Leukocyte toll-like receptor expression in end-stage kidney disease.

BACKGROUND: End-stage renal disease (ESRD) is simultaneously associated with inflammation, impaired immunity and increased susceptibility to microbial infections. Innate immune cells, monocytes and polymorphonuclear leukocytes (PMN) recognize pathogens via toll-like receptors (TLR) triggering phagocytosis, cellular activation and secretion of inflammatory cytokines. Data on expression and function of TLRs in ESRD are limited. METHODS: Blood samples from 21 stable ESRD patients and 21 normal controls were processed for TLR2, TLR4, TLR7 and TLR 9 expression on monocytes and PMN by flow cytometry. TLR activity was examined by determining the response to TLR4 and TLR2 ligands. RESULTS: The ESRD group exhibited significant upregulation of TLR2 and TLR4 (but not TLR7 or TLR 9) expressions on monocytes and of TLR4 on PMN. This was coupled with heightened cytokine production in response to TLR4 activation with lipopolysaccharide. However, the response to TLR2 stimulation with peptidoglycan was unchanged in the ESRD group. CONCLUSIONS: Monocyte TLR2 and TLR4 and neutrophil TLR4 expressions and TLR4 activity are increased hemodialysis patients, representing another dimension of ESRD-associated inflammation.

Comparison of fatty acid contents of erythrocyte membrane in hemodialysis and peritoneal dialysis patients.

OBJECTIVE: Membrane fatty acid composition plays an important role in the cellular function. Erythrocyte fatty acid composition mirrors that of myocardium and is influenced by diet. Earlier studies have shown significant alterations of membrane fatty acid composition in ethnically mixed patients with end-stage renal disease. Given the impact of ethnic and dietary factors, we sought to examine membrane fatty acid composition in an ethnically homogeneous end-stage renal disease population residing in a coastal region of Korea with high fish consumption. DESIGN: Cross-sectional study. SETTING: Outpatient facility at Dong-A University Hospital, Busan, Republic of Korea. PATIENTS: We recruited 15 stable hemodialysis patients, 14 peritoneal dialysis patients, and 10 age- and gender-matched normal controls. Patients with significant malnutrition, short duration of dialysis, recent infection, malignancy, or liver disease were excluded. Dietary intake and use of omega-3 fatty acid supplements were determined. MAIN OUTCOME MEASURE: Erythrocyte membrane fatty acid contents measured by gas chromatography. RESULTS: Palmitoleic acid and alpha-linolenic acid levels were lower, whereas oleic acid, linoleic acid, and arachidonic acid levels were higher in patients with end-stage renal disease compared with the control group. Total monounsaturated fatty acids (palmitoleic acid and oleic acid) were significantly higher in peritoneal dialysis than in hemodialysis patients. Eicosapentaenoic acid and omega-3 docosapentaenoic acid were significantly higher, but total omega-6 fatty acids, omega-6/omega-3, and arachidonic acid/eicosapentaenoic acid ratios were significantly lower in hemodialysis patients consuming omega-3 supplements compared with those who did not. CONCLUSION: Patients with end-stage renal disease exhibited significant alterations in erythrocyte membrane fatty acids, which were partially modified by the dialysis modality and omega-3 fatty acid supplementation.

Effects of dietary salt on intrarenal angiotensin system, NAD(P)H oxidase, COX-2, MCP-1 and PAI-1 expressions and NF-kappaB activity in salt-sensitive and -resistant rat kidneys.

BACKGROUND: Chronic consumption of a high-salt diet causes hypertension (HTN) and renal injury in Dahl salt-sensitive (SSR) but not salt-resistant rats (SRR). These events are, in part, mediated by oxidative stress and inflammation in the kidney and vascular tissues. Activation of the angiotensin II type 1 (AT(1)) receptor plays an important role in the pathogenesis of oxidative stress and inflammation in many hypertensive disorders. However, the systemic renin-angiotensin system (RAS) is typically suppressed in salt-sensitive HTN. This study was designed to test the hypothesis that differential response to a high-salt diet in SSR versus SRR may be related to upregulation of tissue RAS and pathways involved in inflammation and reactive oxygen species (ROS) production. METHODS AND RESULTS: SSR and SRR were studied 3 weeks after consumption of high- (8%) or low-salt (0.07%) diets. The SSR consuming a low-salt diet exhibited significant increases in AT(1) receptor, cyclooxygenase (COX) 2, plasminogen activator inhibitor (PAI) and phospho-I kappaB in the kidney as compared to those found in SRR. The high-salt diet resulted in severe HTN and proteinuria (in SSR but not SRR) and marked elevations of renal tissue monocyte chemoattractant protein 1, p22(phox), NADPH oxidase subunit 4, angiotensin-II-positive cell count, infiltrating T cells and macrophages and further increases in AT(1) receptor, COX-2, PAI-1 and phospho-I kappaB in the SSR group. The high-salt diet significantly lowered plasma renin activity (PRA) in SRR but not in the SSR. COX-1 abundance was similar on the low-salt diet and rose equally with the high-salt diet in both groups. Among subgroups of animals fed the low-salt diet, kidney glutathione peroxidase (GPX) abundance was significantly lower in the SSR than SRR. The high-salt diet raised GPX and mitochondrial superoxide dismutase (SOD) abundance in the SRR kidneys but failed to do so in SSR. Cu/Zn-SOD abundance was similar in the subgroups of SSR and SRR fed the low-salt diet. The high-salt diet resulted in downregulation of Cu/Zn-SOD in SSR but not SRR. CONCLUSIONS: Salt sensitivity in the SSR is associated with upregulations of the intrarenal angiotensin system, ROS-generating and proinflammatory/profibrotic proteins and an inability to raise antioxidant enzymes and maximally suppress PRA in response to high salt intake. These events can contribute to renal injury with high salt intake in SSR.

Effect of renal injury-induced neurogenic hypertension on NO synthase, caveolin-1, AKt, calmodulin and soluble guanylate cyclase expressions in the kidney.

Single injection of a small quantity of phenol into the cortex of one kidney in rats results in development of persistent hypertension (HTN) which is thought to be mediated by activation of renal afferent and efferent sympathetic pathways and sodium retention. Nitric oxide (NO) plays a major role in regulation of renal vascular resistance, tubular Na(+) reabsorption, pressure natriuresis, and thereby systemic arterial pressure. The present study was performed to test the hypothesis that chronic renal injury-induced HTN may be associated with dysregulation of NO system in the kidney. Accordingly, urinary NO metabolite (NO(x)) and cGMP excretions as well as renal cortical tissue (right kidney) expressions of NO synthase (NOS) isoforms [endothelial, neuronal, and inducible NOS, respectively (eNOS, nNOS, and iNOS)], NOS-regulatory factors (Caveolin-1, phospho-AKt, and calmodulin), and second-messenger system (soluble guanylate cyclase [sGC] and phosphodiesterase-5 [PDE-5]) were determined in male Sprague-Dawley rats 4 wk after injection of phenol (50 mul of 10% phenol) or saline into the lower pole of left kidney. The phenol-injected group exhibited a significant elevation of arterial pressure, marked reductions of urinary NO(x) and cGMP excretions, downregulations of renal tissue nNOS, eNOS, Phospho-eNOS, iNOS, and alpha chain of sGC. However, renal tissue AKt, phospho-AKT, Calmodulin, and PDE-5 proteins were unchanged in the phenol-injected animals. In conclusion, renal injury in this model results in significant downregulations of NOS isoforms and sGC and consequent reductions of NO production and cGMP generation by the kidney, events that may contribute to maintenance of HTN in this model.

Expression of NOX-I, gp91phox, p47phox and P67phox in the aorta segments above and below coarctation.

Aorta coarctation results in hypertension (HTN) in the arterial tree proximal to stenosis and, as such, provides an ideal model to discern the effects of different levels of blood pressure on the vascular tissue in the same animal. Compelling evidence has emerged supporting the role of oxidative stress as a cause of HTN. However, whether or not HTN (independent of the circulating humoral factors) can cause oxidative stress is less certain. NAD(P)H oxidase isoforms are the main source of reactive oxygen species (ROS) in the vascular tissues. We therefore compared the expressions of NOX-I, gp91phox and the regulatory subunits of the enzyme in the aorta segments residing above and below coarctation in rats with abdominal aorta banding. Rats were studied 4 weeks after aorta banding above the renal arteries or sham operation. Subunits of NAD(P)H oxidase and its NOX-I isoform as well as endothelial NO synthase (eNOS) and nitrotyrosine (footprint of NO oxidation by superoxide) were measured in the aorta segments above and below coarctation. The gp91phox, p47phox, and p67phox subunits of NAD(P)H oxidase, NOX-I isoform, eNOS and nitrotyrosine were markedly increased in the aorta segment above coarctation (hypertensive zone), but were virtually unchanged in the segment below coarctation. Since, excepting blood pressure, all other conditions were constant, the upregulation of NAD(P)H oxidase isoforms and the increased NO oxidation in the aorta segment above, but not below, coarctation prove that HTN, per se, independent of circulating mediators can cause oxidative/nitrosative stress in the arterial wall. These observations suggest that HTN control may represent a specific form of antioxidant therapy for hypertensive disorders.

Protective effects of estrogen on gender-specific development of diet-induced hypertension.

Dietary and humoral factors are thought to be involved in the development of hypertension. This study investigated the interaction between diet and gonadal hormone status in the development and reversibility of hypertension. Normal male and female and ovariectomized (OVX) female Fischer rats were placed on either a high-fat (primarily saturated), refined carbohydrate (sucrose) (HFS) or a low-fat, complex carbohydrate (LFCC) diet at 2 mo of age, and body weight and systolic blood pressure (BP) were measured. Male and OVX female rats were initially on the diets for 7 mo, whereas normal female rats were on the diets for 2 yr. After this initial phase, a group of rats from each of the normal HFS groups were converted to the LFCC diet for a period of 1 mo (males) and 2 mo (females). The OVX females were subcutaneously implanted with a 0.5-mg estradiol (E2) pellet for 1 mo. A significant rise in arterial BP occurred within 12 mo in female and only 2 mo in male rats on the HFS diet, exceeding 140 mmHg after 24 and 7 mo, respectively. Conversion from the HFS to the LFCC diet led to a normalization of BP in both female and male rats. HFS diet-induced hypertension was accelerated by OVX in female rats, approaching the pattern seen in male rats. The effect of OVX was completely reversed by E2 replacement. BP did not significantly change in any of the LFCC groups at any time point, and E2 replacement had no effect on BP in the OVX LFCC group. All HFS groups had significantly greater body weight, with differences occurring sooner in the male and OVX rats compared with the female rats. Diet modification resulted in a partial but significant reduction of body weight, but E2 replacement did not. These results demonstrate that long-term consumption of HFS diet induces hypertension in both genders and is reversible by diet modification. Hypertension is significantly delayed in females with functional ovaries. This protection is lost by OVX and restored by estrogen replacement. Thus hormone status contributes to the delayed onset of diet-induced hypertension in females compared with males.

Down-regulation of renal endothelial nitric oxide synthase expression in experimental glomerular thrombotic microangiopathy.

Infection with certain strains of Escherichia coli and endotoxemia results in renal glomerular thrombotic microangiopathy (TMA) characterized by endothelial swelling and prominent glomerular microthrombus formation. Nitric oxide (NO) is an endogenous biologic modulator with diverse physiologic functions including vasodilation and inhibition of platelet adhesion and aggregation. NO is synthesized from conversion of L-arginine to L-citrulline by a family of NO synthases (NOS), which include constitutive and inducible isoforms. Indirect evidence supports the hypothesis that TMA is associated with depressed intrarenal NO production. However, the effect of TMA on renal tissue NOS expression has not been fully elucidated. We studied rats with TMA induced by iv bolus injection of high dose (20 mg/kg) E. coli endotoxin. Subgroups of six animals each were sacrificed before or at 30, 90, 180, 360, and 720 minutes after the administration of endotoxin. Renal histology and tissue expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) were examined. Additionally, we examined the effect of endotoxin on glomerular NO production, and eNOS and iNOS protein expression in vitro. Glomerular capillary thrombosis developed by 180 minutes after endotoxin administration in approximately half of the animals. The glomeruli without thrombotic lesions apparent by light microscopy disclosed early signs of TMA characterized by endothelial swelling, platelet accumulation/adhesion, and patchy fibrinogen deposition. These morphologic changes were associated with a marked reduction of renal tissue eNOS expression beyond 180 minutes after the endotoxin administration. The fall in eNOS expression was coupled with a significant rise in iNOS protein abundance, which was expressed largely by glomerular circulating neutrophils and endothelial cells, peritubular vascular endothelium, and collecting ducts of cortex and medulla. In vitro incubation of isolated glomeruli with endotoxin also resulted in a marked reduction in eNOS expression and a significant rise in iNOS content. Administration of E. coli endotoxin leads to a sustained fall in renal eNOS expression both in vivo and in vitro. The associated decline in intrarenal endothelial NO production/availability may result in renal vasoconstriction and a hypercoagulative state, which may contribute to the pathogenesis of endotoxin-induced TMA.

Induction of oxidative stress by glutathione depletion causes severe hypertension in normal rats.

Several recent studies have shown that certain forms of genetic or acquired hypertension are associated with oxidative stress and that animals with those types of hypertension respond favorably to antioxidant therapy. We hypothesize that oxidative stress may cause hypertension via (among other mechanisms) enhanced oxidation and inactivation of nitric oxide (NO). To test this hypothesis, Sprague-Dawley rats were subjected to oxidative stress by glutathione (GSH) depletion by means of the GSH synthase inhibitor buthionine sulfoximine (BSO, 30 mmol/L in drinking water) for 2 weeks. The control group was given drug-free drinking water. In parallel experiments, subgroups of animals were provided vitamin E-fortified chow and vitamin C-supplemented drinking water. The BSO-treated group showed a 3-fold decrease in tissue GSH content, a marked elevation in blood pressure, and a significant reduction in the urinary excretion of the NO metabolite nitrate plus nitrite, which suggests depressed NO availability. These characteristics were associated with a significant accumulation in various tissues of nitrotyrosine, which is the footprint of NO inactivation by reactive oxygen species. Administration of vitamin E plus vitamin C ameliorated hypertension, improved urinary nitrate-plus-nitrite excretion, and mitigated nitrotyrosine accumulation (despite GSH depletion) in the BSO-treated animals but had no effect in the control group. In conclusion, GSH depletion resulted in perturbation of the NO system and severe hypertension in normal animals. The effects of BSO were mitigated by concomitant antioxidant therapy despite GSH depletion, which supports the notion that oxidative stress was involved in the pathogenesis of hypertension in this model.

Muscarinic down-regulation of cAMP-stimulated potassium ion secretion by rabbit distal colon.

The sustained effects of the cholinergic agonist carbachol (CCh) on electrolyte transport across the isolated, short-circuited rabbit distal colon were examined in the absence and presence of secretagogue (di-butyryl cyclic-adenosine monophosphate, dB-cAMP). Steady-state, basal absorption of 22Na+, 42K+ (86Rb+), and 36Cl- were not significantly altered by addition of the CCh (10(-4) mmol/l) to the serosal reservoir. Stimulation with dB-cAMP (1.0 mmol/l, serosal) promoted K+ (or Rb+) and Cl- secretion across the colon, without significantly affecting the unidirectional or net fluxes of Na+. Serosal (but not mucosal) addition of CCh to dB-cAMP-stimulated tissues reduced the serosal to mucosal flux of Rb+ (J(Rb)SM) in a concentration-dependent manner with a half-maximum concentration approximately equal 5 micromol/l. Pretreatment with CCh (100 micromol/l, serosal) inhibited dB-cAMP-induced K+ secretion, but had no significant effect on the steady-state unidirectional fluxes of Na+ or Cl-. Serosal histamine (20 micromol/l) also inhibited J(Rb)SM in dB-cAMP-stimulated tissues. Serosal epinephrine (10 micromol/l) promoted a decrease in short-circuit current (Isc) and transepithelial potential (VT) that was mirrored by increases in J(Rb)SM. Both Isc, and VT became more positive and J(Rb)SM was reduced when CCh was added to the epinephrine-stimulated tissues. Serosal muscarine (50 micromol/l) mimicked the CCh-induced inhibition of J(Rb)SM, but serosal nicotine (50 micromol/l) had no effect. In atropine-treated tissues (1 micromol/l, serosal), CCh failed to block dB-cAMP-stimulated increases in J(Rb)SM. The inhibitory action of CCh was observed in tissues that had been pretreated with 50 micromol/l serosal hexamethonium (a ganglionic transmission blocker) or 2 micromol/l serosal tetrodotoxin (a voltage-gated Na+ channel blocker), indicating that the inhibitory action of this cholinergic agonist does not depend on remnant enteric neural pathways. Rubidium ion transport across confluent monolayers of T84 cells was similarly affected by dB-cAMP and CCh, supporting the notion that enteric neural pathways are not required. Serosal charybdotoxin (20 nmol/l) mitigated the inhibitory action of CCh on J(Rb)SM in dB-cAMP-stimulated tissues, suggesting a role for basolateral, Ca2+-dependent K+ channels in the actions of CCh. It is concluded that basolateral muscarinic receptors (and possibly other Ca2+-dependent receptor pathways) of secretory colonocytes mediate the down-regulation of potassium ion secretion by rabbit distal colon, possibly by increasing basolateral membrane K+ conductance.

Atherogenic lipoproteins and tyrosine kinase mitogenic signaling in mesangial cells.

BACKGROUND: Mesangial hypercellularity is a critical early histopathological finding seen in human and experimental glomerular diseases. Hyperlipidemia and the glomerular deposition of atherogenic lipoproteins [for example, low-density lipoprotein (LDL) and its oxidized variants, minimally oxidized/modified LDL (mm-LDL)] are commonly associated with mesangial hypercellularity and the development of glomerular disease. This article reviews signal transduction pathways involved in cell proliferation and provides evidence for the participation of atherogenic lipoproteins in intracellular signaling pathways for mesangial cell proliferation. The mitogenic intracellular signaling pathways are regulated by the activation of a series of transmembrane and cytoplasmic protein tyrosine kinases that converge into the activation of Ras and downstream mitogen-activated protein (MAP) kinase. Activated MAP kinase, through translocating into the nucleus and the activation of various transcription factors and proto-oncogenes, regulates cellular proliferation. METHODS: Murine mesangial cells were stimulated with LDL and mm-LDL and were analyzed for the tyrosine kinase activity, phosphorylation of membrane proteins, activation of Ras and MAP kinase, and cell proliferation. RESULTS: The results indicated that the stimulation of mesangial cells with LDL and, with greater activity, mm-LDL induced the phosphorylation of membrane platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors, activated Ras, and resulted in sustained (up to 24 hr) activation of MAP kinase. LDL/mm-LDL-mediated mesangial cell proliferation and MAP kinase activation were dependent on the activation of tyrosine kinases. CONCLUSIONS: We suggest that the accumulation of LDL and more potently its oxidized forms within the glomerulus, through the activation of membrane receptor tyrosine kinases, activate the Ras and MAP kinase signaling cascade leading to DNA synthesis and subsequent cell proliferation.

Effects of experimental hemosiderosis on pancreatic tissue iron content and structure.

The effects of iron overload on pancreatic iron content and morphology were investigated. Sprague-Dawley rats were randomized into an iron-overloaded group, which received a single subcutaneous injection of 1.2 g/kg elemental iron as iron-dextran complex, and placebo-treated pair-fed controls. Animals were studied after a 10-month observation period. Tissue nonheme iron content was measured, and histologic examination was carried out. Chronic iron-overloaded animals showed significant increases in tissue iron content. There was a statistically significant increase in stainable iron in perivascular, parenchymal, and lymphoid tissue in the iron-overloaded group. Although pancreatic fibrosis was present in the iron-overload group, it was not statistically significant. The iron-overloaded animals showed some islet cell destruction. In contrast, no significant islet cell destruction was seen in the control group. However, the difference was not statistically significant. Moreover, the serum glucose levels were the same in both groups, suggesting that there was no significant impairment of pancreatic endocrine function. Thus, chronic experimental iron overload in rats leads to significant increases in tissue iron content, but no significant morphologic alterations of the pancreas with the dose and route of iron administered in this animal model.

Simultaneous measurement of marinobufagenin, ouabain, and hypertension-associated protein in various disease states.

We have previously demonstrated that a 12 kD hypertension-associated protein (HAP) is elevated in essential hypertension and that this protein has the characteristics of natriuresis, inhibition of Na-K-ATPase, displaces 3H-ouabain from binding sites, and is vasoconstrictive in vitro. In the present study, plasma from 101 patients were examined [25 normals (N)age 50, 7 with acute congestive heart failure (CHF), 24 with chronic renal failure (CRF), on dialysis, 5 with idiopathic hyperaldosteronism (PA) and 27 with essential hypertension, untreated (EHT)]. Plasma was extracted with 32% acetonitrile, then analyzed by DELFIA for marinobufagenin and ouabain. In addition, from 32 patients (6 N <50, 6 N >50, 5 CHF, 5 CRF, 6 EHT, and 4 PA) SDS gradient gels were obtained. The 12 kD bands were extracted, analyzed for Na-K-ATPase inhibition, marinobufagenin and ouabain, and compared to 14 kD and 21 kD bands. Marinobufagenin was found to be elevated in CRF, EHT, PA and CHF. Ouabain was increased only in PA. When the relative optical densities of the 12 kD and 21 kD bands were contrasted, CRF, PA, and EHT were found to be increased and CHF to be decreased in the 12 kD band, with no discernible changes in the 21 kD bands. Following extraction of the bands, Na-K-ATPase inhibitory activity measured 38% in 18 pooled 12 kD bands, with essentially no activity found in the 14 kD or 21 kD bands. Thus only the 12 kD HAP band possessed all of the attributes of natriuretic hormone.

Erythropoietin ameliorates anemia of cisplatin induced acute renal failure.

Daily intraperitoneal injections of recombinant human erythropoietin (EPO) were administered for 9 days to Sprague-Dawley rats with cisplatin induced acute renal failure (ARF/ EPO group). A group of placebo treated rats with cisplatin induced acute renal failure (ARF group) was used as the control group. As expected, administration of cisplatin to rats caused ARF that was marked by a significant rise in serum creatinine and a significant reduction in creatinine clearance in both groups. The placebo treated ARF animals showed a significant fall in hematocrit, erythrocyte count, reticulocyte count, and hemoglobin concentration. These abnormalities were averted by EPO therapy in the ARF/EPO group. Therefore, when compared to the ARF group, the ARF/EPO group had higher hematocrits (45.8+/-0.6% vs 36.1+/-0.7%, SEM), reticulocyte counts (7.7+/-0.3% vs 3.4+/-0.6%, SEM), erythrocyte counts (7.7+/-0.2 x 10(12)/L vs 6.3+/-0.2 x 10(12)/L, SEM), and hemoglobin levels (16.6+/-0.3 g/dl vs 13.3+/-0.3 g/dl, SEM). The differences were all statistically significant (p < 0.05). Plasma EPO was found to be higher in the ARF/EPO group (reflecting exogenously administered hormone) than in the ARF group, which showed undetectable values. The results suggest that anemia in rats with cisplatin induced ARF can be corrected by EPO therapy.

cAMP-dependent sulfate secretion by the rabbit distal colon: a comparison with electrogenic chloride secretion.

The ability of a Cl-secreting epithelium to support net secretion of an anion other than a halide was investigated with 35SO4 flux measurements across the isolated, short-circuited rabbit distal colon. In most experiments, 36Cl fluxes were simultaneously measured to validate the secretory capacity of the tissues. Serosal addition of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 0.5 mM) stimulated a sustained net secretion of SO4 (about -3.0 nmol.cm-2.h-1 from a 0.20 mM solution) via an increase in the serosal-to-mucosal unidirectional flux, whereas Ca ionophore A-23187 (1 microM, serosal) produced a more transient stimulation of SO4 and Cl secretion. Net adenosine 3',5'-cyclic monophosphate (cAMP)-dependent SO4 and Cl secretion were strongly voltage sensitive, principally through the potential dependence of the serosal-to-mucosal fluxes, indicating an electrogenic transport process. Symmetrical replacement of either Na, K, or Cl inhibited cAMP-dependent SO4 secretion, whereas HCO3-free buffers had no effect on SO4 secretion. Serosal bumetanide (50 microM) or furosemide (100 microM) reduced DBcAMP-stimulated SO4 and Cl secretion, whereas serosal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (50 microM) blocked DBcAMP-induced SO4 secretion while enhancing net Cl secretion and short-circuit current. Mucosal 5-nitro-2-(3-phenylpropylamino)benzoic acid partially inhibited SO4 secretion and completely inhibited Cl secretion. It is concluded that secretagogue-stimulated SO4 secretion, like Cl secretion, may be an electrogenic process mediated by diffusive efflux through an apical anion conductance. Cellular accumulation of SO4 across the basolateral membrane appears to be achieved by a mechanism that is distinct from that employed by Cl.

Plasma membrane calcium ATPase isoform expression in cultured rat mesangial cells.

The maintenance of intracellular ionized calcium (iCa2+) in the submicromolar range is important for mesangial cell (MC) function, and, as in most mammalian cells, plasma membrane Ca(2+)-ATPases (PMCA) play an important role in the homeostatic process. Molecular studies have demonstrated four PMCA isoforms, each with multiple splice variants. The present study examines the expression of PMCA isoforms and calmodulin-binding region splice variants in cultured MC from Sprague-Dawley rats and from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats before and after the onset of hypertension in SHR. Using reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analyses, we have demonstrated PMCA1, -3, and -4, but not PMCA2, to be present in MC from these rat strains. Splice variant analysis revealed PMCA1a and -1b, PMCA3a, -3b, and -3c, and PMCA4a and -4b to be expressed in MC from all three strains. The relative quantities of PMCA1 and PMCA4 mRNA were not different in age-matched SHR vs. WKY rats, correlating with similar iCa2+ measurements. The expression of all three isoforms declined with age in SHR and WKY.

Gene expression of LDL receptor, HMG-CoA reductase, and cholesterol-7 alpha-hydroxylase in chronic renal failure.

BACKGROUND: Chronic renal failure (CRF) is associated with an atherogenic lipid profile and an increased risk of ischaemic cardiovascular disease. The associated hyperlipidaemia is reportedly ameliorated by erythropoietin (Epo) therapy. According to a recent report, rats studied 3 weeks after 5/6 nephrectomy and fed a high-protein diet exhibited increased activities of hepatic HMG-CoA reductase (HMG-CoAR) and cholesterol 7 alpha-hydroxylase (Ch-7 alpha-H), despite normal corresponding mRNA values. DESIGN AND METHODS: This study was designed to examine the effects of naturally progressing CRF of longer duration as well as those of Epo therapy on gene expressions of the key factors involved in hepatic cholesterol metabolism, i.e., LDL receptor (LDLR), HMG-CoAR, and Ch-7 alpha-H. Sprague-Dawley rats were randomized to the CRF group (5/6 nephrectomy), Epo-treated CRF group (given Epo 150 U/kg/twice weekly) and sham-operated, placebo-treated normal controls. They were allowed free access to regular rat chow and studied 6 weeks after surgery. Liver mRNAs and protein mass or activities of the above factors were studied. RESULTS: Plasma cholesterol concentration was significantly increased in the CRF group (P < 0.001) and was modestly lowered (P < 0.05) by Epo therapy. However, microsomal cholesterol concentration and LDLR, HMG-CoAR, and Ch-7 alpha-H mRNA as well as HMG-CoAR activity, and Ch-7 alpha-H and LDLR protein mass measurements were virtually identical in the three groups. Thus, hepatic LDLR, HMG-CoAR, and Ch-7 alpha-H mRNA and protein measurements in rats with CRF were similar to those of the normal control group representing an inappropriate response to the associated hypercholesterolemia. Epo therapy led to partial amelioration of CRF-associated hypercholesterolaemia with no discernible effect on hepatic tissue expression of the above factors.

Up-regulation of renal and vascular nitric oxide synthase in iron-deficiency anemia.

Anemia is frequently associated with increased cardiac output and reduced vascular resistance. The latter has been attributed to reduced inactivation of nitric oxide (NO) by hemoglobin. We hypothesized that in addition to reducing NO inactivation, anemia may up-regulate NO production. To test this hypothesis, male Sprague-Dawley rats with chronic iron-deficiency anemia (produced by multiple phlebotomies and an iron-free diet) were studied. The results were compared with those obtained in a group of normal control animals. The anemic group showed marked increases in urinary excretion, plasma concentration, and renal and aorta tissue contents of NO metabolites (total nitrates and nitrites, NOx). This was accompanied by a significant rise in urinary excretion of cGMP, the second messenger for NO. In addition, NO synthase (NOS) activity and endothelial constitutive (ecNOS) and inducible NOS (iNOS) proteins of the thoracic aorta were markedly increased in the anemic group. Likewise, renal tissue ecNOS and iNOS proteins were greatly increased in the anemic animals. NOS activity and protein values were inversely related to hematocrit and directly related to plasma, tissue and urinary NOx. The constellation of these findings points to an increased NOS expression and NO production as opposed to the mere reduction of NO inactivation in iron-deficiency anemia. Further studies are planned to determine the mechanism of NOS up-regulation in iron-deficiency anemia.

Effects of adenosine infusion on renal function, plasma ANP and ADH concentrations and central hemodynamics in anesthetized pigs.

1. The effect of high-dose adenosine administration on atrial natriuretic peptide (ANP) and antidiuretic hormone (ADH) release is not completely understood, and data concerning the effect of adenosine on renal and systemic hemodynamics in the pig are lacking. Measurements of central hemodynamics, renal blood flow and urine production were made in anesthetized pigs during infusion of adenosine. The relationship between these parameters and the plasma concentrations of ANP, ADH and renal renin production was examined. 2. Adenosine infusion at the rate of 140 mg/kg per minute resulted in a significant decrease in systolic, diastolic and mean arterial blood pressure as well as pulmonary arterial pressure. However, cardiac output and renal blood flow remained unchanged during adenosine infusion. Likewise, heart rate remained unchanged until the end of infusion when it increased significantly, Plasma ANP and ADH concentrations increased significantly within 30 min after adenosine infusion, reaching peak levels at 30 to 60 min. However, despite the significant decrease in arterial blood pressure, renal renin production did not change significantly. 3. The adenosine-induced rise in ANP, which is normally released by atrial stretch, may represent a direct effect of adenosine on the cardiac myocytes. The increase in ADH may be a result of decreased arterial blood pressure triggering stimulatory signals from the aortic arch and carotid body receptors to hypothalamic-pituitary sites of ADH production/release. Urine flow decreased dramatically within 30 min of adenosine infusion. Thus adenosine infusion at the given rate led to marked reduction in systemic and pulmonary arterial pressures without significant change in cardiac output, heart rate and renal blood flow. This was associated with a marked increase in plasma ANP and ADH levels with no significant change in renal renin production despite a marked reduction in arterial blood pressure. 4. Maintenance of renal blood flow despite marked reduction in perfusion pressure suggests that, at high doses, adenosine induces renal vasodilation in pigs as opposed to a combined afferent and efferent vasoconstriction known to occur under different experimental conditions.

Effects of experimental hemosiderosis on intestinal morphology, permeability, and tissue iron content.

Effects of iron overload on intestinal function and structure are unknown and were, therefore, investigated. Sprague-Dawley rats were randomized into an iron-overloaded group, which received a single subcutaneous injection of 1.2 g/kg elemental iron-dextran complex, and placebo-treated pair-fed controls. Animals were studied after a 10-month observation period. Intestinal permeability was assessed by measuring the urinary excretion of lactulose, rhamnose, and mannitol after oral administration. In addition, tissue nonheme iron content was measured, and histologic examination and morphometric measurements were carried out. The chronic iron-overloaded group showed a significant increase in intestine tissue iron content and stainable iron in the submucosa and muscularis propria and adipose tissue of the small intestine and lamina propria and muscularis mucosa of the large intestine. There was a significant decrease in the crypt depths without discernible change in the intestine permeability to any of the markers used. In addition, the iron-overloaded animals showed a significant number of iron-laden cells, which primarily consisted of macrophages, fibroblasts, myocytes, and adipocytes. In contrast, no iron-laden cells were present in tissues obtained from the normal control group. Thus, chronic experimental iron overload in rats leads to significant morphologic, but no permeability, alterations of the alimentary tract.

Effect of hemodialysis on leukocyte adhesion receptor expression.

Hemodialysis with complement-activating membranes such as cuprophane is known to transiently activate leukocytes, leading to increased cellular adhesiveness, pulmonary leukostasis, and reduced functional capacity of monocytes and neutrophils. Clinically, this repetitive cell activation may contribute to the increased morbidity and mortality associated with chronic hemodialysis. To examine the effect of cuprophane hemodialysis on expression of cell-surface proteins involved in leukocyte adhesiveness, we monitored CD11b, CD18, CD14, CD54, and plasma-soluble CD54 in 10 patients during hemodialysis with cuprophan dialyzers. To test the effect of local blood recirculation, in two patients, arterial supply to the dialyzer was accessed from the peripheral arteriovenous fistula and was returned via an indwelling central venous catheter. In an attempt to examine the possible role of membrane-induced complement activation, the results were compared with those seen after incubation with C5a in vitro. Finally, the leukocyte responses to C5a and lipopolysaccharide were measured before and after hemodialysis. Leukocyte expression of CD11b and CD18 increased and CD14 decreased with hemodialysis, while CD54 remained unaltered. Plasma CD54 was markedly elevated before and remained unchanged during hemodialysis. Data obtained with C5a activation in vitro revealed identical changes in CD11b expression as that seen with hemodialysis, suggesting the role of membrane-induced complement activation. Preliminary data obtained using remote arterial and venous access sites showed only a slight increase in CD11b expression in the arterial blood, suggesting that the apparent systemic activation seen with arteriovenous access may be due to recirculation and local activation within the blood access. Finally, dialysis procedure did not impair lipopolysaccharide- or C5a-mediated upregulation of CD11b expression.