Acute renal failure following percutaneous transhepatic cholangiography. A retrospective study.

We analyzed retrospectively the incidence of potential nephrotoxic effects of radiographic contrast material associated with percutaneous transhepatic cholangiography and percutaneous biliary drainage. Of 72 consecutive patients who underwent these procedures, three developed acute renal insufficiency (defined as a rise in the serum creatinine concentration of greater than 2 mg/dL [greater than 180 mumol/L]) following administration of contrast medium. In two patients, abdominal roentgenograms taken after the procedure showed persistently opaque kidneys, indicating that contrast material had gained access to the circulation. Percutaneous transhepatic cholangiography and percutaneous biliary drainage may be associated with nephrotoxic effects of radiographic contrast material, and patients with recognized risk factors may benefit from prophylactic hydration regimens as recommended for other procedures.

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.

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.

Molecular approaches to renal physiology and therapeutics.

The recent development of methods to transfer, mutate, or ablate genes in vivo has provided renal investigators and physicians with powerful tools to explore normal renal physiology, the pathophysiological basis of renal disease, and potential therapeutic interventions. The use of transgenic and knockout mice to produce gain-of-function and loss-of-function mutations, and to create animal models of human hereditary renal diseases, permits unprecedented versatility and power of experimental design. Conditional and inducible gene targeting methods to control the temporal and spatial expression of transgenes offer considerable promise in studying the impact of normal and disease genes in the kidney. In vivo gene transfer of encoding DNAs, antisense DNA and RNA, and cis-element decoys allows manipulation of specific genes in somatic cells. Liposome-mediated, virally mediated, and ex vivo transduced renal cells represent novel approaches to facilitate in vivo gene transfer to the kidney.

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.

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.

Macrophages and chronic renal allograft nephropathy.

In a previous study we demonstrated that macrophage infiltrates stained for thromboxane A synthase (TxAS) correlated inversely with renal function six months after biopsy. We propose that macrophage based inflammation is a cofactor leading to chronic allograft nephropathy. For this study we compared four indices of renal allograft nephropathy with renal survival. The Banff Score of Inflammatory Changes (BSI) is an index of acute inflammation. The Banff Chronic Index (BCI) and Chronic Allograft Damage Index (CADI) are indexes of chronic disease. The Macrophage Index (MI) is the same as the BSI applied only to macrophages. These indices were determined on renal allograft biopsies obtained because of delayed graft function within the first week of transplantation, and for increasing plasma creatinine levels after stable function. All four indices predicted renal survival in the post-biopsy interval. MI predicted renal survival for the entire transplant period. In addition, the presence of TxAS transcripts in the renal allografts was determined using a reverse transcription-polymerase chain reaction-based assay. This confirms previous observations of TxAS in the grafts. This study supports the hypothesis that macrophage derived inflammation is a cofactor for chronic allograft nephropathy.

Acute renal failure produced by combining cyclosporine and brief renal ischemia in the Munich Wistar rat.

To evaluate the combined effects of a brief ischemic insult and cyclosporine, four groups of male Munich Wistar rats were given: a) parenteral cyclosporine (60 mg/kg i.p.) for 4 days following 20 minutes of bilateral renal ischemia, b) the castor oil cyclosporine vehicle in a comparable volume and the same ischemic insult, c) saline in the same volume and ischemia, or d) saline and sham surgery. The cyclosporine animals ate and drank poorly, and therefore the other groups were pair-fed and watered with them. The cyclosporine-ischemia group developed significant renal failure. The other groups exhibited only a mild rise in blood urea nitrogen. Tubular vacuolization was a prominent feature in the cyclosporine and vehicle groups, but not in the saline groups. Vacuolization was correlated with severity of renal impairment. Lipid stains showed that many of the vacuoles contained lipid. Eosinophilic cytoplasmic inclusions were seen only in the cyclosporine or vehicle- (castor oil) treated animals. These findings emphasize the probable functional importance of tubular lesions in cyclosporine-induced acute renal failure, and suggest that the castor oil vehicle of parenteral cyclosporine may have renal effects of its own.

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.

Nitric oxide in renal health and disease.

Nitric oxide (NO) is a labile radical gas that is widely acclaimed as one of the most important molecules in biology. Through covalent modifications of target proteins and redox reactions with oxygen and superoxide radical and transition metal prosthetic groups, NO plays a critical role in many vital biological processes, including the control of vascular tone, neurotransmission, ventilation, hormone secretion, inflammation, and immunity. Moreover, NO has been shown to influence a host of fundamental cellular functions, such as RNA synthesis, mitochondrial respiration, glycolysis, and iron metabolism. NO is formed from L-arginine by NO synthases (NOSs), a family of related enzymes encoded by separate unlinked genes. The different NOS isozymes exhibit disparate tissue and intrarenal distributions and are governed by unique regulatory mechanisms. In the kidney, NO participates in several vital processes, including the regulation of glomerular and medullary hemodynamics, the tubuloglomerular feedback response, renin release, and the extracellular fluid volume. While NO serves beneficial roles as a messenger and host defense molecule, excessive NO production can be cytotoxic, the result of NO's reaction with reactive oxygen and nitrogen species, leading to peroxynitrite anion formation, protein tyrosine nitration, and hydroxyl radical production. Indeed, NO may contribute to the evolution of several commonly encountered renal diseases, including immune-mediated glomerulonephritis, postischemic renal failure, radiocontrast nephropathy, obstructive nephropathy, and acute and chronic renal allograft rejection. Moreover, impaired NO production has been implicated in the pathogenesis of volume-dependent hypertension. This duality of NO's beneficial and detrimental effects has created extraordinary interest in this molecule and the need for a detailed understanding of NO biosynthesis.

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.

Renal H,K-ATPase: structure, function and regulation.

The H,K-ATPase comprises a family of isoenzymes with unique biochemical, pharmacological, and regulatory properties. This review explores recent advances in discovery of the molecular biology, biochemistry, and function of these ion pumps, with particular emphasis on the implications for renal potassium and proton handling. Structure-function correlations governing the ion transport mechanisms, inhibitor binding sites, oligomerization, and transcriptional control of the H,K-ATPase isoforms are examined. Functional studies of H,K-ATPase in renal tubules and cultured epithelial cells are analyzed and related to data concerning the expression and distribution of the H,K-ATPase gene products in the kidney. Functional and molecular biological evidence for adaptive changes in renal H,K-ATPase expression during the evolution of potassium and acid-base disturbances are discussed. Current investigative challenges and avenues for future research of these enzymes are presented.

CCAAT/enhancer binding protein-beta trans-activates murine nitric oxide synthase 2 gene in an MTAL cell line.

Nitric oxide production by nitric oxide synthase 2 (NOS2) has been implicated in epithelial cell injury from oxidative and immunologic stress. The NOS2 gene is transcriptionally activated by lipopolysaccharide (LPS) and cytokines in medullary thick ascending limb of Henle's loop (MTAL) cells and other cell types. The 5'-flanking region of the NOS2 gene contains a consensus element for CCAAT/enhancer binding proteins (C/EBP) at -150 to -142 that we hypothesized contributes to NOS2 trans-activation in the mouse MTAL cell line ST-1. Gel shift assays demonstrated LPS + interferon-gamma (IFN-gamma) induction of C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the complexes were comprised of C/EBPbeta, but not C/EBPalpha, C/EBPdelta, or C/EBPepsilon. NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box exhibited lower activities in response to LPS + IFN-gamma compared with wild-type NOS2 promoter constructs. Overexpression of a C/EBP-specific dominant-negative mutant limited LPS + IFN-gamma activation of the NOS2 promoter. In trans-activation assays, overexpression of C/EBPbeta stimulated basal NOS2 promoter activity. Thus C/EBPbeta appears to be an important trans-activator of the NOS2 gene in the MTAL.

Nitric oxide inhibits transcription of the Na+-K+-ATPase alpha1-subunit gene in an MTAL cell line.

Nitric oxide (NO) has been implicated as an autocrine modulator of active sodium transport. To determine whether tonic exposure to NO influences active sodium transport in epithelial cells, we established transfected medullary thick ascending limb of Henle (MTAL) cell lines that overexpressed NO synthase-2 (NOS2) and analyzed the effects of deficient or continuous NO production [with or without NG-nitro-L-arginine methyl ester (L-NAME) in the culture medium, respectively] on Na+-K+-ATPase function and expression. The NOS2-transfected cells exhibited high-level NOS2 expression and NO generation, which did not affect cell viability or cloning efficiency. NOS2-transfected cells were grown in the presence of vehicle, NG-nitro-D-arginine methyl ester (D-NAME), or L-NAME for 16 h, after which 86Rb+ uptake assays, Northern analysis, or nuclear run-on transcription assays were performed. The NOS2-transfected cells allowed to produce NO continuously (vehicle or D-NAME) exhibited lower rates of ouabain-sensitive 86Rb+ uptake ( approximately 65%), lower levels of Na+-K+-ATPase alpha1-subunit mRNA ( approximately 60%), and reduced rates of de novo Na+-K+-ATPase alpha1-subunit transcription compared with L-NAME-treated cells. These results have uncovered a novel effect of NO to inhibit transcription of the Na+-K+-ATPase alpha1-subunit gene.

Expression and cellular localization of mRNA encoding the "gastric" isoform of H(+)-K(+)-ATPase alpha-subunit in rat kidney.

The distribution of transcripts encoding the gastric H(+)-K(+)-adenosinetriphosphatase (ATPase) alpha-subunit in the normal rat kidney was studied by reverse transcription-polymerase chain reaction (RT-PCR), combined with DNA sequence analysis and renal microdissection, and by nonradioactive in situ hybridization of fixed kidney sections using highly specific molecular probes. RT-PCR products corresponding to the gastric H(+)-K(+)-ATPase alpha-subunit were detected in the cortex, outer and inner medulla, and in isolated cortical (CCD) and inner medullary collecting ducts (IMCD). With digoxigenin-labeled cRNAs derived from the 5' and 3' ends of the gastric H(+)-K(+)-ATPase alpha-subunit cDNA, specific hybridization signal was detected prominently in all the cells of the connecting segment and CCD, the intercalated cells of the outer medullary collecting duct, the IMCD, and the renal pelvic epithelium lining the secondary pouches. Weak labeling was noted in the S3 segment of the proximal tubule, the distal convoluted tubule, and the cortical thick ascending limb of Henle. Hybridization with the sense probes produced no cellular labeling. These data provide the first direct demonstration for the expression and cellular distribution of mRNA encoding the gastric H(+)-K(+)-ATPase alpha-subunit in the normal, potassium-replete kidney, and they provide essential tools for the molecular analysis of renal acid base and potassium transport under physiological and pathophysiological conditions.

Biosynthesis and homeostatic roles of nitric oxide in the normal kidney.

Nitric oxide (NO) is an important molecular mediator of numerous physiological processes in virtually every organ. In the kidney, NO plays prominent roles in the homeostatic regulation of glomerular, vascular, and tubular function. Differential expression and regulation of the NO synthase (NOS) gene family contribute to this diversity of action. This review explores recent advances in the molecular and cell biology of the NOS isoforms and relates these findings to functions of NO in the control of normal renal hemodynamics, the glomerular microcirculation, and renal salt excretion. Newly recognized molecular diversity of the NOS gene products, factors governing NOS isozyme gene expression and catalytic activity, and the intrarenal distribution of the NOS isoforms are examined. Physiological data regarding the complex roles of NO in the control of renal hemodynamics and the glomerular microcirculation are analyzed, and the effects of chronic NOS inhibition on glomerular function and structure are presented. The contributions of NO to renal salt excretion as well as functional and molecular biological evidence for adaptive changes in NOS isoform expression during variations in dietary salt balance are discussed. Current investigative challenges and goals for future research of renal NO biology are presented.

A novel N-terminal splice variant of the rat H+-K+-ATPase alpha2 subunit. Cloning, functional expression, and renal adaptive response to chronic hypokalemia.

The H+-K+-ATPase of renal collecting duct mediates K+ conservation during chronic hypokalemia. K+ deprivation promotes H+-K+-ATPase alpha2 (HKalpha2) gene expression in the medullary collecting duct, the principal site of active K+ reabsorption, suggesting that this isozyme contributes to renal K+ reclamation. We report here that alternative transcriptional initiation and mRNA splicing give rise to distinct N-terminal variants of the HKalpha2 subunit. Sequence analysis and in vitro translation revealed that HKalpha2a corresponds to the known HKalpha2 cDNA (Crowson, M. S., and Shull, G. E. (1992) J. Biol. Chem. 267, 13740-13748), whereas HKalpha2b represents a novel variant truncated by 108 amino acids at its N terminus. HKalpha2b mRNA contains a complex 5'-untranslated region with eight upstream open reading frames, features implicated in translational regulation of other genes. Heterologous expression of HKalpha2b with and without the gastric H+-K+-ATPase beta subunit in HEK 293 cells indicated that this variant encodes a K+ uptake mechanism that is relatively Sch 28080-resistant, partially sensitive to ouabain, and appears to require coexpression with the gastric H+-K+-ATPase beta subunit for optimal functional activity. Northern analysis demonstrated that both subtypes (HKalpha2b > HKalpha2a) are expressed abundantly in distal colon and modestly in proximal colon and kidney. Moreover, the abundance of the two mRNAs increases coordinately among the renal zones, but not in colon, with chronic K+ deprivation. These results demonstrate the potential for complex control of HKalpha2 gene expression by transcriptional and posttranscriptional mechanisms not recognized in other members of the Na+-K+-ATPase/H+-K+-ATPase family.

Ultrastructure of the thick ascending limb of Henle in the rat kidney.

The thick ascending limb of Henle (TAL) in the rat until recently has been considered a morphologically homogeneous structure despite physiologic and biochemical evidence to the contrary. The present study was designed to examine the ultrastructural characteristics of the TAL in the inner cortex and the outer and inner stripes of the outer medulla using qualitative and quantitative transmission electron microscopy. Kidneys of male Sprague-Dawley rats were preserved by in vivo perfusion with glutaraldehyde for light and electron microscopy. The peritubular diameter and cell height were determined by direct measurements on tubule cross sections. Morphometric analyses were performed on montages of tubule cross sections. The peritubular diameter of the TAL was similar in the three regions under investigation, but the TAL cells were taller in the inner stripe than in the inner cortex and outer stripe. Morphometry revealed significant differences between the three regions with respect to the mean tubular cross-sectional area (AT), the surface density (SV), and the surface area per mm of tubule (ST) of apical and basolateral plasma membranes, and the volume density (VV) of mitochondria. The major morphologic division appeared to be between the inner stripe segment and the remainder of the TAL. These findings document the presence of significant morphologic heterogeneity of the rat TAL.

Coordinated regulation of intracellular K+ in the proximal tubule: Ba2+ blockade down-regulates the Na+,K+-ATPase and up-regulates two K+ permeability pathways.

To avoid large changes in cell K+ content and volume during variations in Na+,K+-ATPase activity, Na+-transporting epithelia must adjust the rate of K+ exit through passive permeability pathways. Recent studies have shown that a variety of passive K+ transport mechanisms may coexist within a cell and may be functionally linked to the activity of the Na+,K+-ATPase. In this study, we have identified three distinct pathways for passive K+ transport that act in concert with the Na+,K+-ATPase to maintain intracellular K+ homeostasis in the proximal tubule. Under control conditions, the total K+ leak of the tubules consisted of discrete Ba2+-sensitive (approximately 65%), quinine-sensitive (approximately 20%), and furosemide-sensitive (approximately 10%) pathways. Following inhibition of the principal K+ leak pathway with Ba2+, the tubules adaptively restored cell K+ content to normal levels. This recovery of cell K+ content was inhibited, in an additive manner, by quinine and furosemide. Following adaptation to Ba2+, the tubules exhibited a 30% reduction in Na+-K+ pump rate coupled with an increase in K+ leak by means of the quinine-sensitive (approximately 70%) and furosemide-sensitive (approximately 280%) pathways. Thus, the proximal tubule maintains intracellular K+ homeostasis by the coordinated modulation of multiple K+ transport pathways. Furthermore, these results suggest that, like Ba2+, other inhibitors of K+ conductance will cause compensatory changes in both the Na+-K+ pump and alternative pathways for passive K+ transport.