A new role for complement in experimental membranous nephropathy in rats.

The only established role for complement in mediating immunologic renal disease involves elaboration of leukochemotactic factors and neutrophil-dependent glomerular injury. In the passive Heymann nephritis (PHN) model of experimental membranous nephropathy, rats injected with sheep antibody to rat proximal tubular brush border antigen (Fx1A) form subepithelial deposits of sheep IgG and rat complement (C3), and develop heavy proteinuria after 5 d without glomerular inflammatory changes. To study the role of complement in mediating proteinuria in PHN, 16 rats were treated daily with cobra venom factor from before antibody injection to maintain C3 levels at < 10% of pretreatment values and compared to 16 untreated controls. Proteinuria at 5 d was abolished in C3-depleted rats (4 +/- 1, controls 70 +/- 15 mg/d, P < 0.001), although renal deposition of 125I-labeled antibody ws the same in both groups (188 +/- 35 vs. 191 +/- 22 microgram IgG/2 kidneys, P > 0.5). Nephritogenic doses of both the noncomplement-fixing F(ab')2 portion and the gamma 2 subclass of anti-Fx1A IgG produced subepithelial deposits of immunoglobulin without C3, but proteinuria did not occur despite glomerular deposition of up to 70 microgram/2 kidneys of gamma 2. However, glomerular deposition of as little as 60 microgram of gamma 1 produced C3 fixation in vivo and heavy proteinuria. No neutrophil exudate could be detected histologically in PHN from the time of antibody injection through development of proteinuria. Proteinuria in five PHN rats depleted of neutrophils to < 200/mm3 with antineutrophil serum was not reduced compared to six controls with normal neutrophil counts (34 +/- 9.6 vs. 25 +/- 10.4 mg/d, P > 0.5). These results demonstrate that proteinuria in the PHN model of membranous nephropathy is complement-dependent and strongly suggest a neutrophil-independent mechanism. Thus a new role for the complement system in mediating immunologic glomerular injury is identified.     

Effect of alterations in glomerular charge on deposition of cationic and anionic antibodies to fixed glomerular antigens in the rat

Reduction of the negative charge of the glomerular capillary wall alters its charge- and size-selective properties. To investigate the effect of alteration in glomerular charge properties on antibody localization, we prepared cationic and anionic fractions of antibodies to subepithelial and glomerular basement membrane (GBM) antigens, and compared their deposition in normal rats and rats treated with protamine sulfate or aminonucleoside of puromycin to reduce capillary wall charge. IgG antibodies were eluted from kidneys of rats with active Heymann's nephritis (AICN), passive Heymann's nephritis (PHN), or anti-GBM nephritis (NTN), separated into cationic and anionic fractions, and radiolabeled with iodine 125 or iodine 131. Relative antibody content of each fraction was determined by incubation with an excess of glomerular antigen. Varying amounts of cationic and anionic IgG eluted from kidneys of rats with AICN or PHN were injected into 24 normal or protamine sulfate-treated rats. Glomerular binding of all antibodies was highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 4 hours was 1.08 +/- 0.07 for AICN eluate and 0.37 +/- 0.04 for PHN eluate. The ratios were not significantly different in animals pretreated with protamine sulfate (1.15 +/- 0.06 and 0.44 +/- 0.06, respectively; P greater than 0.05). Varying amounts of cationic and anionic IgG eluted from kidneys of rats with NTN were injected into 10 normal rats and four rats treated with aminonucleoside of puromycin. Glomerular binding of antibody was again highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 1 hour was 1.03 +/- 0.06, and was not significantly altered in rats treated with aminonucleoside of puromycin (1.05 +/- 0.03, P greater than 0.5). Proteinuria in PHN rats was also unaffected by treatment with protamine sulfate for 5 days (controls: 68 +/- 21 mg/day; protamine sulfate-treated: 65 +/- 14 mg/day; n = 25, P greater than 0.08). These results demonstrate that treatment to reduce glomerular polyanion does not significantly alter the ratio of cationic to anionic antibodies to fixed glomerular antigens that deposit in the glomerulus, or reduce proteinuria caused by deposition of antibody to a fixed subepithelial antigen.     

Experimental glomerulonephritis in the isolated perfused rat kidney.

The development of immune deposits on the subepithelial surface of the glomerular capillary wall was studied in isolated rat kidneys perfused at controlled perfusion pressure, pH, temperature, and flow rates with recirculating oxygenated perfusate containing bovine serum albumin (BSA) in buffer and sheep antibody to rat proximal tubular epithelial cell brush border antigen (Fx1A). Control kidney were perfused with equal concentrations of non-antibody immunoglobulin (Ig)G. Renal function was monitored by measuring inulin clearance, sodium reabsorption, and urine flow as well as BSA excretion and fractional clearance. Perfused kidneys were studied by light, immunofluorescence, and electron microscopy. All kidneys perfused with anti-Fx1A developed diffuse, finely granular deposits of IgG along the glomerular capillary wall by immunofluorescence. Electron microscopy revealed these deposits to be localized exclusively in the subepithelial space and slit pores. Similar deposits were produced in a nonrecirculating perfusion system, thereby excluding the formation of immune complexes in the perfusate caused by renal release of tubular antigen. Control kidneys perfused with nonantibody IgG did not develop glomerular immune deposits. Renal function and BSA excretion were the same in experimental and control kidneys. Glomerular deposits in antibody perfused kidneys were indistinguishable from deposits in rats injected with anti-Fx1A or immunized with Fx1A to produce autologous immune complex nephropathy. These studies demonstrate that subepithelial immune deposits can be produced in the isolated rat kidney by perfusion with specific antibody to Fx1A in the absence of circulating immune complexes. In this model deposits result from in situ complex formation rather than circulating immune complex deposition.     

Leukotriene D4 is a mediator of proteinuria and glomerular hemodynamic abnormalities in passive Heymann nephritis.

We assessed the role of leukotrienes (LTs) in Munich-Wistar rats with passive Heymann nephritis (PHN), an animal model of human membranous nephropathy. 10 d after injection of anti-Fx1A antibody, urinary protein excretion rate (Upr) in PHN was significantly higher than that of control. Micropuncture studies demonstrated reduced single nephron plasma flow and glomerular filtration rates, increased transcapillary hydraulic pressure difference, pre- and postglomerular resistances, and decreased ultrafiltration coefficient in PHN rats. Glomerular LTB4 generation from PHN rats was increased. Administration of the 5-LO activating protein inhibitor MK886 for 10 d markedly blunted proteinuria and normalized glomerular hemodynamic abnormalities in PHN rats. An LTD4 receptor antagonist SK&F 104353 led to an immediate reduction in Upr and to reversal of glomerular hemodynamic impairment. Ia(+) cells/glomerulus were increased in PHN rats. In x-irradiated PHN rats, which developed glomerular macrophage depletion, augmented glomerular LT synthesis was abolished. Thus, in the autologous phase of PHN, LTD4 mediates glomerular hemodynamic abnormalities and a hemodynamic component of the accompanying proteinuria. The synthesis of LTD4 likely occurs directly from macrophages or from macrophage-derived LTA4, through LTC4 synthase in glomerular cells.     

Inhibition of cyclooxygenases reduces complement-induced glomerular epithelial cell injury and proteinuria in passive Heymann nephritis

In the passive Heymann nephritis (PHN) model of rat membranous nephropathy, complement induces glomerular epithelial cell injury and proteinuria, which is partially mediated by eicosanoids. Glomerular cyclooxygenase (COX)-1 and -2 are up-regulated in PHN and contribute to prostanoid generation. In the current study, we address the role of COX isoforms in proteinuria, using the nonselective COX inhibitor indomethacin and the COX-2-selective inhibitor 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone (DFU). Four groups of rats with PHN were treated twice daily, from day 7 through 14 with vehicle, 1 mg/kg DFU, 10 mg/kg DFU, or 2 mg/kg indomethacin. Vehicle-treated rats with PHN showed significant proteinuria on day 14 (163 +/- 15 mg/d, n = 19), compared with normal rats (10 +/- 4 mg/d, n = 3, p < 0.001). Treatment with DFU (1 or 10 mg/kg) reduced proteinuria significantly (by ~33%), compared with vehicle, but to a lesser extent than indomethacin (56% reduction). Glomerular eicosanoid generation was reduced significantly in the DFU and indomethacin groups, compared with vehicle. There were no significant differences among vehicle- or DFU-treated groups in [(3)H]inulin clearance, or in glomerular expression of COX-1 and -2. DFU did not affect the autologous immune response. In cultured rat glomerular epithelial cells, COX inhibition reduced complement-induced cytotoxicity, and this reduction was reversed by the thromboxane A(2) analog 9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin F(2alpha) (U46619). Thus, in experimental membranous nephropathy, selective inhibition of COX-2 reduces proteinuria, without adversely affecting renal function. However, inhibition of both COX-1 and -2 is required to achieve a maximum cytoprotective and antiproteinuric effect.     

Changes in glomerular hemodynamic response to angiotensin II after subacute renal denervation in rats.

We examined the changes in glomerular hemodynamics produced by angiotensin II (AII) in both normal Munich-Wistar rats and rats which were unilaterally renal denervated (measured kidney) 4-6 d prior to the measurement periods. Measurements of glomerular dynamics were performed in a control period after plasma volume expansion and during infusion of 11 ng X 100 g body wt-1 X min-1 of AII. The glomerular hydrostatic pressure gradient increased from 38 +/- 1 to 49 +/- 1 mmHg in denervated rats compared with a lesser response in controls (from 39 +/- 1 to 45 +/- 1 mmHg, P less than 0.05). Single nephron plasma flow decreased from 213 +/- 17 to 87 +/- 4 nl X min-1 X g kidney wt (KW)-1 in denervated kidneys versus a more modest decrease in control kidneys (from 161 +/- 9 to 102 +/- 5 nl X min X gKW-1). These changes were due to a greater increase in both afferent and efferent arteriolar resistance after AII infusion in denervated compared with control kidneys. Glomerular AII receptor maximum binding was 1,196 +/- 267 fmol/mg protein in denervated kidneys compared with 612 +/- 89 fmol/mg protein (P less than 0.01) in controls with no change in receptor affinity. We conclude the subacute unilateral renal denervation results in renal vasodilation, denervation magnifies the vasoconstrictive effect of AII infusion on glomerular hemodynamics, and the observed increased response to AII after denervation is associated with increases in glomerular AII receptors.     

The role of adenosine in glycine-induced glomerular hyperfiltration in rats.

Ingestion of a high-protein diet or infusion of amino acids induces glomerular hyperfiltration and hyperemia. We have investigated the role of endogenous adenosine in glycine-induced hyperfiltration. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured in conscious chronically instrumented rats. Glycine (3.7 mg/min, i.v.; n = 6) significantly increased GFR and ERPF from 0.92 +/- 0.07 to 1.13 +/- 0.08 and 3.28 +/- 0.24 to 3.69 +/- 0.19 ml/min.100 g, respectively. In the presence of adenosine deaminase (ADA, 2 U/kg.min, n = 6), glycine-induced glomerular hyperfiltration and hyperemia were blunted. The small changes in GFR (from 0.86 +/- 0.06 to 0.90 +/- 0.10 ml/min.100 g) and ERPF (from 3.60 +/- 0.57 to 3.83 +/- 0.53 ml/min x 100 g) were not statistically significant. Erythro-9-(2-hydroxy-3-nonyl) adenosine hydrochloride (100 micrograms/kg.min, n = 6), an ADA inhibitor, reversed the effect of ADA. Injection of 8-phenyltheophylline (10 mg/kg, n = 6), an adenosine A1 receptor antagonist that alone did not affect GFR, abolished the glycine-induced glomerular hyperfiltration (GFR from 1.02 +/- 0.08 to 0.93 +/- 0.08 ml/min.100 g, P > .05). 8-phenyltheophylline, which itself decreased ERPF, also significantly decreased the ERPF response to glycine (3.47 +/- 0.26 to 2.78 +/- 0.14 ml/min x 100 g). Thus, endogenous adenosine, acting at adenosine A1 receptors, plays an important role in the glomerular hyperfiltration and hyperemia induced by glycine.     

Experimental Membranous Glomerulonephritis in Rats: QUANTITATIVE STUDIES OF GLOMERULAR IMMUNE DEPOSIT FORMATION IN ISOLATED GLOMERULI AND WHOLE ANIMALS

Quantitation of immune deposit formation in glomeruli and correlation with immunohistologic and functional changes has been accomplished only in models of anti-glomerular basement membrane antibody-induced nephritis, or indirectly in immune complex disease by measuring radiolabeled antigen deposition. The kinetics of subepithelial immune deposit formation and the relationship between the quantity of antibody deposited and proteinuria are defined here for the first time in an established model of membranous immune complex nephritis (passive Heymann nephritis) induced by a single intravenous injection of ¹²⁵I-labeled sheep immunoglobulin (Ig)G antibody to rat tubular brush border antigen (Fx1A). Measurement of antibody deposition in glomeruli (GAb) isolated from rats injected with 10 mg of anti-Fx1A demonstrated a mean of 12 μg GAb in 4 h, which increased linearly to 48 μg in 5 d. GAb represented only 20 and 44% of total kidney antibody binding at these times. Proteinuria occurred only after 4-5 d of antibody deposition in rats with total kidney antibody binding exceeding ~200 μg/2 kidneys. Steroid treatment and vasoactive amine blockade did not significantly alter the quantity or localization of immune deposits. It was also demonstrated that isolated rat glomeruli specifically bound nephritogenic quantities of anti-Fx1A in vitro within hours. Analysis of the quantitative aspects of glomerular antibody deposition in vivo and glomerular antibody binding in vitro provides additional evidence that subepithelial immune deposits in passive Heymann nephritis may form in situ by reaction of free antibody with antigenic constitutents of the normal rat glomerulus. The observed kinetics of deposit formation differ markedly from those in anti-glomerular basement membrane disease and suggest a role for factors in addition to antigen-antibody interaction in determining this unique pattern of glomerular immune deposit formation.     

Glomerular actions of endothelin in vivo.

In Munich-Wistar rats, a micropipette was inserted into a first-order branch of the left main renal artery and continuously infused with human/porcine endothelin (0.4 ng/min). Micropuncture measurements revealed substantial differences within the cortical microcirculation of the same left kidney: SNGFR was some 35% lower in glomeruli exposed to endothelin compared with non-endothelin-perfused glomeruli (P less than 0.005). Similarly, glomerular plasma flow rate was some 38% lower in the endothelin-exposed glomeruli (P less than 0.001). The hypoperfusion and hypofiltration in the endothelin-exposed glomeruli reflected an increase in resistances in the afferent and efferent arterioles. There was no difference in the value of the glomerular capillary ultrafiltration coefficient between the two populations of glomeruli. We also studied kidneys that underwent 25 min of renal artery clamping 48 h before study. Antiendothelin antibody infused into one of the branches of the main renal artery ameliorated the vasoconstriction characteristic of postischemic nephrons: within the cortical microcirculation, the SNGFR in glomeruli exposed to antiendothelin antibody was 27.0 +/- 3.1 nl/min as compared with 17.4 +/- 1.7 measured in glomeruli not perfused with the antibody (P less than 0.001). Similarly, glomerular plasma flow rate was higher in the glomeruli exposed to antiendothelin antibody (128.7 +/- 14.4 nl/min vs. 66.6 +/- 5.6, P less than 0.005). Resistances in both the afferent and efferent arterioles were substantially lower in the antibody-exposed glomeruli. It is, therefore, suggested that endothelin, presumably released from damaged endothelium, may play an important intermediary role in the hypoperfusion and hypofiltration observed in postischemic kidneys.     

Quantitative studies of in situ immune complex glomerulonephritis in the rat induced by planted, cationized antigen

Cationized human IgG can bind to the rat glomerular basement membrane (GBM), act as planted antigen, and induce in situ immune complex formation accompanied by severe glomerulonephritis. Perfusion of highly cationized human IgG (isoelectric point {more than} 9.5) via the left renal artery resulted in preferential localization within the perfused kidney (up to 56 percent of dose injected); after intravenous administration, only 4 percent was bound to the kidneys. The planted antigen was localized along the glomerular capillary walls and was accessible for antibody administered intravenously 1 h after perfusion, when virtually no antigen remained in the circulation. Persistence of cationized human IgG in the perfused kidney was markedly prolonged when complexed with antibody; one-half the cationized human IgG was still present after 12 d. There was a difference in the disappearance rates of antigen and antibody, as cationized human IgG was removed faster from the kidney than the antibody, the binding of which remained almost unchanged during the first week. Renal perfusion of a minimum of 20 μg of cationized human IgG, followed by intravenous injection of antibody, regularly induced severe glomerulonephritis with a proteinuria of at least 100 mg/24 h. The degree and the persistence of proteinuria induced depended on the dose of cationized human IgG perfused. Experiments using radiolabeled antigen and antibody showed that after renal perfusion of 20 μg cationized human IgG, 11.1 μg was kidney bound at the time of antibody injection. At the onset of proteinuria, 4.0 μg of antigen and 31.9 μg of anti-human IgG antibody were present in the perfused kidney. Immunofluorescence revealed immune deposits consisting of cationized human IgG and rabbit IgG (anti-human IgG) along the GBM. The staining pattern was linear (confluent) during the first 2 d and became granular during the course of the disease. Electronmicroscopically, a prominent finding was the accumulation of dense deposits, mainly in the subepithelial space and beneath the slit pores.     

Role of the terminal complement pathway in experimental membranous nephropathy in the rabbit.

Our recent observations of a complement-mediated, cell-independent mechanism of altered glomerular permeability in rat membranous nephropathy suggested a possible role for the terminal complement pathway in the mediation of proteinuria in certain forms of glomerular disease. To directly determine whether the membranolytic terminal complement components (C5b-C9) are involved in glomerular injury, we studied the development of proteinuria in normal and C6-deficient (C6D) rabbits, in both of which a membranous nephropathy-like lesion develops early in the course of immunization with cationized bovine serum albumin (cBSA) (pI 8.9-9.2). C6 hemolytic activity of C6D was 0.01% that of control rabbits. After 1 wk of daily intravenous injections of cBSA, proteinuria developed in 71% of controls (median 154, range 1-3,010 mg/24 h, n = 24), whereas none of C6D were proteinuric (median 6, range 2-12 mg/24 h, n = 12, P less than 0.01). After 1 wk of cBSA, both groups had qualitatively identical glomerular deposits of BSA, rabbit IgG, and C3 on immunofluorescence microscopy, predominantly subepithelial electron-dense deposits on electron microscopy, and minimal glomerular inflammatory cell infiltration of glomeruli. Glomeruli were isolated from individual animals after 1 wk of cBSA and deposits of rabbit IgG antibody were quantitated by a standardized in vitro assay using anti-rabbit IgG-125I. Rabbit IgG deposits were found to be similar in control (29.8 +/- 13.2, range 12.7-48.6 micrograms anti-IgG/2,000 glomeruli, n = 6) and C6D rabbits (32.6 +/- 13.8, range 16.8-48.8 micrograms anti-IgG/2,000 glomeruli, n = 5, P greater than 0.05). After 2 wk, coincident with a prominent influx of mononuclear cells and neutrophils, proteinuria developed in C6D rabbits. These results document, for the first time, a requirement for a terminal complement component in the development of immunologic glomerular injury. Since the only known action of C6 is in the assembly of the membrane attack complex, these observations suggest that the membranolytic properties of complement may contribute to glomerular damage.     

Effects of cyclosporin A on glomerular barrier function in the nephrotic syndrome.

1. To elucidate the mechanisms by which cyclosporin A diminishes proteinuria, we studied 20 patients with severe nephrotic syndrome. Biopsy-established pathologies included minimal change disease (n = 5), membranous glomerulopathy (n = 6), membranoproliferative glomerulonephritis (n = 5) and focal segmental glomerulosclerosis (n = 4). Before, at the end of a 90 day course of cyclosporin A, and finally 1 month after stopping cyclosporin A we determined 24 h protein excretion. Measurements of glomerular filtration rate, effective renal plasma flow, fractional clearance rates of albumin and immunoglobulins with different charges and the transglomerular sieving of uncharged dextrans of broad size distribution were used to study the effects of cyclosporin A on renal perfusion and the glomerular filtration barrier. The findings were analysed with a theoretical model of solute transport. 2. Among the different forms of glomerulopathy the response to low-dose cyclosporin A (trough levels 32.0-36.9 ng/ml) varied markedly. In minimal change disease, proteinuria decreased from 9.5 +/- 3.1 to 1.3 +/- 0.2 g/24 h (mean +/- SEM, P less than 0.01). This response was due to restoration of the charge selectivity of the glomerular barrier. The depressed value of the glomerular permeability coefficient also returned to normal. Glomerular filtration rate, effective renal plasma flow and renal vascular resistance did not change. Proteinuria returned after stopping cyclosporin A, although it did not reach pretreatment levels. In membranous glomerulopathy, proteinuria fell from 9.9 +/- 1.5 to 1.8 +/- 0.3 g/24 h (P less than 0.01). Changes in protein excretion and dextran sieving were compatible with an increase in glomerular permselectivity and a decrease in filtrate flow through the 'shunt' pathway. Glomerular filtration rate was maintained, although effective renal plasma flow fell significantly. Proteinuria relapsed after stopping cyclosporin A. In membranoproliferative glomerulonephritis and focal segmental glomerulosclerosis proteinuria did not respond to cyclosporin A, although cyclosporin A exerted important haemodynamic effects. 3. In minimal change disease and membranous glomerulopathy cyclosporin A exerts its beneficial effects on proteinuria through changes in the properties of the glomerular barrier, resulting in increased charge and size selectivity, respectively.     

Renin-angiotensin system inhibition reduces glycine-induced glomerular hyperfiltration in conscious rats.

It has been reported that high protein intake or amino acid infusion-induced glomerular hyperfiltration are accompanied by an elevation of plasma renin activity and renal renin mRNA. We therefore investigated the effect of inhibition of the renin-angiotensin system by SK&F 108566, a novel, nonpeptide angiotensin II (AII) receptor antagonist, or by enalapril, an angiotensin converting enzyme inhibitor, on glycine-induced hyperfiltration. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured by inulin and p-aminohippurate clearances in conscious chronically instrumented rats. Glycine infusion (3.7 mg/min i.v.; n = 8) significantly increased GFR by 27% (from 1.09 +/- 0.53 to 1.38 +/- 0.08 ml/min.100 g), ERPF by 22% (2.96 +/- 0.30 to 3.61 +/- 0.32 ml/min.100 g) and significantly decreased effective renal vascular resistance by 22% [from 25.4 +/- 2.9 to 20.8 +/- 2.5 mm Hg/(ml/min.100 g)]. SK&F 108566 (30 micrograms/kg.min) or enalapril (1 mg/kg), at doses which inhibited the pressor effects of AII or AI, respectively, but had no significant influence on base-line GFR and ERPF, significantly attenuated the glycine-induced glomerular hyperfiltration and hyperemia. In the presence of SK&F 108566 or enalapril, glycine resulted in only small, statistically insignificant changes in GFR (from 1.07 +/- 0.03 to 1.10 +/- 0.04 and from 1.19 +/- 0.03 to 1.21 +/- 0.08 ml/min.100 g, respectively), ERPF (from 3.27 +/- 0.21 to 3.53 +/- 0.26 and from 3.57 +/- 0.11 to 3.41 +/- 0.38 ml/min.100 g, respectively) and effective renal vascular resistance [from 21.2 +/- 1.9 to 19.2 +/- 1.6 and from 18.4 +/- 0.9 to 20.2 +/- 2.2 mm Hg/(ml/min.100 g], respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Basic fibroblast growth factor augments podocyte injury and induces glomerulosclerosis in rats with experimental membranous nephropathy.

Podocyte injury is believed to contribute to glomerulosclerosis in membranous nephropathy. To identify the factors involved, we investigated the effects of basic fibroblast growth factor (bFGF), a cytokine produced by podocytes, on rats with membranous nephropathy (passive Heymann nephritis [PHN]). All rats received a daily i.v. bolus of 10 microg bFGF or vehicle from days 3-8 after PHN induction. In proteinuric PHN rats on day 8, bFGF injections further increased proteinuria. Podocytes of bFGF-injected PHN rats showed dramatic increases in mitoses, pseudocyst formation, foot process retraction, focal detachment from the glomerular basement membrane, and desmin expression. bFGF injections in PHN rats did not alter antibody or complement deposition or glomerular leukocyte influx. bFGF-injected PHN rats developed increased glomerulosclerosis when compared with control PHN rats. Also, bFGF induced proteinuria and podocyte damage in rats injected with 10% of the regular PHN-serum dose. None of these changes occurred in bFGF-injected normal rats, complement-depleted PHN rats or rats injected with 5% of the regular PHN serum dose. These divergent bFGF effects were explained in part by upregulated glomerular bFGF receptor expression, induced by PHN serum. Thus, bFGF can augment podocyte damage, resulting in increased glomerular protein permeability and accelerated glomerulosclerosis. This bFGF action is confined to previously injured podocytes. Release of bFGF from glomerular sources (including podocytes themselves) during injury may represent an important mechanism by which podocyte damage is enhanced or becomes self sustained.     

Involvement of endothelium-derived relaxing factor in the pressure control of renin secretion from isolated perfused kidney.

Using isolated rat kidneys perfused at controlled pressure, we examined a potential role of endothelium-derived relaxing factor (EDRF) in the pressure control of renin secretion. We found that stimulation of EDRF release by acetylcholine (1 mumol/liter) increased mean perfusate flow rates from 15.0 +/- 0.5 to 18.0 +/- 0.5 ml/min per g and average renin secretion rates from 3.5 +/- 0.5 to 16.0 +/- 2.0 ng angiotensin I/h per min per g at a perfusion pressure of 100 mmHg (mean +/- SEM, n = 6). Those effects of acetylcholine were significantly reduced during inhibition of EDRF formation with NG-nitro-L-arginine (100 mumol/liter), but they were not affected with the cyclooxygenase inhibitor indomethacin (10 mumol/liter). Lowering of the perfusion pressure from 100 mmHg to 40 mmHg resulted in an increase of average renin secretion rates from 3.5 +/- 0.5 to 79 +/- 12 ng AngI/h per min per g under control conditions (n = 8), and to 171 +/- 20 ng AngI/h per min per g in the presence of 10 mumol/liter acetylcholine (n = 3). The rise of renin secretion in response to a reduction of the renal artery pressure was markedly attenuated with inhibitors of EDRF formation such as NG-nitro-L-arginine (100 mumol/liter) and related compounds. During inhibition of EDRF formation, addition of sodium nitroprusside (10 mumol/liter) increased mean perfusate flow rates from 12.0 +/- 0.5 to 23.0 +/- 2.0 ml/min per g and average renin secretion rates from 2.0 +/- 0.5 to 18.0 +/- 1.5 ng AngI/h per min per g at 100 mmHg (n = 5). Lowering of the perfusion pressure from 100 mmHg to 40 mmHg under those conditions increased average renin secretion rates to 220 +/- 14 ng AngI/h per min per g (n = 5). Taken together, our findings suggest that EDRF and related activators of soluble guanylate cyclase stimulate renin secretion from isolated kidneys, predominantly at lower perfusion pressure. Moreover, pressure control of renin secretion appears to require the tonical stimulation by intrarenal EDRF.     

Reactive oxygen species during ischemia-reflow injury in isolated perfused rat liver.

The hypothesis that intracellular generation of reactive oxygen species in hepatocytes or reticuloendothelial cells may cause ischemia-reperfusion injury was tested in isolated perfused livers of male Fischer rats. GSSG was measured in perfusate, bile, and tissue as a sensitive index of oxidative stress. After a preperfusion phase of 30 min, the perfusion was stopped (global ischemia) for various times (30, 120 min) and the liver was reperfused for another 60 min. The bile flow (1.48 +/- 0.17 microliters/min X gram liver weight), the biliary efflux of total glutathione (6.54 +/- 0.94 nmol GSH eq/min X g), and GSSG (1.59 +/- 0.23 nmol GSH eq/min X g) recovered to 69-86% after short-term ischemia and to 36-72% after 2 h of ischemia when compared with values obtained from control livers perfused for the same period of time. During reperfusion, the sinusoidal efflux of total glutathione (16.4 +/- 2.1 nmol GSH eq/min X g) and GSSG (0.13 +/- 0.05 nmol GSH eq/min X g) did not change except for an initial 10-30-s increase during reperfusion washout. No increased GSSG secretion into bile was detectable at any time during reperfusion. The liver content of total glutathione (32.5 +/- 3.5 nmol GSH eq/mg protein) and GSSG (0.27 +/- 0.09 nmol GSH eq/mg protein) did not change significantly during any period of ischemia or reperfusion. We conclude, therefore, that at most only a minor amount of reactive oxygen species were generated during reperfusion. Thus, reactive oxygen species are unlikely to cause ischemia/reperfusion injury in rat liver by lipid peroxidation or tissue thiol oxidation.     

Albuminuria is not an aggravating factor in experimental focal glomerulosclerosis and hyalinosis.

It is widely known that the severity of glomerular sclerosis is proportional to the degree and chronicity of proteinuria and that the degenerative changes of glomerular epithelial cells that are associated with overflow albuminuria can be experimentally induced by the injection of large quantities of heterologous albumin. Such evidence suggests that autologous albuminuria per se may have a harmful effect on the kidneys. To examine the cause and effect relationship between renal lesions and albuminuria, we produced Adriamycin-induced experimental focal glomerular sclerosis in Nagase analbuminemic (NA) rats and control Sprague-Dawley (SD) rats and observed both the renal functional and histologic changes for 20 weeks. At week 4 after injection of Adriamycin glomerular epithelial lesions including foot process fusion were similarly revealed by an electron microscopic study in both groups in spite of the presence of a large difference in the amount of proteinuria (SD rats: 491 +/- 84 mg/day, NA rats: 43 +/- 30 mg/day) and albuminuria (SD rats: 383 +/- 73 mg/day, NA rats: 2 +/- 1 mg/day). At week 20, a light microscopic study showed the same degree of glomerular sclerosis and hyalinosis and tubulointerstitial changes associated with a decrease in inulin clearance in both groups. The increased glomerular accumulation of immunoglobulin M or complement 3 and glomerular trapping of aggregated human immunoglobulin G were also similar between the SD and NA groups. In summary, renal destruction of Adriamycin-nephropathy was not dependent on the degree of albuminuria. These results suggest that albuminuria is not an aggravating factor in focal glomerulosclerosis.     

Proteolytic enzyme treatment reduces glomerular immune deposits and proteinuria in passive Heymann nephritis

We investigated the effect of proteolytic enzyme treatment on the course of passive Heymann nephritis (PHN). PHN was induced by intravenous injection of Heymann antibody into Sprague Dawley rats. Protease-treated rats received intraperitoneal chymopapain and subtilisin. In rats given subnephritogenic doses of Heymann antibody (5 or 10 mg, insufficient to cause proteinuria), glomerular immune deposits were assessed by immunofluorescence and electron microscopy. In rats given 5 mg Heymann antibody and treated with protease in the heterologous phase of the disease (days 1-7), fewer animals were positive for rabbit IgG and rat IgG, as determined by immunofluorescence on day 12, compared with controls (p less than 0.01). Rats given 10 mg Heymann antibody and treated on days 1-5 were less frequently positive for rabbit IgG on day 5 than controls (p less than 0.05). When treatment was given on days 6-12 (autologous phase), fewer rats had glomerular rabbit and rat IgG compared with controls (p less than 0.025). Protease treatment of rats given nephritogenic doses of Heymann antibody (greater than or equal to 40 mg, causing proteinuria) did not result in significant differences in immunofluorescence deposits. However, protease treatment significantly reduced the number of electron dense deposits at all doses of antibody (p less than 0.01). Furthermore, rats given 60 mg Heymann antibody followed by enzyme treatment in the heterologous phase (days 1-7) or throughout the autologous phase (days 6-18) had significantly reduced protein excretion during the autologous phase compared with control rats (p less than 0.05). After onset of significant proteinuria on day 15 in rats given 40 mg Heymann antibody and treated from day 15 until day 25, there was significantly less (p less than 0.05) proteinuria on days 21-22 and 24-25 than in control rats; thus, enzymes could reverse proteinuria. In normal rats, administration of proteases did not have significant effects on urinary protein excretion, serum creatinine, or renal morphology, nor did protease affect anti-rabbit IgG antibody production in rats injected with Heymann antibody. The overall results indicate that proteolytic enzyme treatment can prevent or remove glomerular immune deposits and can prevent or reverse proteinuria.     

Effect of fatty acids on glucose production and utilization in man.

Since the initial proposal of the glucose fatty acid cycle, considerable controversy has arisen concerning its physiologic significance in vivo. In the present study, we examined the effect of acute, physiologic elevations of FFA concentrations on glucose production and uptake in normal subjects under three controlled experimental conditions. In group A, plasma insulin levels were raised and maintained at approximately 100 microU/ml above base line by an insulin infusion, while holding plasma glucose at the fasting level by a variable glucose infusion. In group B, plasma glucose concentration was raised by 125 mg/100 ml and plasma insulin was clamped at approximately 50 microU/ml by a combined infusion of somatostatin and insulin. In group C, plasma glucose was raised by 200 mg/100 ml above the fasting level, while insulin secretion was inhibited with somatostatin and peripheral glucagon levels were replaced with a glucagon infusion (1 ng/min X kg). Each protocol was repeated in the same subject in combination with a lipid-heparin infusion designed to raise plasma FFA levels by 1.5-2.0 mumol/ml. With euglycemic hyperinsulinemia (study A), lipid infusion caused a significant inhibition of total glucose uptake (6.3 +/- 1.3 vs. 7.4 +/- 0.6 mg/min X kg, P less than 0.02). Endogenous glucose production (estimated by the [3-3H]glucose technique) was completely suppressed both with and without lipid infusion. With hyperglycemic hyperinsulinemia (study B), lipid infusion also induced a marked impairment in glucose utilization (6.2 +/- 1.1 vs. 9.8 +/- 1.9 mg/min X kg, P less than 0.05); endogenous glucose production was again completely inhibited despite the increase in FFA concentrations. Under both conditions (A and B), the percentage inhibition of glucose uptake by FFA was positively correlated with the total rate of glucose uptake (r = 0.69, P less than 0.01). In contrast, when hyperglycemia was associated with relative insulinopenia and hyperglucagonemia (study C), thus simulating a diabetic state, lipid infusion had no effect on glucose uptake (2.9 +/- 0.2 vs. 2.6 +/- 0.2 mg/min X kg) but markedly stimulated endogenous glucose production (1.4 +/- 0.5 vs. 0.5 +/- 0.4 mg/min X kg, P less than 0.005). Under the same conditions as study C, a glycerol infusion producing plasma glycerol levels similar to those achieved with lipid-heparin, enhanced endogenous glucose production (1.5 +/- 0.5 vs. 0.7 +/- 0.6 mg/min X kg, P less than 0.05). We conclude that, in the well-insulinized state raised FFA levels effectively compete with glucose for uptake by peripheral tissues, regardless of the presence of hyperglycemia. When insulin is deficient, on the other hand, elevated rates of lipolysis may contribute to hyperglycemia not by competition for fuel utilization, but through an enhancement of endogenous glucose output.     

Abnormal sodium handling occurs in the isolated perfused kidney of the nephrotic rat

1. In order to examine the handling of sodium by the nephrotic kidney when separated from the immediate influences of renal nerves and humoral factors, kidneys were taken from nephrotic rats (puromycin aminonucleoside) and studied over a range of perfusion pressures using the isolated perfused kidney technique. 2. When perfused with medium containing 6.7 g/dl albumin, the nephrotic kidneys performed differently from controls with a reduction in sodium excretion at all pressures [(mean +/- SEM) 1.14 +/- 0.43 vs 4.20 +/- 0.69 mumol/min at 105 mmHg (14 kPa); 6.32 +/- 1.56 vs 44.60 +/- 5.30 mumol/min at 150 mmHg (20 kPa)]. Renal vascular resistance, inulin clearance, fractional sodium excretion and fractional lithium excretion were also reduced. 3. When kidneys were perfused without oncotic agent these differences between nephrotic and control kidneys remained. Perfusion with medium containing 10 g/dl albumin, designed to prevent glomerular filtration, abolished the difference in vascular resistance between the two groups. Captopril had no effect on the sodium retention or vascular resistance of nephrotic kidneys. 4. It was concluded that (a) the isolated nephrotic kidney demonstrates increased avidity for sodium, (b) the abnormality of sodium handling is not dependent on the presence of altered oncotic forces, and (c) the alteration in vascular resistance is conditional upon glomerular filtration.