Role of nitric oxide in rat nephrotoxic nephritis: comparison between inducible and constitutive nitric oxide synthase

Nitric oxide (NO), generated by inducible NO synthase (iNOS) in migrating macrophages, is increased in glomerulonephritis. This study investigates the effect of NO inhibition on rat nephrotoxic nephritis (NTN) to clarify the role of NO production in glomerular damage. NTN was induced in Sprague Dawley rats by an injection of an anti-glomerular basement membrane (GBM) antibody. Urinary nitrite excretion and nitrite release from kidney slices (5.47 +/- 1.19 versus 2.15 +/- 0.73 nmol/mg protein, NTN versus Control, P < 0.05) were increased in NTN on day 2. Glomerular macrophage infiltration and intercellular adhesion molecule (ICAM)-1 expression increased from day 2. iNOS expression was increased in interstitial macrophages. Glomerular endothelial cell NOS (ecNOS) expression evaluated by counting immunogold particles along GBM was suppressed (0.06 +/- 0.02 versus 0.35 +/- 0.04 gold/micron GBM, P < 0.0001). Glomerular damage developed progressively. NG-nitro-L-arginine methyl ester (L-NAME), which inhibits both iNOS and ecNOS and aminoguanidine (AG), a relatively selective inhibitor for iNOS, equally suppressed nitrite in urine and renal tissue. Glomerular ICAM-1 expression and macrophage infiltration were reduced by L-NAME, but not by AG. Expression of ecNOS was significantly increased by L-NAME (0.91 +/- 0.08, P < 0.0001 versus NTN), but slightly by AG (0.18 +/- 0.04). AG significantly and L-NAME slightly attenuated the glomerular damage at day 4. In conclusion, suppression of iNOS prevents glomerular damage in the early stage of NTN. Treatment by L-NAME reduces macrophage infiltration by suppression of ICAM-1 expression, which may be explained by an increase in ecNOS expression.     

Role of thromboxane A2, endothelin-1 and endothelin-3 in rat nephrotoxic serum nephritis

To clarify the role of thromboxane A2 (TxA2), endothelin-1 (ET-1) and endothelin-3 (ET-3) in the progression of glomerular injury in accelerated nephrotoxic serum nephritis (NTN) in the rat, we studied the expression of ET-1 and ET-3 at the kidney by immunohistochemical method and examined the effect of a novel TxA2 receptor antagonist, S-1452. The S-1452-treated group showed significantly lowered 24-hr proteinuria and milder glomerular cell proliferation and lobulation than the non-treated group (NT group) on experimental day 10. There was no significant difference in the glomerular polymorphonuclear cell (PMN) exudation between the 2 groups. Immunofluorescent findings revealed that ET-1 and ET-3 were seen along the glomerular capillary wall and partly in the mesangial area in all rats of the NTN group. The degree and positive rate of ET-1 and ET-3 staining were significantly higher in the NTN group than in the S-1452 group. These findings suggest that TxA2 may be an important mediator in the development of NTN, and that TxA2 receptor antagonist may be useful for the reduction of glomerular injury in this type of nephritis. In addition, local production of ET may contribute to the development of this nephritis.     

Molecular cloning of cDNA for nonhepatic mitochondrial arginase (arginase II) and comparison of its induction with nitric oxide synthase in a murine macrophage-like cell line

Arginase exists in two isoforms. Liver-type arginase (arginase I) is expressed almost exclusively in the liver and catalyzes the last step of urea synthesis, whereas the nonhepatic type (arginase II) is expressed in extrahepatic tissues. Arginase II has been proposed to play a role in down-regulation of nitric oxide synthesis. A cDNA for human arginase II was isolated. A polypeptide of 354 amino acid residues including the putative NH2-terminal presequence for mitochondrial import was predicted. It was 59% identical with arginase I. The arginase II precursor synthesized in vitro was imported into isolated mitochondria and proteolytically processed. mRNA for human arginase II was present in the kidney and other tissues, but was not detected in the liver. Arginase II mRNA was coinduced with nitric oxide synthase mRNA in murine macrophage-like RAW 264.7 cells by lipopolysaccharide. This induction was enhanced by dexamethasone and dibutyryl cAMP, and was prevented by interferon-gamma. Possible roles of arginase II in NO synthesis are discussed.     

Arginase II downregulates nitric oxide (NO) production and prevents NO-mediated apoptosis in murine macrophage-derived RAW 264.7 cells

Excess nitric oxide (NO) induces apoptosis of some cell types, including macrophages. As NO is synthesized by NO synthase (NOS) from arginine, a common substrate of arginase, these two enzymes compete for arginine. There are two known isoforms of arginase, types I and II. Using murine macrophage-like RAW 264.7 cells, we asked if the induction of arginase II would downregulate NO production and hence prevent apoptosis. When cells were exposed to lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), the inducible form of NOS (iNOS) was induced, production of NO was elevated, and apoptosis followed. When dexamethasone and cAMP were further added, both iNOS and arginase II were induced, NO production was much decreased, and apoptosis was prevented. When the cells were transfected with an arginase II expression plasmid and treated with LPS/IFN-gamma, some cells were rescued from apoptosis. An arginase I expression plasmid was also effective. On the other hand, transfection with the arginase II plasmid did not prevent apoptosis when a NO donor SNAP or a high concentration (12 mM) of arginine was added. These results indicate that arginase II prevents NO-dependent apoptosis of RAW 264.7 cells by depleting intracellular arginine and by decreasing NO production.     

Myofibroblasts and the progression of experimental glomerulonephritis

We have studied the sequential morphological changes that took place in the kidneys of 8 rats with nephrotoxic serum nephritis (NTN). Rats underwent kidney biopsies at different time intervals (days 7, 15, 30, 90 and 120). The tissues were processed for light microscopy as well as immunohistochemistry for inflammatory cellular infiltrate as well as for the components of the extracellular matrix (ECM) and myofibroblasts (cells expressing alpha-smooth muscle actin, alpha-SMA). Nephrotic rats developed severe proteinuria, impaired renal function as well as progressive renal scarring. However, the natural history of NTN was heterogeneous with some rats recovering (n = 5) and other progressing to end-stage renal failure (n = 3). The heterogeneous nature of the glomerulonephritis has established that those with a good outcome had a stabilisation, with some resolution, of the deposited ECM and of the scarring process. By contrast, rats with a poor outcome had a progressive increase in glomerular as well as interstitial ECM. Cells expressing alpha-SMA (myofibroblasts) were detected in the glomeruli as well as in the interstitium of nephritic rats. Changes in the expression of cells expressing alpha-SMA paralleled those of the components of the ECM in particular fibronectin. alpha-SMA immunostain was the best predictor of progression. Early glomerular alpha-SMA immunostain (days 7 and 30) was a strong predictor of the subsequent development of glomerulosclerosis and renal dysfunction. The predictive value of interstitial alpha-SMA immunostain on days 7 for subsequent tubulo-interstitial scarring and renal insufficiency was also strong and exceeded that of other histological or immunohistochemical parameters of scarring. This study establishes the natural history of experimental renal scarring and identifies a renal cell type, the myofibroblast, as a useful marker of progression. It also suggests a role for myofibroblasts in the progression of glomerulosclerosis and tubulo-interstitial fibrosis.     

Abrogation of glomerular injury in nephrotoxic nephritis by continuous infusion of interleukin-6

Interleukin-6 (IL-6) has been reported to have pro- and anti-inflammatory effects. It has also been shown to cause mesangial cell proliferation in vitro and has been suggested as a mediator of injury in proliferative nephritis. We have assessed the effects of continuous infusion of human recombinant (hr) IL-6, by osmotic minipump, on the degree of glomerular injury, and on glomerular and interstitial cell proliferation, in the accelerated autologous phase of nephrotoxic nephritis. Two groups of rats were pre-immunized with 1 mg of normal rabbit IgG in Freund's complete adjuvant. One week later, nephritis was induced by an intravenous injection of 1 ml of rabbit nephrotoxic serum. One day before the induction of nephritis, group 1 (N = 9) was subcutaneously implanted with osmotic minipumps filled with 50 micrograms (200 microliters) of IL-6 (equivalent to a dose of 6 micrograms/day), while in group 2 (N = 11) the minipumps were filled with 200 microliters of normal saline. In group 3 (N = 6) normal rats were infused with 50 micrograms of IL-6 alone. The rats were killed seven days after implantation of minipumps. The administered hrIL-6 was detectable in the circulation within the pathophysiological range, and induced a hepatic acute phase response, as assessed by alpha 2-macroglobulin levels. Continuous treatment with IL-6 resulted in a significant reduction in albuminuria (from 195 +/- 37 mg/20 hr to 60 +/- 15 mg/20 hr on day 1, and from 494 +/- 52 mg/20 hr to 238 +/- 30 mg/20 hr on day 7, P < 0.002) and in the prevalence of glomerular capillary thrombosis (from 19 +/- 3% to 5 +/- 1%, P < 0.002). There was also a reduction in macrophage infiltration (ED1 + ve cells from 524 +/- 34 to 466 +/- 14 per 50 glomeruli, P < 0.02) and activation (ED3 + ve cells from 106 +/- 13 to 42 +/- 5 per 50 glomeruli, P < 0.002). Immunohistology showed fewer interstitial Ia + ve cells (OX3 and OX4) in the IL-6 treated group. Similar results were obtained in a second set of experiments in which the IL-6 treatment was extended until day 14. Kidney sections taken from nephritic rats infused with IL-6 showed no increase in glomerular or interstitial cell proliferation when stained with antibodies to proliferating cell nuclear antigen. There was no difference in the deposition of rabbit IgG or rat IgG along the glomerular basement membrane (GBM), and the titer of rat anti-rabbit IgG was similar in the IL-6 and control treated rats. Infusion of IL-6 alone in normal rats had no functional or pathological effects. In conclusion, these results show that IL-6 has powerful anti-inflammatory effects in a rat model of anti-GBM nephritis, and does not induce mesangial cell proliferation in vivo.     

Ultrastructure, immunofluorescence, western blot, and PCR analysis of eight isolates of Encephalitozoon (Septata) intestinalis established in culture from sputum and urine samples and duodenal aspirates of five patients with AIDS

ONO-5046 is a potent, specific and intravenously active inhibitor of neutrophil elastase. To examine the role of elastase in glomerulonephritis, we tested the effects of ONO-5046 on nephrotoxic serum (NTS) nephritis in a rat model of the disease in humans. Rats were administered ONO-5046 or phosphate-buffered saline (PBS) intraperitoneally 24 hours prior to injection of NTS, and they were then given equal doses of ONO-5046 or PBS three hours and 1, 2, 3, 4, 5 and 6 days later. Compared with the control groups, ONO-5046 significantly reduced proteinuria and hematuria, and suppressed the formation of crescentic glomeruli in a dose-dependent manner. Our results suggest that neutrophil elastase participates in NTS nephritis by degrading glomerular basement membrane proteins, and that the elastase inhibitor, ONO-5046, suppresses crescentic formation and glomerular injury caused by elastase.     

Drug misuse in a special clinic patient population in Glasgow

Defibrination with ancrod in nephrotoxic nephritis in rabbits. In rabbits with nephrotoxic nephritis, defibrination with ancrod provided protection when administered during the autologous phase, after extensive glomerular fibrin deposition had occurred and crescents and renal failure were developing. When further glomerular fibrin deposition was prevented by defibrination, deposited fibrin was rapidly removed, indicating that glomerular fibrin-clearing mechanisms are retained in crescentic nephritis. Defibrination had no effect on the extent of glomerular C3 deposition or on the amount of proteinuria.     

L-arginine deficiency causes suppression of nonadrenergic noncholinergic nerve-mediated smooth muscle relaxation: role of L-citrulline recycling

Studies were performed on the internal anal sphincter (IAS) smooth muscle strips obtained from opossums (Didelphis virginiana). Isometric tension and L-arginine levels of the tissues were measured under basal conditions, in the presence of electrical field stimulation (EFS) and after treatment with different concentrations of arginase. For the nonadrenergic noncholinergic nerve stimulation, short trains (4 sec) as well as continuous EFS were used. During continuous EFS, after the initial IAS relaxation, the response began to fade within several min to approximately 80% recovery of the basal tone. We also examined the influence of L-arginine and L-citrulline on these responses. For some studies, the tissues were pretreated with L-glutamine (an inhibitor of L-citrulline uptake), L-glutamate or N(G)-hydroxy-L-arginine (an inhibitor of arginase). Interestingly, the basal levels of L-arginine were found to be significantly higher in the IAS (tonic smooth muscle) than in the rectal (phasic smooth muscle) smooth muscle. Arginase caused a concentration-dependent attenuation of the IAS relaxation caused by EFS. L-Citrulline and L-arginine were equipotent in reversing the attenuation. Both arginase (60 min pretreatment) and continuous EFS (tissues collected at the time of maximal recovery of the basal IAS tone after the initial relaxation) caused significant decreases in L-arginine levels. The decreases in the levels of L-arginine were restored by the exogenous administration of either L-arginine or L-citrulline. The restoration of L-arginine levels by L-citrulline but not by L-arginine was selectively blocked by L-glutamine. Furthermore, the IAS relaxation, attenuated by arginase was unaffected by L-glutamine but was restored by N-hydroxy-L-arginine pretreatment. The studies suggest that L-citrulline-L-arginine recycling may play a significant role in the maintenance of IAS relaxation in response to nonadrenergic noncholinergic nerve stimulation.     

Immunocytochemical detection of active DNA synthesis by in vivo labelling with anti-bromodeoxyuridine antibodies in glomeruli of rats with nephrotoxic serum nephritis

We studied DNA synthesis in rats with nephrotoxic serum nephritis (NTSN), a model of a glomerular disease, using in vivo labelling with 5-bromo-2'-deoxyuridine (BrdUrd). NTSN was induced by intravenous injection of subnephritogenic doses of rabbit anti-rat GBM antiserum into male Sprague-Dawley rats. Each rat received a single injection of the DNA precursor, 3H-thymidine analog (BrdUrd), ten min before the tissues were removed. For immunocytochemical detection of DNA synthesis, semithin sections were prepared at various intervals (4 h up to 84 days) after pulse labelling. Using a monoclonal anti-BrdUrd antibody, BrdUrd-incorporated DNA-synthesizing cells were noted in the proliferative zone of the gastric mucosa at all times. In NTSN, BrdUd incorporated DNA-synthesizing cells were detected in the glomeruli from 4h through 28 days after inoculation, with the peak occurring at days 2 to 4. On those days, up to half of the glomeruli showed BrdUrd-incorporated cells, with 8 cells per glomerulus as a maximum. From days 7 to 28, few glomerular cells incorporated BrdUrd, and none did so after day 28. The majority of the BrdUrd-incorporated cells were endothelial. These results suggest that active DNA synthesis by glomerular endothelial cells occurs during a short period of the heterologous phase in this model, and that the lack of mesangial cell proliferation might explain the self-limiting nature of this model. By using in vivo labelling with BrdUrd, we were also able to easily and accurately detect active DNA synthesis without consideration of the normal cell renewal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional gel electrophoresis of rat islet proteins. Interleukin 1 beta-induced changes in protein expression are reduced by L-arginine depletion and nicotinamide

Interleukin (IL)-1 beta-mediated damage to beta-cells in isolated islets of Langerhans depends upon de novo synthesis of proteins that have not been fully identified. Further, IL-1 beta-induced and tumor necrosis factor alpha-induced islet damage partly depends on the intracellular production of the nitric oxide (NO) radical. IL-1 beta has also been reported to induce the synthesis of cellular defense proteins, e.g., heme-oxygenase and heat shock proteins 70 and 90. Nicotinamide, while in itself inactive, inhibited IL-1 beta-induced NO production in a time- and dose-dependent manner. To enable the identification of IL-1 beta-induced proteins with possible protective and deleterious effects, we characterized the effects of IL-1 beta, nicotinamide, and NO synthesis inhibition by L-arginine depletion on rat islet protein expression detected by high-resolution two-dimensional gel electrophoresis. More than 1,600 proteins were reproducibly detected in control rat islets. Incubation with IL-1 beta-, nicotinamide-, or L-arginine-depleted control medium upregulated 29, 3, and 1 protein, respectively, and downregulated 4, 0, and 1 protein, respectively. Addition of nicotinamide and L-arginine depletion reduced the upregulation of 16 and 20 IL-1 beta-induced proteins, respectively. The identity of these proteins is under study. The demonstrated changes in protein expression caused by IL-1 beta +/- nicotinamide and L-arginine depletion may form the basis for identification of proteins with possible protective and deleterious roles in the initial beta-cell destruction in insulin-dependent diabetes mellitus.     

Expression of endothelial and inducible nitric oxide synthase in human glomerulonephritis

The presence of nitric oxide (NO) in the kidney has been implicated in the pathogenesis of human glomerulonephritis. However, the exact type of glomerular cells that express NO synthase (NOS) and the NOS isoform involved in the local production of NO has not been identified in the human diseased kidney. We examined the expression of three isoforms of NOS, inducible NOS (iNOS), endothelial NOS (eNOS) and brain NOS (bNOS) in the renal tissue of patients with IgA nephropathy (IgAN, N = 10), lupus nephritis (LN, N = 5), membranous nephropathy (MN, N = 5) and minimal change nephrotic syndrome (MCNS, N = 5). Sections were immunostained and the correlation between the expression of each NOS and the degree of glomerular injury in that section was also examined. Normal portions of surgically resected kidneys served as controls. eNOS was present in glomerular endothelial cells and endothelium of cortical vessels in the control and diseased kidneys. iNOS was localized in mesangial cells, glomerular epithelial cells and infiltrating cells in the diseased glomeruli, whereas immunostaining for iNOS was hardly detected in control kidneys. In addition, the expression pattern of eNOS in each glomerulus was the reverse of that of iNOS. In IgAN and LN, the extent of staining for eNOS correlated negatively with the degree of glomerular injury, while the extent of staining for iNOS correlated positively with the degree of glomerular injury in the same tissues. bNOS was not detected in normal or nephritic glomeruli. Our results indicate the presence of a NO pathway in human diseased kidney, and suggest that NO derived from eNOS and iNOS may be involved in the progression of renal diseases and that NO derived from each NOS may play an important role in different way in human inflamed glomeruli.     

Therapeutic effects of prostacyclin analog on crescentic glomerulonephritis of rat

Prostacyclin (PGI2) is known to have a relaxative action on vascular smooth muscle, an inhibitory action against platelet activation and neutrophil function. Previous studies showed the preventive effects of PGI2 on lupus nephritis and Thy-1 nephritis, although the mechanism has not been clarified. Glomerular endothelial expression of intercellular adhesion molecule-1 (ICAM-1) is up-regulated in experimental and human glomerular diseases, and is known to facilitate leukocyte infiltration into the glomeruli, which ultimately induces the various glomerular injuries. In the present study, we evaluated the therapeutic effects of PGI2 on a rat model for crescentic glomerulonephritis and investigated its putative mechanism in relation to ICAM-1-mediated leukocyte recruitment. Wistar-Kyoto (WKY) rats were injected with nephrotoxic serum and received continuous intraperitoneal infusion of PGI2. PGI2 dramatically decreased proteinuria (123.0 +/- 18.8 vs. 31.6 +/- 4.5), crescent formation and deposition of fibrinogen in the glomeruli, while the deposition of rabbit IgG, rat IgG and rat C3 along the capillary walls was not changed. Furthermore, intraglomerular expression of ICAM-1 and infiltration of macrophages were significantly suppressed by administration with PGI2. In contrast, influx of CD4 or CD8 positive cells was not altered. The present results suggest that PGI2 shows the preventive effects on experimental crescentic glomerulonephritis by inhibiting intraglomerular coagulation and ICAM-1-mediated macrophage-glomerular endothelial cell adhesive pathway.     

Deletion polymorphism in the angiotensin-converting enzyme gene as a thrombophilic risk factor after hip arthroplasty

Recent work has suggested a possible role for nitric oxide (NO) in the development of hepatic encephalopathy (HE). In this study, we examined the effect of ammonia and manganese, factors implicated in the pathogenesis of HE, on the transport of arginine (a precursor of NO) into primary cultures of astrocytes. Treatment with 5 mM ammonia for 1-4 days produced a maximal (53%) increase in L-arginine uptake at 3 days when compared to untreated cells. Kinetic analysis following 4-day treatment with 5 mM ammonia revealed an 82% increase in the Vmax and a 61% increase in the Km value. Similar analysis with 100 microM manganese showed a 101% increase in Vmax and a 131% increase in the Km value. These results suggest that both manganese and ammonia alter L-arginine uptake by modifying the transporter for arginine. A decrease of 32% in the non-saturable component of L-arginine transport was also observed following treatment with ammonia. When cultures were treated separately with 5 mM ammonia and 100 microM manganese for 2 days, the uptake of L-arginine increased by 41% and 57%, respectively. Combined exposure led to no further increase in uptake. Our results suggest that ammonia and manganese may contribute to the pathogenesis of HE by influencing arginine transport and thus possibly NO synthesis in astrocytes.     

The utilization of dipeptides containing L-arginine by chicken macrophages

L-Arginine is the precursor of NO, a cytotoxic agent of macrophages. Studies were carried out to determine whether dipeptides containing arginine can be utilized by lipopolysaccharide (LPS)-activated avian macrophages for NO production. A chicken macrophage cell line, the HD11 cell, was used in all experiments. Peptidase activities were observed in fetal bovine serum (FBS) and macrophage serum free medium (Mac-SFM). Therefore, the utilization of dipeptides by macrophages was examined using Dulbecco's modified Eagle medium (D-MEM), a chemically defined medium, in short-term culture without FBS. Nitrite accumulation in the culture medium was used as the indicator of NO production. At concentrations of 0.15 mM in the culture media, L-leucinyl-L-arginine was 89% as effective as L-arginine in providing substrate for NO production. L-Argininyl-L-leucine was 38% as effective as L-arginine. The effectiveness increased to 93 and 58%, respectively, when the concentrations of dipeptides and arginine were 1.0 mM. Both values were slightly higher in a second experiment (97 and 70%, respectively). L-Lysine (10 mM) inhibited nitrite formation from all three sources of L-arginine. In studies of initial rates of transport by HD11 cells in Hanks Balanced Salts solution (HBSS), both L-argininyl-L-leucine and L-leucinyl-L-arginine inhibited arginine uptake. As lysine and arginine share a common transporter for cationic amino acids and are known to compete for transport, these studies suggest that the peptides were hydrolyzed extracellularly, yielding arginine that was transported into the cell where it served as a substrate for NO synthesis.     

Modulation of glomerular hypertension defines susceptibility to progressive glomerular injury

The fawn-hooded rat constitutes a spontaneous model for chronic renal failure with early systemic and glomerular hypertension, proteinuria (UpV) and high susceptibility to development of focal and segmental glomerular sclerosis (FGS). It has been argued that uninephrectomy (UNX) accelerates the development of glomerular injury by aggravation of glomerular hypertension and by an independent effect to promote glomerular enlargement. The present study was performed to further delineate the importance of these parameters for the development of FGS. At the age of eight weeks male rats were UNX and randomly assigned to either control (CON), enalapril (ENA) or Nw-nitro L-arginine methyl ester (NAME) treatment. In all groups glomerular hemodynamic studies were performed four weeks post-UNX. Systemic blood pressure and UpV were monitored for 4 to 12 weeks post-UNX. Kidneys were then prepared for morphologic study. ENA treatment achieved control of both systemic and glomerular hypertension, maintenance of glomerular hyperfiltration and hyperperfusion, increased ultrafiltration coefficient(Kf), and long-term protection against UpV and FGS. NAME rats showed aggravation of both systemic and glomerular hypertension, decreased renal perfusion and filtration with reduced Kf, and high filtration fraction. The incidence of FGS in NAME and CON groups was similar at 8 and 12 weeks post-UNX, respectively. Glomerular enlargement was present in CON and ENA rats, but did not correlate with injury, while glomerular tuft size was lowest in NAME rats, which displayed prominent glomerular injury. Systemic blood pressure correlated strongly with glomerular capillary pressure. We conclude that systemic and glomerular hypertension govern the development of UpV and FGS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved survival and amelioration of nephrotoxic nephritis in intercellular adhesion molecule-1 knockout mice

Intercellular adhesion molecule-1 (ICAM-1) expression is upregulated in nephrotoxic nephritis, a model of human rapidly progressive glomerulonephritis. To evaluate the pathogenetic relevance of ICAM-1 in this model, nephrotoxic nephritis was induced in ICAM-1 knockout mice and genetic controls. Mice were preimmunized with rabbit IgG in complete Freund's adjuvant. Seven days later they received rabbit anti-mouse glomerular basement membrane IgG. The early humoral immune responses (levels of circulating mouse anti-rabbit IgG, glomerular deposition of rabbit and mouse IgG and mouse C3c) were not altered in ICAM-1 knockout mice. During 28 d of follow-up, 3 of 19 control nephritic mice and 0 of 16 ICAM-1 knockout mice died. Proteinuria was high in nephritic control mice (means 10 to 12 mg/24 h at all time points investigated) and significantly reduced in nephritic ICAM-1 knockout mice (means <4.4 mg). Mean serum creatinine rose from 29 micromol/L at day -7 to 48 micromol/L (day 28) in nephritic control mice. This increase in serum creatinine was significantly lower in ICAM-1 knockout mice: 27 (day -7) and 36 micromol/L (day 28). Histologic analysis at day 28 revealed that ICAM-1 deficiency in nephrotoxic nephritis mice led to significantly reduced glomerular crescent formation (2+/-3% in ICAM-1 knockout mice versus 13+/-8% in nephritic controls) and tubulointerstitial injury (score 0.4+/-0.4 versus 2.0+/-1.1). By immunohistochemistry, ICAM-1 deficiency in nephritic mice led to significantly reduced (peri-)glomerular and/or interstitial macrophage influx, alpha-smooth muscle actin expression, and type IV collagen accumulation. These data indicate that ICAM-1 is a central mediator of glomerular and tubulointerstitial injury in murine nephrotoxic nephritis.     

Pediatric antifungal therapy

We have recently reported the synthesis of urea from ammonia, glutamine and arginine in enterocytes of postweaning pigs. The present study was conducted to determine the compartmentation and kinetics of urea cycle enzymes in these cells. Carbamoyl phosphate synthase I (CPS I) and ornithine carbamoyltransferase (OCT) were located exclusively in mitochondria, whereas argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) were found in the cytosol. Arginase isozymes were present in both the cytosol and mitochondria of enterocytes, and differed in their sensitivity to heat inactivation. Except for OCT, Vmax values of urea cycle enzymes were much lower in enterocytes than in the liver of pigs, and vice versa for their Km values. Because of a low rate of ureagenesis in enterocytes compared with the liver, intestinal urea cycle enzymes may function primarily to synthesize citrulline. The co-localization of CPS I and OCT and a high activity of OCT in enterocyte mitochondria favors the intestinal synthesis of citrulline from ammonia, HCO3- and ornithine. Low activities of cytosolic ASS and ASL minimize the conversion of citrulline into arginine and therefore, the recycling of citrulline into ornithine via arginase in postweaning-pig enterocytes. These kinetic properties of intestinal urea cycle enzymes maximize the net synthesis of citrulline from glutamine and explain the release of large amounts of citrulline by the pig small intestine. The two compartmentally separated arginase isozymes in enterocytes may play an important role in regulating the intestinal metabolism of proline, nitric oxide and polyamines.     

Dietary L-arginine in renal disease

The amino acid L-arginine is a substrate for at least three products involved extensively in tissue injury and fibrosis. L-arginine is metabolized to L-proline, a major constituent of the collagen that makes up fibrotic extracellular matrix. L-arginine is a precursor for polyamines, which are required for proliferative responses characteristic of many renal disease. L-arginine is also the sole substrate for generation of nitric oxide (NO) which, produced in large quantities by macrophages, has been implicated in tissue injury. On the other hand, NO produced in small quantities by endothelium is a critical vasodilator. Given the importance of elevated intraglomerular pressure in renal injury, it is perhaps not surprising that dietary L-arginine supplementation increase NO generation and is beneficial in reducing intraglomerular pressure and subsequent disease. Other data, based on the therapeutic effects of low protein diets, have suggested that L-arginine restriction limits NO-mediated glomerular injury and greatly reduces matrix accumulation, consistent with the idea that limitation of substrate effectively diminishes injurious NO levels, polyamine synthesis, and collagen production.