The antiinflammatory effects of essential fatty acid deficiency in experimental glomerulonephritis. The modulation of macrophage migration and eicosanoid metabolism

Dietary polyunsaturated fatty acid modulation exerts a beneficial effect in immune-mediated glomerulonephritis. To elucidate the mechanisms underlying this phenomenon, the effects of essential fatty acid (EFA) deficiency on the heterologous phase of nephrotoxic nephritis in rats (induced by the injection of a rabbit antiglomerular basement membrane antibody) were studied. The heterologous phase of nephrotoxic nephritis was characterized by an invasion of leukocytes into the glomerulus. Polymorphonuclear neutrophils predominated early on (3 h), whereas macrophages predominated at 24 and 72 h. EFA deficiency selectively prevented the influx of macrophages into the glomerulus. The invasion of polymorphonuclear neutrophils, in contrast, was unaffected. The influx of leukocytes into the glomerulus during nephritis was accompanied by a marked enhancement (10- to 40-fold) in glomerular thromboxane and leukotriene B4 production. EFA deficiency largely attenuated this change. Renal dysfunction during the heterologous phase of nephritis was manifested as azotemia, polyuria, sodium retention, and proteinuria. With EFA deficiency, polyuria, azotemia, and sodium retention were not seen. Proteinuria was reduced by approximately 85%. To address whether the lack of macrophage migration into the glomerulus in the context of nephritis with EFA deficiency might be due to a functional defect in macrophage migration, the chemotactic responsiveness of EFA-deficient macrophages was examined. EFA-deficient macrophages displayed normal chemotactic migration toward activated C. In sum, EFA deficiency prevents the invasion of macrophages into the glomerulus in nephrotoxic nephritis and attenuates the accompanying metabolic and functional alterations, but does not affect macrophage chemotactic responsiveness. Alterations in macrophage elicitation and lipid mediator generation by inflamed glomeruli thus appear to be central to the salutary effect of dietary polyunsaturated fatty acid modification on glomerulonephritis.     

Essential fatty acid deficiency impairs macrophage spreading and adherence. Role of arachidonate in cell adhesion

Dietary polyunsaturated fatty acid manipulation exerts a strikingly protective effect in models of tissue inflammation and injury. A critical element of this effect appears to revolve around leukocyte trafficking but underlying mechanisms are ill understood. In the current study it was observed that essential fatty acid (EFA) deficiency markedly impaired the capacity of resident macrophages to spread and adhere. This effect was not a simple function of the alteration of membrane fatty acid composition. Elicited EFA-deficient macrophages were equally adherent to elicited control cells, despite the fact that they were equally EFA-deficient relative to resident EFA-deficient cells. With respect to the mechanism underlying defective macrophage adherence in EFA deficiency, no change in the expression of cell surface adherence molecules (Fc receptor, Mac-1, or LFA-1) was noted with the deficiency state. Also, an adherence defect could not be induced in normal cells pharmacologically with cyclooxygenase blockade, lipoxygenase blockade, or a platelet-activating factor receptor antagonist. In contrast, phospholipase inhibition was able to induce a spreading and adherence defect in resident macrophages similar to that seen with EFA deficiency. Using several phospholipase inhibitors, a correlation between phospholipase inhibition and impairment of adherence was observed. Adding back exogenous fatty acids to cells after phospholipase inhibition demonstrated that normal adherence was reconstituted with arachidonate. This alteration in macrophage spreading and adherence with EFA deficiency may be an important component of the anti-inflammatory effect of dietary polyunsaturated fatty acid manipulation. Additionally, these results suggest that arachidonate may be an intracellular mediator of leukocyte adherence.     

Increase of Soluble Casein during Storage of Sterilized Concentrated Skim Milk

We previously demonstrated that a methionine-threonine-supplemented low (8.5%) casein diet (8.5CMT) reduced symptoms such as proteinuria in nephritic rats without severe protein malnutrition. In this study, we examined whether or not L-arginine supplementation to 8.5CMT would exacerbate proteinuria and other symptoms in nephritic rats. Male Wistar rats with glomerulonephritis induced by a single intravenous injection of nephrotoxic serum were fed either a 20% casein diet (control), 8.5% casein diet, 8.5CMT, or L-arginine-supplemented 8.5CMT (8.5CMTA) for 16 days. The 8.5CMTA, as compared with the 8.5CMT, aggravated proteinuria and glomerulonephritis. Administration of L-N^G-nitroarginine methyl ester, an inhibitor of nitric oxide synthase, to 8.5CMTA-fed nephritic rats by drinking water for 14 days canceled the adverse effect of L-arginine on proteinuria and histopathological damage in glomeruli. These results suggest that the supplementation of L-arginine makes exacerbation via nitric oxide production in glomerulonephritis.     

Arginine as an exacerbating factor for glomerulonephritis in rats fed a methionine-threonine-supplemented low casein diet.

We previously demonstrated that a methionine-threonine-supplemented low (8.5%) casein diet (8.5CMT) reduced symptoms such as proteinuria in nephritic rats without severe protein malnutrition. In this study, we examined whether or not L-arginine supplementation to 8.5CMT would exacerbate proteinuria and other symptoms in nephritic rats. Male Wistar rats with glomerulonephritis induced by a single intravenous injection of nephrotoxic serum were fed either a 20% casein diet (control), 8.5% casein diet, 8.5CMT, or L-arginine-supplemented 8.5CMT (8.5CMTA) for 16 days. The 8.5CMTA, as compared with the 8.5CMT, aggravated proteinuria and glomerulonephritis. Administration of L-N(G)-nitroarginine methyl ester, an inhibitor of nitric oxide synthase, to 8.5CMTA-fed nephritic rats by drinking water for 14 days canceled the adverse effect of L-arginine on proteinuria and histopathological damage in glomeruli. These results suggest that the supplementation of L-arginine makes exacerbation via nitric oxide production in glomerulonephritis.     

Metabolic and functional alterations in macrophages induced by essential fatty acid deficiency

Essential fatty acid (EFA) deficiency has been shown to protect against the glomerulonephritis in a murine model of systemic lupus erythematosus. Since macrophages are an important cellular constituent of the inflammatory lesion, the effects of EFA deficiency on the eicosanoid metabolism and function of these cells were determined. EFA-deficient macrophages exhibited a depletion of phospholipid arachidonate and an accumulation of 20:3(n-9); phosphatidylinositol was the phospholipid most affected. When these macrophages were stimulated with unopsonized zymosan, they produced markedly less leukotriene C4 and B4 than control macrophages. EFA-deficient macrophages also synthesized leukotriene C3 from endogenous 20:3(n-9). No leukotriene B3 was detected. In contrast to the effects on leukotriene production, prostaglandin and thromboxane production were only minimally affected by EFA deficiency. When challenged with zymosan, EFA-deficient macrophages released less arachidonate relative to control macrophages and released half again as much 20:3(n-9) as arachidonate. Release of arachidonate from phosphatidylcholine in the EFA-deficient cells was highly selective for arachidonate; however, release of arachidonate from phosphatidylinositol was depressed relative to control and was not selective. Incubation of macrophages with exogenous arachidonate and 20:3(n-9) established that 20:3(n-9) decreased leukotriene C4 and B4 synthesis from arachidonate but did not affect prostaglandin production. To determine the functional effects of the deficiency state, receptor-mediated pinocytosis and phagocytosis were also examined in EFA-deficient cells. EFA-deficient macrophages exhibited a marked reduction in receptor-mediated pinocytosis. Phagocytosis, however, was unaffected by the deficiency state. These effects on macrophage eicosanoid metabolism and function may comprise a significant component of the anti-inflammatory effect of EFA deficiency.     

Effect of essential fatty acid deficiency on the lipid composition of the Yoshida ascites hepatoma (AH 130) and of the liver and blood plasma from host and normal rats.

In order to study the response of a poorly differentiated tumor to nutritional manipulation, the Yoshida ascites hepatoma (AH 130) was grown in rats fed an essential fatty acid (EFA)-deficient diet and in rats fed a control diet. Hepatomas, livers, and blood plasma from host rats and normal rats were studied as to the effects of EFA deficiency on the lipid composition. Normal rats fed an EFA-deficient diet showed an increased concentration of triglycerides and cholesteryl esters in the liver and a reduced level of total phospholipids in plasma. Host rats fed the EFA-deficient diet showed a lower concentration of triglycerides in the liver when compared with the host rats fed a control diet. In addition, EFA-deficient host rats had reduced levels of plasma free fatty acids and triglycerides. These latter were markedly high in host rats under normal dietetic conditions. As compared to the livers of either host rats or normal rats fed the control diet, the Yoshida hepatoma cells had a lower content of total phospholipids and free fatty acids as well as a higher level of free cholesterol; they also showed a typical fatty acid pattern in their phospholipids. The main characteristics of this pattern were a high content of oleic and palmitoleic acids and a low level of C20 and C22 polyunsaturated fatty acids. Exposure of Yoshida hepatoma cells to an EFA-deficient environment resulted in a decrease in the concentration of total phospholipids and free fatty acids and in changes in the fatty acid composition similar to those observed in the livers of normal and host rats. These changes suggest that, under the experimental conditions used, the Yoshida hepatoma cells are responsive to EFA deficiency.     

Intraglomerular fibronectin in rat experimental glomerulonephritis.

To clarify the mechanisms of glomerular pericapillary fibronectin deposition in human membranous nephropathy and mesangial proliferative glomerulonephritis, intraglomerular fibronectin distribution was examined by light and electron microscopy using the experimental rat models of Heymann and nephrotoxic serum nephritis. As previously demonstrated by immunofluorescence microscopy (Pettersson and Colvin 1978; Ikeya et al. 1985, 1986), fibronectin was distributed in the mesangial areas and occasionally on percicapillary walls of normal glomeruli, while in nephrotoxic serum nephritis and Heymann nephritis, fibronectin was diffusely located along glomerular capillary walls as well as in the mesangium. By immunoelectron microscopy using the immunogold technique, fibronectin was also noted in the mesangial areas and the lamina densa of the glomerular basement membrane (GBM) in normal glomeruli. In nephrotoxic serum nephritis, fibronectin was seen around mesangial cells situated between endothelial cells and the GBM, suggesting that pericapillary fibronectin in nephrotoxic serum nephritis reflects mesangial extension. However, in Heymann nephritis, it was found uniformly in the lamina rara interna, lamina densa and lamina rara externa of the GBM, indicating no specific relation to glomerular cells. When sections of normal and both experimental nephritis kidneys were incubated with fluorescein isothiocyanate conjugated with rat plasma fibronectin, a linear pattern of fluorescein staining along the glomerular capillary walls was observed in Heymann nephritis but not in normal or nephrotoxic serum nephritic rats. The GBM in Heymann nephritis would thus appear to have an affinity for plasma fibronectin. Based on the above findings, fibronectin in the GBM of rats with Heymann nephritis may reasonably be concluded to originate from the plasma.     

Maternal essential fatty acid deficiency depresses serum leptin levels in suckling rat pups

Dietary lipid quantity and quality have recently been shown to affect serum leptin levels in adult rats. Moreover, suckling pups from dams fed a high fat diet had increased serum leptin levels. The aim of the present study was to analyze the influence of essential fatty acid (EFA) deficiency on serum leptin levels in dams and their pups during the suckling period. For the last 10 days of gestation and throughout lactation, pregnant rats were fed a control or an EFA-deficient (EFAD) diet. The levels of leptin and EFA in the serum of the dams and pups were analyzed 1, 2, and 3 weeks after delivery. In parallel, serum levels of glucose and corticosterone were analyzed in the pups. Low serum leptin levels were found in the control lactating dams during the entire lactation period compared with the age-matched nonlactating animals. The leptin concentrations in the lactating dams fed the EFAD diet were lower compared with those fed the control diet. The serum leptin levels of suckling pups from dams on the EFAD diet were markedly decreased compared with controls (P < 0.05). The reduced serum leptin levels could not be explained by nutritional restriction as evaluated by serum levels of glucose and corticosterone. These results indicate the importance of the EFA composition of the maternal diet for serum leptin levels in both dams and pups. EFA deficiency in lactating dams may cause long-term effects on the pups through dysregulation of leptin and leptin-dependent functions. -- Korotkova, M., B. Gabrielsson, L. A. Hanson, and B. Strandvik. Maternal essential fatty acid deficiency depresses serum leptin levels in suckling rat pups. J. Lipid Res. 2001. 42: 359--365.     

Essential fatty acid deficiency in mice impairs lactose digestion

Essential fatty acid (EFA) deficiency in mice induces fat malabsorption. We previously reported indications that the underlying mechanism is located at the level of the intestinal mucosa. We have investigated the effects of EFA deficiency on small intestinal morphology and function. Mice were fed an EFA-deficient or control diet for 8 wk. A 72-h fat balance, the EFA status, and small intestinal histology were determined. Carbohydrate absorptive and digestive capacities were assessed by stable isotope methodology after administration of [U-(13)C]glucose and [1-(13)C]lactose. The mRNA expression and enzyme activity of lactase, and concentrations of the EFA linoleic acid (LA) were measured in small intestinal mucosa. Mice fed the EFA-deficient diet were markedly EFA-deficient with a profound fat malabsorption. EFA deficiency did not affect the histology or proliferative capacity of the small intestine. Blood [13C6]glucose appearance and disappearance were similar in both groups, indicating unaffected monosaccharide absorption. In contrast, blood appearance of [13C]glucose, originating from [1-(13)C]lactose, was delayed in EFA-deficient mice. EFA deficiency profoundly reduced lactase activity (-58%, P<0.01) and mRNA expression (-55%, P<0.01) in mid-small intestine. Both lactase activity and its mRNA expression strongly correlated with mucosal LA concentrations (r=0.77 and 0.79, respectively, P<0.01). EFA deficiency in mice inhibits the capacity to digest lactose but does not affect small intestinal histology. These data underscore the observation that EFA deficiency functionally impairs the small intestine, which in part may be mediated by low LA levels in the enterocytes.     

Essential fatty acid deficiency inhibits the in vivo generation of leukotriene B4 and suppresses levels of resident and elicited leukocytes in acute inflammation

Essential fatty acid (EFA) deficiency exerts an anti-inflammatory effect in several models of inflammation. In an effort to understand underlying mechanisms, the effect of EFA deficiency on the generation of eicosanoids and the elicitation of leukocytes in a model of acute inflammation was examined. Acute inflammation was induced by the i.p. injection of zymosan in mice. The injection of zymosan in normal mice was followed by a short burst of eicosanoid synthesis lasting 2 hr. Leukotriene (LT)B4, LTC4, LTD4, and LTE4, thromboxane B2, and 6-keto-prostaglandin F1 alpha were detected using high pressure liquid chromatography and specific radioimmunoassays. This initial phase of eicosanoid production was followed by a more prolonged infiltration of leukocytes (predominantly polymorphonuclear neutrophils (PMN)) lasting 48 hr with little eicosanoid synthesis. When challenged with zymosan, EFA-deficient mice exhibited a marked decrease in the production of eicosanoids during the early phase. No LTB could be detected at all. The number of resident peritoneal macrophages in EFA-deficient mice was also substantially decreased, and the influx of PMN during the inflammatory response was markedly diminished. In order to establish that the generation of eicosanoids during the early phase of this model of acute inflammation played a causal role in the later infiltration of PMN, the effect of the mixed lipoxygenase/cyclooxygenase inhibitor, BW755C, on LTB formation and PMN influx in this model of inflammation was assessed in control animals. BW755C completely blocked LTB synthesis and inhibited the subsequent influx of PMN. In conclusion, EFA deficiency inhibits eicosanoid generation, depresses levels of resident macrophages, and markedly diminishes the influx of PMN in the acute inflammatory response. The decrease in PMN influx appears to result from the inhibition of the antecedent generation of LTB.     

Masugi's nephritis: preparation of an active nephrotoxic serum

An active nephrotoxic serum (NTS) was prepared by immunization of rabbits with isolated renal glomeruli of rats and Freund's complete adjuvant. The glomeruli were isolated from the renal cortex by the sieving procedure. Intravenous injection of the NTS to rats induced the development of marked proteinuria and the nephrotic syndrome. Histological and electron microscopic studies of renal tissue performed by days 5 and 15 since the first injection of the NTS revealed extracapillary proliferative glomerulonephritis. IgG depositions on the basal membranes of the glomerular capillaries were linear on day 5 and granular on day 15 since the first injection of the NTS.     

Glomerular lysozyme binding in protein-overload proteinuria

Binding of the cationic molecule lysozyme to the glomerular basement membrane and to the glomerular epithelial cell coat was investigated in the glomerulus of normal female Wistar rats and in rats in which heavy proteinuria was induced by the daily administration of 1 g of bovine serum albumin. In normal rats the binding of lysozyme to the anionic groups in the glomerular basement membrane and the cell coat had no effect on the ultrastructure of the glomerular epithelial cell, in particular the foot processes were unchanged. In the proteinuric rats the lysozyme-binding to the glomerular basement membrane and the epithelial cell coat was completely lost in the damaged glomeruli. In the apparently normal glomeruli present in these proteinuric animals binding was similar to that seen in normal rats. These results suggest that in protein-overload proteinuria there is a loss of glomerular anion and hence a reduction in the glomerular charge barrier. This may account, at least in part, for the increased glomerular leak of negatively charged serum albumin in this experimental model of proteinuria.     

Effect of dietary essential fatty acid deficiency on Ca(2+)-ATPase activity in microsomal fraction of rat submandibular gland.

1. The effect of dietary essential fatty acid (EFA) deficiency on Ca(2+)-ATPase activity of rat submandibular gland microsomal fraction was studied. 2. The specific activity of Ca(2+)-ATPase per milligram of microsomal protein was depressed about 35% in rats fed the EFA-deficient diet as compared with that in those fed the control diet. 3. Lineweaver-Burk plots for Ca(2+)-ATPase activity showed no significant differences in Km values for Ca2+ and ATP, but the Vmax was decreased in the EFA-deficient rats. 4. The above results suggest that depression of the Ca(2+)-ATPase activity in rats fed the EFA-deficient diet is probably due to the decrease in the Vmax of the enzyme.     

Abrogation of mercuric chloride-induced nephritis in the Brown Norway rat by treatment with antibodies against TNFalpha

HgCl(2) induces an autoimmune disease in the Brown Norway rat characterized by synthesis of autoantibodies (mainly, anti-GBM Abs), severe proteinuria and interstitial nephritis. Also, HgCl(2)- injected rats develop glomerular cell infiltrates consisting of ED1(+) cells (monocyte/macrophage), starting on day 4 and reaching a maximum on day 8. Treatment with anti-TNF-alpha antiserum had preventative effects as it reduced the urinary protein levels to close to the normal range and also blocked the influx of inflammatory cells in the renal glomeruli and interstitium, but circulating anti-GBM and lineal glomerular IgG deposits were unmodified. In addition, whole isolated glomeruli from HgCl(2)-induced nephritis secreted TNF-alpha commencing on day 8, being maximally detected on day 11 and preceding, between 2 to 3 days, the development of proteinuria. The administration of anti-TNF-alpha antiserum or anti-alpha4 integrin mAb completely abrogated the synthesis of TNF-alpha in glomeruli isolated from the respective treated groups of animals, in addition to the proteinuria. Taken together our results confirm that TNF-alpha plays an important role in the induction and development of HgCl(2)-induced nephritis and highlights the pathogenic importance of the local release of TNF in those renal diseases in which prominent glomerular macrophage accumulation is a constant feature.     

Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid-supplemented diet (+EFA) or from rats fed an essential fatty acid-deficient diet (-EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the ?EFA preparations were one-half or less than the values for the +EFA preparations. Basal, F^?, or glucagon-stimulated adenylate cyclase activities were significantly lower in EFA-deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon-stimulated adenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA-deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in ?EFA livers. When glucagon was used in vitro at 1–1,000 nM, stimulation of adenylate cyclase remained lower in EFA-deficient membranes, but extent of stimulation above basal activity was larger in ?EFA membranes than in +EFA. Total Na⁺, K⁺ (Mg²⁺)-ATPase from EFA-depleted LPM exhibited significantly higher values of apparent K_m and V_max. 5′-Nucleotidase activity, in contrast, was considerably decreased in EFA-deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane-bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.     

Effects of infusion of some prostaglandins in essential fatty acid-deficient and normal rats

Infusion of 1 mg/kg per day of prostaglandin E(1) (PGE(1)) for 2 and 7 wk failed to correct the dermal signs of essential fatty acid (EFA) deficiency in rats despite the known conversion of EFA to certain prostaglandins. PGE(1) caused no significant changes in serum cholesterol, triglycerides, or phospholipids or in liver neutral lipids in EFA-deficient or normal rats. In normal rats epinephrine-induced lipolysis was greater in fat pads from infused than from untreated rats. The effect on epinephrine-induced lipolysis was greater after the 7 wk infusion than after the 2 wk infusion. The 7 wk infusion also lowered plasma free fatty acid (FFA) concentrations. Infusion of PGE(2) and PGF(2alpha) in combination for 4 wk had no significant effect on either dermal signs of EFA deficiency, lipolysis, or plasma FFA concentrations.     

Interleukin-1 production by mononuclear leukocytes and mesangial cells in experimental nephrotoxic nephritis

We investigated the intensiveness of the cell proliferation, number of mononuclear leucocytes (MNL) and production of interleukin-1 (IL-1) in glomerular cell cultures of rats with nephrotoxic serum (NTS) nephritis and control (C) rats. Five days after intravenous injection of nephrotoxic serum from rabbits the glomeruli were isolated, treated with collagenase and cultured over a period of 20 days. In cultures the number and intensity of mesangial cell proliferation in the NTS group were significantly higher than in the C group. Thus, the intensive mesangial cell proliferation in experimental nephrotoxic serum nephritis is related to infiltration of glomerulus by MNL of bone marrow origin with increased production of IL-1.     

ASK1 inhibitor treatment suppresses p38/JNK signalling with reduced kidney inflammation and fibrosis in rat crescentic glomerulonephritis

Activation of p38 mitogen‐activated protein kinase (MAPK) and c‐Jun amino terminal kinase (JNK) is prominent in human crescentic glomerulonephritis. p38 and JNK inhibitors suppress crescentic disease in animal models; however, the upstream mechanisms inducing activation of these kinases in crescentic glomerulonephritis are unknown. We investigated the hypothesis that apoptosis signal‐regulating kinase 1 (ASK1/MAP3K5) promote p38/JNK activation and renal injury in models of nephrotoxic serum nephritis (NTN); acute glomerular injury in SD rats, and crescentic disease in WKY rats. Treatment with the selective ASK1 inhibitor, GS‐444217 or vehicle began 1 hour before nephrotoxic serum injection and continued until animals were killed on day 1 (SD rats) or 14 (WKY rats). NTN resulted in phosphorylation (activation) of p38 and c‐Jun in both models which was substantially reduced by ASK1 inhibitor treatment. In SD rats, GS‐444217 prevented proteinuria and glomerular thrombosis with suppression of macrophage activation on day 1 NTN. In WKY rats, GS‐444217 reduced crescent formation, prevented renal impairment and reduced proteinuria on day 14 NTN. Macrophage activation, T‐cell infiltration and renal fibrosis were also reduced by GS‐444217. In conclusion, GS‐444217 treatment inhibited p38/JNK activation and development of renal injury in rat NTN. ASK1 inhibitors may have therapeutic potential in rapidly progressive glomerulonephritis.     

Improvement of Hyperlipidemia and Proteinuria without Noticeable Growth Retardation by Feeding a Methionine and Threonine Supplemented Low-casein Diet to Nephritic Rats

We have previously demonstrated that low-casein diets supplemented with cystine and threonine reduced hyperlipidemia and proteinuria in nephritic rats without noticeable protein malnutrition. In the present study, we examined whether or not a low-casein diet supplemented with methionine, sulfur amino acid other than cystine, and threonine would ameliorate the symptoms without protein malnutrition in rats with nephrotoxic serum nephritis by feeding experimental diets for 10 days. A methionine-threonine-supplemented 8.5% casein diet (8.5 CMT), when compared with a basal 20% casein diet, improved hypoalbuminemia as well as hyperlipidemia and proteinuria without noticeable growth retardation and fatty liver induction in nephritic rats. Fecal bile acid excretion and microsomal cholesterol 7α-hydroxylase activity were enhanced by 8.5CMT feeding. These results suggest that amino acid-balanced low protein diet would have a beneficial effect on the symptoms of nephritis. They also suggest that the hypocholesterolemic action of 8.5CMT may be, at least in part, due to increased fecal bile acid excretion accompanied by elevated microsomal cholesterol 7α-hydroxylase activity.