished by Sch-28080 indicates an effect of this agent on the Na(+)-K(+)-ATPase. ATPase assays in membranes, or lysed cells, demonstrated sensitivity to ouabain but not Sch-28080. Thus the inhibitory effect of Sch-28080 was dependent on cell integrity. (86)Rb(+)-uptake studies without bumetanide demonstrated that ouabain inhibited activity by only 50%. Addition of Sch-28080 (200 microM) blocked all residual activity. Intracellular ATP declined after Sch-28080 (200 microM) but recovered after removal of this agent. In conclusion, high concentrations of Sch-28080 inhibit K(+)-ATPase activity in mouse IMCD-3 (mIMCD-3) cells as a result of ATP depletion.

alpha-MSH inhibits induction of C/EBPbeta-DNA binding activity and NOS2 gene transcription in macrophages.

BACKGROUND: alpha-Melanocyte-stimulating hormone (alpha-MSH) is an endogenous tridecapeptide that exerts anti-inflammatory actions and abrogates postischemic renal injury in rodents. alpha-MSH inhibits lipopolysaccharide (LPS)-induced gene expression of several cytokines, chemokines, and nitric oxide synthase-2 (NOS2), but the molecular mechanisms underlying these effects have not been clearly defined. To test the hypothesis that alpha-MSH inhibits the expression of inducible trans-activating factors involved in NOS2 regulation, we used RAW 264.7 macrophage cells to examine the effects of alpha-MSH on the activation of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer binding protein-beta (C/EBPbeta), trans-acting factors known to be involved in LPS + interferon (IFN)-gamma induction of the NOS2 gene. METHODS: Gel shift assays were performed to identify NF-kappaB and C/EBP DNA binding activities in LPS + IFN-gamma-treated RAW 264.7 cells in the presence and absence of alpha-MSH. NOS2 promoter assays were conducted to identify the effects of alpha-MSH on LPS + IFN-gamma-mediated induction of NOS2 transcription. RESULTS: Gel shift assays demonstrated LPS + IFN-gamma induction of NF-kappaB and C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the C/EBP complexes were comprised of C/EBPbeta, but not C/EBPalpha, C/EBPdelta, or C/EBPepsilon. alpha-MSH (100 nmol/L) inhibited the LPS + IFN-gamma-mediated induction of nuclear DNA binding activity of C/EBPbeta, but not that of NF-kappaB (in contrast to reports in other cell types), as well as the activity of a murine NOS2 promoter-luciferase construct. In contrast, alpha-MSH (100 nmol/L) had no effect on the induction of NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box. CONCLUSIONS: These data indicate that alpha-MSH inhibits the induction of C/EBPbeta DNA binding activity and that this effect is a major mechanism by which alpha-MSH inhibits the transcription of the NOS2 gene. The inability of alpha-MSH to inhibit LPS + IFN-gamma induction of NF-kappaB in murine macrophage cells, which contrasts with inhibitory effects of the neuropeptide in other cell types, suggests that cell-type-specific mechanisms are involved.

Protein-protein interactions controlling nitric oxide synthases.

Nitric oxide (NO) biosynthesis is tightly regulated by a variety of mechanisms ranging from transcriptional to post-translational controls. Calmodulin has long been known to be an allosteric modulator of the three major NO synthases (NOS). Recent studies indicate that other proteins directly associate with NOS isoforms and regulate their activity or spatial distribution in the cell. Several proteins residing in or recruited to plasmalemmal caveolae of endothelial cells serve as allosteric regulators of endothelial NOS (eNOS). Caveolins, the resident scaffolding proteins of caveolae, and calmodulin undergo reciprocal Ca2+-dependent association and dissociation with eNOS in the caveolar membrane that inhibits (caveolins) and activates (calmodulin) eNOS activity. Other caveolar proteins appear to contribute to the eNOS-membrane complex, including the bradykinin B2 receptor, the angiotensin AT1 receptor, the CAT1 arginine transporter, and Hsp90. Direct interactions of a variety of proteins bearing PDZ domains with the PDZ domain of neuronal NOS (nNOS) have been shown to influence the subcellular distribution and/or activity of the enzyme in brain and muscle. One of these proteins, PSD-93, co-localizes with a subpopulation of nNOS in the macula densa. Although considerable emphasis has been placed on transcription as the principal step of regulation for inducible NOS (iNOS), our laboratory has recently defined a regulatory interaction of iNOS with Rho family GTPases. While the role of protein-eNOS interactions in the control of vascular tone has been increasingly clarified, the interactions and regulatory importance of protein association with nNOS and iNOS in the vasculature and kidney remains to be explored.

How will gene therapy apply to the kidney in the 21st century?

Nephrology is entering the age of genomics-based drug discovery and development. Once only a theoretical objective, gene therapy is now being tested in various diseases. New and substantially improved vector systems and related technologies are undergoing development, many have shown promise in animal studies, and some are now being used in clinical trials. Recent advances in the molecular basis for renal diseases, organ transplant rejection, and hypertension have led to preclinical tests of gene therapeutic approaches. The most impressive of these strategies will likely soon be studied in the clinic. This review details recent advances in gene therapy technology and highlights potential novel applications of gene therapy in the treatment of renal diseases and hypertension. While the manufacture and widespread use of gene therapy products as conventional pharmaceuticals for renal diseases and hypertension may seem to be a goal for the remote future, much of the needed genetic information, technology, and intellectual resources are rapidly becoming available.

Localization and regulation of nitric oxide synthase isoforms in the kidney.

Nitric oxide synthases (NOS), which comprise a multi-gene family, play important roles in a variety of physiological and pathophysiological processes in the kidney. The three major NOS isoforms are expressed in a cell type--specific manner and are subject to complex and distinct control mechanisms. Although knowledge about the intrarenal distribution and regulation of the major NOS isoforms has been expanding, recent advances in the molecular details of the structure, function, and regulation of the NOS genes and the enzymes they encode have added considerable complexity to the effort. Molecular biological studies have identified alternative splice variants of NOS1 and NOS2 that appear to be subject to unique regulation and may encode functionally distinct proteins. The renal distribution of these new variants has yet to be explored in detail. In addition, newly discovered transcriptional and posttranscriptional control mechanisms, including alternative promoter usage, protein-protein interactions, and phosphorylation events, for the three major NOS isoforms await characterization in renal cells. This review highlights the current state of knowledge about the distribution and regulation of the NOS isoforms in the kidney, and identifies new opportunities for further renal investigation.