The repetitive activation of extracellular signal-regulated kinase is required for renal regeneration in rat

In this study, we investigated the activation of p42 extracellular signal-regulated kinase (ERK2) during renal regeneration after HgCl2-induced acute renal failure (ARF) in rat. ERK2 activation was observed at 5 and 29 hr after HgCl2 injection, respectively. The tyrosine phosphorylation of hepatocyte growth factor receptor (c-MET) occurred between 2.5 and 5 hr after the treatment. On the other hand, the phosphorylation of epidermal growth factor receptor (EGFR) was transiently observed at 29 hr after the injection. The peak of ornithine decarboxylase activity as a marker of G1 phase was at 10 hr, and subsequently the labeling index of proliferating cell nuclear antigen as a marker of S phase increased at 53 hr. These results indicate that the repetitive activation of ERK2 related to the phosphorylation of c-MET and EGFR is required for the renal regeneration in HgCl2-induced ARF of rat.     

Thrombin increases clusterin mRNA in glomerular epithelial and mesangial cells

Clusterin, a multifunctional protein with complement blocking activity, and fibrin, a product of thrombin's enzymatic activity, are present in the kidney during acute and chronic renal failure. The role of thrombin in regulating clusterin mRNA in the kidney is not known. The effect of thrombin on clusterin mRNA expression was examined in rat glomerular mesangial and glomerular epithelial cells, and cultured human renal proximal tubular epithelial cells by northern blot. Thrombin (10(-8) M) increased clusterin mRNA levels two- to fourfold in glomerular mesangial, glomerular epithelial, and proximal tubule epithelial cells. This was a specific effect of thrombin receptor activation because peptides corresponding to the tethered ligand of the thrombin receptor were also able to increase clusterin mRNA levels. Epidermal growth factor, insulin-like growth factor-1, and transforming growth factor-beta 1 had little or no effect on clusterin mRNA levels. The protein kinase C inhibitor RO-32-0432 (1 microM) inhibited the thrombin-induced increase in clusterin mRNA, suggesting that thrombin receptor activation may regulate renal clusterin mRNA levels through protein kinase C.     

Influence of chronic undernutrition on oxygen consumption of rats during exercise

The current study was undertaken to examine the effects of dithiothreitol (DDT), a sulfhydryl-reducing agent and heavy metal chelator, on the course of heavy metal-induced acute renal failure in the rat. Groups of rats in metabolic cages received uranyl nitrate (UN) alone, UN plus DTT, mercuric chloride (HgCl2) alone, and HgCl2 plus DTT. UN injected alone produced azotemia, decreased creatinine clearance, and rising fractional sodium excretion over the 48 hr of study. These effects of UN on renal function were not observed when DTT was administered 30 min after UN injection. Qualitatively similar results were obtained with HgCl2-induced acute renal failure. Groups of rats were killed at 6 hr after UN plus DTT, HgCl2 alone, or HgCl2 plus DTT; and determinations of plasma renin activity (PRA) and renin activities of the superficial and deep juxtaglomerular apparatus (JGA) were performed. PRA's and JGA renins were increased in animals receiving either UN or HgCl2 alone, but not in the rats receiving both DTT and UN or HgCl2. The effect of DTT on distribution of 203Hg was also examined. Treatment with DTT did not alter the renal accumulation of 203Hg, suggesting that this agent does not act by limiting renal exposure to the heavy metals. Thus, DTT ameliorates the course of heavy metal-induced ARF, and this effect is associated with prevention of heavy metal-induced alterations in sodium excretion and renin-angiotensin system activity.     

Cloning, functional analysis and cell localization of a kidney proximal tubule water transporter homologous to CHIP28

The localization and transporting properties of a kidney protein homologous to human erythrocyte protein CHIP28 was evaluated. The cDNA encoding rat kidney protein CHIP28k was isolated from a rat renal cortex cDNA library. A 2.8-kb cDNA was identified which contained an 807 bp open reading frame encoding a 28.8 kD protein with 94% amino acid identity to CHIP28. in vitro translation of CHIP28k cDNA in rabbit reticulocyte lysate generated a 28-kD protein; addition of ER-derived microsomes gave a 32-kD transmembrane glycoprotein. Translation of truncated RNA demonstrated glycosylation of residue Asn42 which is predicted to lie between the first and second transmembrane domains. Expression of in vitro transcribed mRNA encoding CHIP28k in Xenopus oocytes increased oocyte osmotic water permeability (Pf) from (4 +/- 1) x 10(-4) to (33 +/- 4) x 10(-4) cm/s at 10 degrees C; the increase in oocyte Pf was weakly temperature dependent and inhibited by HgCl2. Two-electrode voltage clamp measurements indicated that CHIP28k was not permeable to ions. Oocyte Pf also increased with expression of total mRNA from kidney cortex and papilla; the increase in Pf with mRNA from cortex, but not kidney papilla, was blocked by coinjection with excess antisense CHIP28k cRNA. In situ hybridization of a 150 base cRNA antisense probe to tissue sections from rat kidney showed selective CHIP28k localization to epithelial cells in proximal tubule and thin descending limb of Henle. Pf in purified apical membrane vesicles from rat and human proximal tubule, and in proteoliposomes reconstituted with purified protein, was very high and inhibited by HgCl2; stripping of apical vesicles with N-lauroylsarcosine enriched a 28-kD protein by 25-fold and yielded a vesicle population with high water, but low urea and proton permeabilities. CHIP28k identity was confirmed by NH2-terminus sequence analysis. These results indicate that CHIP28k is a major and highly selective water transporting protein in the kidney proximal tubule and thin descending limb of Henle, but not collecting duct.     

Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity

Postischemic injury in recipients of 3-7-d-old renal allografts was classified into sustained (n = 19) or recovering (n = 20) acute renal failure (ARF) according to the prevailing inulin clearance. Recipients of optimally functioning, long-standing allografts and living donors undergoing nephrectomy served as functional (n = 14) and structural controls (n = 10), respectively. Marked elevation above control of fractional clearance of dextrans of graded size was consistent with transtubular backleak of 57% of filtrate (inulin) in sustained ARF. No backleak was detected in recovering ARF. To explore a structural basis for backleak, allograft biopsies were taken intraoperatively, 1 h after reperfusion in all recipients, and again on day 7 after transplant in a subset (n = 10). Electron microscopy revealed disruption of both apical and basolateral membranes of proximal tubule cells in both sustained and recovering ARF, but cell exfoliation and tubule basement membrane denudation were negligible. Histochemical analysis of membrane-associated adhesion complexes confirmed an abnormality of proximal but not distal tubule cells, marked in sustained ARF but not in recovering ARF. Staining for the zonula occludens complex (ZO-1) and adherens complex (alpha, beta, and gamma catenins) revealed diminished intensity and redistribution of each cytoskeletal protein from the apico-lateral membrane boundary. We conclude that impaired integrity of tight junctions and cell-cell adhesion in the proximal tubule provides a paracellular pathway through which filtrate leaks back in sustained allograft ARF.     

Fasting suppresses pulsatile luteinizing hormone (LH) secretion and enhances orderliness of LH release in young but not older men

BACKGROUND: Studies identifying genes that are differentially expressed following induction of acute ischemic injury have been useful in delineating the pathophysiology of acute renal failure. METHODS: A differential cDNA library screening technique was used to identify genes that are differentially expressed in rat kidney following induction of acute ischemic renal injury. RESULTS: Levels of mRNA with a high homology to that coding for Siva, a human proapoptotic protein, were increased approximately 4.5-fold in kidneys obtained from rats within 12 hours following ischemia, compared to kidneys from sham-operated rats. A partial cDNA sequence for the rat protein (rat Siva) was determined that overlaps 92% of the human open reading frame. The cDNA sequence predicts a protein 177 amino acids in length with 76% homology to human Siva. Levels of rat Siva in kidneys were elevated at one, five and seven days post-ischemia were not different from those in kidneys from sham-operated controls. In situ hybridization demonstrated that rat Siva mRNA was expressed in cells lining damaged sections in the S3 segment of the proximal tubule at 12 hours and one day post-ischemia. At five and seven days, Siva mRNA was located in epithelial cells of regenerating tubules including in papillary proliferations. TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells colocalized with cells containing Siva mRNA. CD27, the receptor for Siva was localized by immunohistochemistry to sloughed cells in the lumens of damaged S3 segments at 12 hours post-ischemia and to cells within papillary proliferations at five days post-injury. CONCLUSIONS: Siva that is produced within the kidney could be a mediator of apoptosis post-ischemia via an interaction with CD27.     

Acute renal failure syndromes in human immunodeficiency virus infection

Two major groups of renal complications in human immunodeficiency virus (HIV) disease are a spectrum of disorders that result in potentially reversible acute renal failure, primarily acute tubular necrosis (ATN), and HIV-associated nephropathy (HIVAN), predominantly focal and segmental glomerulosclerosis (FSGS), leading to end-stage renal disease (ESRD). Fluid-electrolyte and acid-base derangements frequently encountered in acquired immune deficiency syndrome (AIDS) are major risk factors for the development of acute renal failure (ARF). HIVAN is an unusual form of poorly responsive glomerular disease characterized by nephrotic syndrome, FSGS, and a rapid fulminant progression to ESRD. ARF syndromes encountered in HIV patients are diverse in nature; many are similar to that in non-HIV subjects, whereas some are more common and unique. In general, HIV disease patients with ARF are younger and much sicker. Although ATN secondary to ischemic and toxic injuries is the commonest ARF syndrome, urinary obstruction is a rare cause of severe renal failure. In many AIDS patients afflicted with complicated infections and multi-organ failure, ATN is a terminal event, whereas in others treated aggressively, ARF is associated with good prognosis. In our large comparative study of severe ARF, recovery of renal function and mortality were determined by patient's general hemodynamic status, and not by the presence or absence of HIV infection. The prognosis of hemolytic uremic and thrombotic thrombocytopenic purpura syndromes often observed in HIV patients is much worse than in non-HIV patients. The syndrome of crystalluria-induced ARF is common, and protease inhibitor induced disease is confined to HIV patients.     

Hypertrophy of rabbit proximal tubule cells is associated with overexpression of TGF beta

The expression of transforming growth factor beta (TGF beta) in proximal tubule cells from rabbit kidney cortex after uninephrectomy (UNX) was investigated. Cell protein and [3H]leucine incorporation in these cells were significantly increased, while cell number was decreased, at two weeks following UNX. At this time period after UNX, we found that proximal tubule cells showed a dramatic increase of cytoplasmic immunostaining with a pan-specific anti-TGF beta antibody. This was accompanied by a 3-fold increase in TGF beta 1 mRNA expression in these cells. Furthermore, proximal tubule cells from two-week uninephrectomized rabbits secrete about 2-fold higher TGF beta bioactivity to the cell conditioned medium compared to cells from sham-operated animals. Addition of anti-TGF beta 1, beta 2, beta 3 neutralizing antibody increased the growth of the former cells, and it abolished cell hypertrophy. These results indicate that hypertrophy of proximal tubule cells during compensatory renal growth is associated with overexpression of TGF beta.     

A non-aversive approach to reducing hospital absconding in a head-injured adolescent boy

Human envenomation caused by bee or wasp stings has been reported to cause acute renal failure (ARF), usually due to acute tubular necrosis (ATN), as a frequent complication. The pathogenetic mechanisms of ATN occurring in these accidents are still unclear. In the present study, female Wistar rats weighing 150-200 g were injected intravenously with Africanized bee venom at a dose of 0.4 microl/100 g body weight and used in functional and light microscopy studies. The animals were divided into two groups: the early group was studied 3-8 h after inoculation, and the late group was studied 24-30 h thereafter. The animals showed ARF characterized by reduction of glomerular filtration rate with increasing levels of plasma creatinine. They also showed increased fractional sodium and potassium excretions, suggesting changes in the proximal portion of the nephron. The water transport through collecting tubules was reduced, with consequent diuresis, indicating functional changes in the distal portion of the nephron. These functional changes were more marked in the early group, with recovery tending to occur after 24 h. Albuminuria was also observed in this group. Light microscopy showed ATN mainly in cortex and outer medulla, with isolated necrosis in cells or small groups of cells and cast formation in the distal and collecting tubules. After 24 h frequent mitotic figures were found in the tubular epithelium. The observed ARF was due to ATN which in turn was probably caused by multiple effects, mainly hemodynamic changes secondary to cardiotoxicity and systemic vasodilation caused by the venom, myohemoglobinuria, and the direct action of the venom on tubular cells.     

Expression of fetal kidney growth factors in a kidney tumor line: role of FGF2 in kidney development

To identify growth factors which may play a role in kidney organogenesis, we have analyzed culture supernatants from the pediatric kidney tumor cell line G401. G401 cells were found to secrete fibroblast growth factor 2 (FGF2), a potent mitogen for mesenchymal cells, OP-1/BMP7, an epithelial cell growth inhibitor, and midkine (MK). Northern blotting confirmed expression of FGF2, OP-1/BMP7 and MK mRNA, as well as Wnt5A mRNA in G401 cells. In situ hybridization and immunocytochemistry on human fetal kidney demonstrated FGF2 expression in epithelial cells of the branching ureteric bud epithelium, nephron precursors ("S-shaped bodies"), proximal tubule epithelium and the parietal epithelium of the glomerulus. FGF2 protein in condensed "caps" of induced mesenchymal cells was also detected by immunocytochemistry. FGF2 protein was found to be concentrated in nuclei, particularly in proximal tubule epithelial cells. Recombinant FGF2 was found to act as a mitogen on primary mouse fetal kidney cell cultures. The results demonstrate G401 cells secrete a variety of fetal kidney growth factors and that FGF2 may act as a mitogen for fetal kidney cells and thus could play a role in the morphogenesis of the kidney.     

Cyclic GMP and cGMP-binding phosphodiesterase are required for interleukin-1-induced nitric oxide synthesis in human articular chondrocytes

This study addressed the role of guanylyl cyclase (GC) and phosphodiesterase (PDE) in interleukin (IL)-1 activation of human articular chondrocytes. The GC inhibitors LY83583 and methylene blue dose-dependently inhibited IL-1-induced nitric oxide (NO) production, inducible NO synthase (iNOS) protein, and mRNA expression. These effects of GC inhibition were consistent with the rapid induction of cGMP by IL-1, which reached maximal levels after 5 min. The effects of GC inhibitors were selective as they did not reduce IL-1-induced cyclooxygenase II protein and mRNA. An inhibitor specific for soluble GC did not affect IL-1-induced NO production, and activators of soluble GC did not induce NO. However, the expression of iNOS mRNA was induced by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), activators of particulate GC, indicating that particulate rather than soluble guanylyl cyclases were involved in iNOS induction. The expression of iNOS mRNA and the production of NO were induced by a slowly hydrolyzable analog of cGMP, 8-bromo-cGMP, but not by nonhydrolyzable analog, dibutyryl cGMP, suggesting that PDE rather than cGMP-dependent protein kinase mediates the cGMP effects. Chondrocytes contained extensive cGMP PDE activity. This had PDE5 biochemical features and an inhibitor profile consistent with PDE5. Furthermore, the nonisoformspecific PDE inhibitor IBMX and PDE5-specific inhibitors suppressed IL-1-induced NO release and iNOS mRNA expression. PDE5 mRNA was constitutively expressed in chondrocytes. In addition to increasing PDE5 activities, IL-1 treatment reduced the sensitivity of PDE5 to several pharmacological inhibitors by up to 50-fold. In summary, inhibitors of either GC or PDE5 prevented IL-1 induction of iNOS; IL-1 increased the rates of both cGMP generation and hydrolysis; and exogenous PDE hydrolyzable cGMP analog induced iNOS and NO. These results suggest that increased cGMP metabolic flux is sufficient to induce iNOS, and GC and PDE5 activities are required for IL-1 induction of iNOS expression via increases in coupled cGMP synthesis and hydrolysis.     

Time course of growth factor expression in mercuric chloride acute renal failure

BACKGROUND: Renal EGF expression decreases in varying models of acute renal failure (ARF). We found previously that the loss of distal tubular EGF during gentamicin ARF is strongest in the cortex, where proximal tubular injury was most severe. To gain more insight into the mechanism underlying this apparent anatomical association, renal growth factor expression was investigated during mercuric chloride ARF, in which proximal tubular injury is most severe in the outer stripe of the outer medulla (OSOM). METHODS: Endogenous renal growth factor expression was investigated by RNA hybridization and by immunohistochemistry in a rat model of mercuric chloride ARF. In addition we determined temporal and spatial profiles of tubular injury, cell proliferation, and mononuclear cell infiltration during the 3-week observation period. RESULTS: Serum creatinine values were maximal 2 days after treatment and were again normalized at day 6. Tubular injury was most severe in the PST and maximal at day 2. Cell proliferation was also higher in the PST and maximal at day 4. Three weeks after treatment, normal renal morphology was restored. Increased numbers of mononuclear cells appeared transiently in the renal interstitium from day 1 on. Most of these cells were macrophages and T lymphocytes; macrophages surrounded preferentially the severely injured PST in the OSOM. In analogy to gentamicin ARF, renal EGF and IGF-I gene expression were decreased early in the setting of mercuric chloride ARF. The decrease in distal tubular EGF staining was most pronounced in the OSOM, i.e. the anatomical area where mercuric-chloride-induced proximal tubular injury was most severe. CONCLUSIONS: Renal EGF and IGF-I gene expression decreases strongly during mercuric chloride ARF. The spatial association between the initial decrease of distal tubular EGF expression and the zone of major proximal tubular injury could originate from metabolic alterations secondary to oxygen starvation. A possible role of mononuclear cells remains to be determined.     

Decreased expression of mitochondrial-derived H2O2 and hydroxyl radical in cytoresistant proximal tubules

Increased production of reactive oxygen metabolites (ROM) can contribute to the initiation phase of nephrotoxic and ischemic acute renal failure (ARF). However, whether altered ROM expression also exists during the maintenance phase of ARF has not been adequately assessed. Since diverse forms of tubular injury can initiate a "cytoresistant state," this study tested whether a down-regulation of ROM expression might develop in the aftermath of acute tubular damage, potentially limiting renal susceptibility to further attack. To test this hypothesis, rats were subjected to either mild myohemoglobinuria (glycerol injection) or bilateral ureteral obstruction and 24 hours later, cytoresistant proximal tubular segments (PTS) were isolated to assess ROM expression. PTS from sham operated rats were used to establish normal values. Both sets of cytoresistant PTS manifested approximately 75% reductions in H2O2 levels, as assessed by the phenol red/horseradish peroxidase technique (P < 0.01 to 0.001). A 40% reduction in hydroxyl radical (.OH) levels was also observed (salicylate trap method), thereby substantiating decreased oxidant stress in cytoresistant PTS. Catalase, glutathione peroxidase, and free iron levels were comparable in control and cytoresistant PTS, suggesting that decreased H2O2 production (such as by mitochondria) was the cause of the decreased oxidant stress. To test this latter hypothesis, H2O2 expression by control and cytoresistant PTS was assessed in the presence of respiratory chain inhibitors. Although site 1 and site 3 inhibition markedly suppressed H2O2 production in control PTS, they had no impact on H2O2 production in cytoresistant PTS, implying that production at these sites was already maximally suppressed. Correlates of the decreased mitochondrial H2O2 production were improvements in cell energetics (increased ATP/ADP ratios with Na ionophore treatment) and approximately 40 to 90% increases in PTS/renal cortical glutathione content. We conclude that: (1) proximal tubule H2O2/.OH expression can be downregulated during the maintenance phase of ARF; (2) this seemingly reflects a decrease in mitochondrial ROM generation; and (3) the associated improvements in glutathione content and/or cellular energetics could conceivably contribute to a post-injury cytoresistant state.     

Regulation of mitogen-activated protein kinase phosphatase-1 induction by insulin in vascular smooth muscle cells. Evaluation of the role of the nitric oxide signaling pathway and potential defects in hypertension

In this study, we examined the regulation of mitogen-activated protein kinase phosphatase (MKP-1) expression by insulin in primary vascular smooth muscle cell cultures. Insulin caused a rapid time- and dose-dependent induction of MKP-1 mRNA and protein expression. Blockade of nitric-oxide synthase (NOS) with NG-monomethyl-L-arginine acetate, and cGMP with RpcGMP, completely inhibited MKP-1 expression. Insulin-mediated MKP-1 expression was preceded by inducible NOS (iNOS) induction and cGMP production. Blockade of phosphatidylinositol 3-kinase (PI3-kinase) signaling with wortmannin inhibited insulin-mediated iNOS protein induction, cGMP production, and MKP-1 expression. To evaluate potential interactions between NOS and the mitogen-activated protein kinase (MAPK) signaling pathways, we employed PD98059 and SB203580, two specific inhibitors of ERKs and p38 MAPK. These inhibitors abolished the effect of insulin on MKP-1 expression. Only PD98059 inhibited insulin-mediated iNOS protein induction. Vascular smooth muscle cells from spontaneous hypertensive rats exhibited a marked decrease in MKP-1 induction due to defects in insulin-induced iNOS expression because of reductions in PI3-kinase activity. Treatment with sodium nitroprusside and 8-bromo-cGMP restored MKP-1 mRNA expression to levels comparable with controls. We conclude that insulin-induced MKP-1 expression is mediated by PI3-kinase-initiated signals, leading to the induction of iNOS and elevated cGMP levels that stimulates MKP-1 expression.     

Differential expression of inducible nitric oxide synthase messenger RNA along the longitudinal and crypt-villus axes of the intestine in endotoxemic rats

OBJECTIVES: To characterize the mechanisms leading to excessive production of nitric oxide within the gut as a consequence of endotoxemia. We sought to: a) determine the time course of inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) expression in the intestine after challenging rats with lipopolysaccharide (LPS); and b) investigate whether there is differential expression of iNOS in enterocytes along the longitudinal or crypt-villus axes of the intestine in rats after LPS administration. DESIGN: Prospective, randomized, unblinded study. SETTING: Research laboratories at a large university-affiliated medical center. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: At T = 0 hr, rats were injected with O111:B4 Escherichia coli LPS (5 mg/kg) or a similar volume of the saline vehicle. At various time points thereafter, samples of duodenum, jejunum, ileum, colon, and liver were harvested for subsequent extraction of RNA. In some cases, populations of enterocytes enriched in either crypt or villus cells were harvested from the ileum. In some studies, rats were injected with cycloheximide (25 mg i.p.) 15 mins before being challenged with LPS or dexamethasone (2 mg i.p.) 30 mins before being injected with LPS. MEASUREMENTS AND MAIN RESULTS: iNOS mRNA was undetectable in ileal tissue from rats under basal conditions, but was evident by T = 1 hr and was maximal at T = 2 hrs after injection of LPS. Thereafter, ileal iNOS mRNA concentrations decreased and were undetectable again at T = 24 hrs. At T = 2 hrs after LPS injection, there was marked expression of iNOS mRNA in the ileum, whereas much lower concentrations of iNOS mRNA were detected in the jejunum and colon, and no iNOS mRNA was detected in the duodenum. At T = 3 hrs after LPS injection, expression of iNOS mRNA was up-regulated in both villus and crypt cells, although LPS-induced iNOS mRNA was more prominent in the former than the latter cell type. Pretreatment of rats with dexamethasone virtually abrogated the expression of iNOS mRNA in ileal samples obtained 3 hrs after the injection of LPS. Prior treatment of rats with the protein synthesis inhibitor, cycloheximide, also blunted LPS-induced iNOS mRNA expression. CONCLUSIONS: LPS-induced iNOS expression is differentially regulated along both the longitudinal and crypt villus axes of the intestinal mucosa, being most prominent in the villus cells of the ileum. LPS-induced iNOS expression is blunted by pretreating rats with dexamethasone or cycloheximide. The latter finding suggests that LPS-induced expression of iNOS mRNA in the gut requires new protein synthesis. Differential regulation of nitric oxide production along the longitudinal and crypt-villus axes of the gut may be a determinant of the pattern of sepsis-induced intestinal damage.