A founder mutation in COL4A3 causes autosomal recessive Alport syndrome in the Ashkenazi Jewish population.

Alport syndrome is an inherited progressive nephropathy arising from mutations in the type IV collagen genes, COL4A3, COL4A4, and COL4A5. Symptoms also include sensorineural hearing loss and ocular lesions. We determined the molecular basis of Alport syndrome in a non-consanguineous Ashkenazi Jewish family with multiple affected females using linkage analysis and next generation sequencing. We identified a homozygous COL4A3 mutation, c.40_63del, in affected individuals with mutant alleles inherited from each parent on partially conserved haplotypes. Large-scale population screening of 2017 unrelated Ashkenazi Jewish samples revealed a carrier frequency of 1 in 183 indicating that COL4A3 c.40_63del is a founder mutation which may be a common cause of Alport syndrome in this population. Additionally, we determined that heterozygous mutation carriers in this family do not meet criteria for a diagnosis of Thin Basement Membrane Nephropathy and concluded that carriers of c.40_63del are not likely to develop benign familial hematuria.

Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K+ channel, ROMK.

Mutations in the Na-K-2Cl cotransporter (NKCC2), a mediator of renal salt reabsorption, cause Bartter's syndrome, featuring salt wasting, hypokalaemic alkalosis, hypercalciuria and low blood pressure. NKCC2 mutations can be excluded in some Bartter's kindreds, prompting examination of regulators of cotransporter activity. One regulator is believed to be ROMK, an ATP-sensitive K+ channel that 'recycles' reabsorbed K+ back to the tubule lumen. Examination of the ROMK gene reveals mutations that co-segregate with the disease and disrupt ROMK function in four Bartter's kindreds. Our findings establish the genetic heterogeneity of Bartter's syndrome, and demonstrate the physiologic role of ROMK in vivo.

Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III.

Analysis of patients with inherited hypokalaemic alkalosis resulting from salt-wasting has proved fertile ground for identification of essential elements of renal salt homeostasis and blood-pressure regulation. We now demonstrate linkage of this phenotype to a segment of chromosome 1 containing the gene encoding a renal chloride channel, CLCNKB. Examination of this gene reveals loss-of-function mutations that impair renal chloride reabsorption in the thick ascending limb of Henle's loop. Mutations in seventeen kindreds have been identified, and they include large deletions and nonsense and missense mutations. Some of the deletions are shown to have arisen by unequal crossing over between CLCNKB and the nearby related gene, CLCNKA. Patients who harbour CLCNKB mutations are characterized by hypokalaemic alkalosis with salt-wasting, low blood pressure, normal magnesium and hyper- or normocalciuria; they define a distinct subset of patients with Bartter's syndrome in whom nephrocalcinosis is absent. These findings demonstrate the critical role of CLCNKB in renal salt reabsorption and blood-pressure homeostasis, and demonstrate the potential role of specific CLCNKB antagonists as diuretic antihypertensive agents.

Increased lipid fluidity in synaptosomes from brains of hyperosmolal rats.

Taurine, a product of sulfur amino acid metabolism, is important in cerebral osmoregulation. To understand the adaptive changes in transport which accompany different hyperosmolal states, we determined lipid composition and fluorescence anisotropy of synaptosomal liposomes from rats with chronic hypernatremic dehydration (CHD), streptozocin-induced (STZ) diabetes, and insulin treated diabetes. Induction of CHD increased serum osmolality, and enhanced in vitro synaptosomal taurine uptake (P < 0.01, n = 3, vs. control). Fluorescence anisotropy studies showed that the fluidity of lipids from CHD synaptosomes was higher than control (P < 0.05, n = 3). STZ-diabetes resulted in hyperglycemia, increased serum osmolality, and stimulated synaptosomal taurine uptake (P < 0.01, n = 3, vs. control). Insulin treatment of diabetic rats restored serum osmolality and taurine transport to control values. The fluidity of diabetic rat brain synaptosomal lipids was significantly higher than control (P < 0.05, n = 3); fluidity was normalized by insulin administration to diabetic rats. Total fatty acid, cholesterol, and cholesterol/phospholipid molar ratio of CHD, STZ, and insulin treated diabetic rats were similar to control. However, the ratio of saturated to unsaturated fatty acids was decreased in hyperosmolal states. This suggests that adaptive increases in cerebral taurine transport during hyperosmolality may result from a direct effect on membrane composition that alters fluidity and permits enhanced transmembrane flux of osmoprotective molecules.

Cerebral taurine transport is increased during streptozocin-induced diabetes in rats.

Taurine is a cerebral osmolyte whose intracellular content changes in parallel with plasma osmolality. We conducted experiments to assess whether cerebral taurine transport is modified during chronic hyperglycemia. Rats with STZ-induced diabetes were studied after 1 wk of sustained hyperglycemia. Cerebral taurine uptake in synaptosomes (metabolically active nerve terminal vesicles) was measured using a rapid filtration technique. The synaptosomes were isolated by homogenization of the brain and purification on discontinuous Ficoll gradients (n = 8 synaptosome preparations). Diabetic rats (n = 13) displayed a 15-25% increase in synaptosomal taurine uptake compared with normoglycemic control animals (n = 12) at all time points assayed between 5 and 120 min. Thus, after a 30-min incubation, cerebral taurine uptake increased from a control level of 3.53 +/- 0.23 to 4.10 +/- 0.24 mumol/mg protein (n = 10) in hyperglycemic rats, P less than 0.03. The magnitude of the plasma-to-brain cell taurine gradient was unchanged in diabetic animals. The intrasynaptosomal taurine concentration (approximately 2 microM) and taurine efflux from the synaptosomes were no different in hyperglycemic versus control rats; efflux amounted to less than 2.5% of the uptake value at corresponding time points. Maximal brain taurine uptake under both control and experimental hyperglycemic conditions required the presence of external Na+ and Cl-. Synaptosomal taurine transport was reduced by competing beta-amino acids such as beta-alanine, beta-aminoisobutyric acid, and hypotaurine (P less than 0.01). Addition of oubain and the anionic binding site inhibitors, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid and 4,4'diisothio-cyanatostilbene-2,2'-disulfonic acid, also decreased cerebral taurine uptake under normoglycemic and hyperglycemic conditions (P less than 0.01). The increased synaptosomal taurine uptake by diabetic rats was not a result of generalized membrane dysfunction because glycine transport was not elevated in hyperglycemic rats. The enhanced transport rate was attributable to a 35 and 81% increase in the Vmax of the high- and low-affinity taurine transporters, respectively (P less than 0.01), without significant change in the Km of the carrier systems. Treatment of hyperglycemic rats (n = 5) with ultra-long-acting insulin to normalize the serum glucose concentration restored synaptosomal taurine uptake to the level observed in normoglycemic controls. The effect of insulin was attributable to correction of hyperglycemia, because addition of insulin (500 mU/ml) to the in vitro assay system did not alter synaptosomal taurine uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetics, Pharmacodynamics, and Safety of Lisinopril in Pediatric Kidney Transplant Patients: Implications for Starting Dose Selection.

Hypertension in pediatric kidney transplant recipients contributes to long-term graft loss, yet treatment options--including angiotensin-converting enzyme inhibitors--are poorly characterized in this vulnerable population. We conducted a multicenter, open-label pharmacokinetic (PK) study of daily oral lisinopril in 22 children (ages 7-17 years) with stable kidney transplant function. Standard noncompartmental PK analyses were performed at steady state. Effects on blood pressure were examined in lisinopril-naïve patients (n = 13). Oral clearance declined in proportion to underlying kidney function; however, in patients with low estimated glomerular filtration rate (30-59 ml/min per 1.73m(2)), exposure (standardized to 0.1 mg/kg/day dose) was within the range reported previously in children without a kidney transplant. In lisinopril-naïve patients, 85% and 77% had a ≥ 6 mmHg reduction in systolic and diastolic blood pressure, respectively. Lisinopril was well tolerated. Our study provides initial insight on lisinopril use in children with a kidney transplant, including starting dose considerations.

Nephrotoxicity of allopurinol is enhanced in experimental hypertension.

Hyperuricemia is present in 20-40% of pediatric and adult patients with essential hypertension. This metabolic abnormality may represent an additional risk factor for the development of cardiovascular disease. Therefore, we performed the following studies to determine 1) whether hyperuricemia is more prevalent in the spontaneously hypertensive rat (SHR) and 2) whether allopurinol treatment has a beneficial effect on the development of hypertension in this strain, based on its capacity to lower the serum uric acid concentration and to act as an antioxidant agent. SHR and control Wistar-Kyoto (WKY) rats were assigned to two groups, one given tap water to drink and the other provided water containing allopurinol (400 mg/l) to furnish an approximate daily dose equal to 100 mg/kg body wt. This treatment was maintained for 15 weeks. The serum uric acid levels were similar in untreated SHR and WKY rats (1.85 +/- 0.10 versus 1.66 +/- 0.14 mg/dl; p = 0.28). In the control WKY rat strain, allopurinol therapy did not adversely affect weight gain or hematocrit and did not cause an increase in mortality. It resulted in a moderate decrement in kidney function (creatinine clearance: allopurinol-treated group 0.32 +/- 0.09 versus control group 0.46 +/- 0.04 ml/min/100 g body wt, in conjunction with mild-to-moderate tubulointerstitial inflammation (allopurinol-treated group 0.9 +/- 0.4 versus control group 0).(ABSTRACT TRUNCATED AT 250 WORDS)

Dyslipoproteinemia in murine systemic lupus erythematosus.

Dyslipoproteinemia, a feature of systemic lupus erythematosus may contribute to premature atherosclerosis. In order to develop an experimental model for this dyslipoproteinemia we measured plasma concentrations of lipoproteins in juvenile NZB/W (lupus) and NZB/B (control) mice. Additionally to evaluate the effects of a diet rich in n - 3 fatty acids we measured lipoprotein concentrations in mice on normal or menhaden oil-enriched diets. The lupus mice had elevated triglycerides compared to the controls, similar to that seen in human SLE patients (161 +/- 31 vs 113 +/- 13 mg/dl, P less than 0.003). In contrast, the menhaden oil diet fed NZB/W mice had triglycerides similar to the NZB/B control fed group. In the NZB/W murine SLE model, dyslipoproteinemia is an early sign of disease as has been shown in man, therefore this model will be useful in elucidating the mechanism of dyslipoproteinemia in SLE.

Immunohistochemical localization of taurine in rat renal tissue: studies in experimental disease states.

Taurine, a sulfur amino acid, is present in abundant amounts in cells throughout the body. The kidney regulates taurine balance by modulating proximal tubule reabsorption in response to fluctuations in dietary intake of this nutrient. There is no information about the localization of taurine within the kidney in normal and diseased renal parenchyma. Therefore, using an antibody to a taurine-glutaraldehyde-BSA conjugate, we examined the distribution of taurine in renal tissue. Normal rats, those with streptozocin diabetes, puromycin aminonucleoside nephropathy, bilateral ureteral ligation, and 5/6 nephrectomy were studied. In normal animals, taurine was found primarily in medullary tubules, with minimal staining of proximal tubules and glomeruli. There was increased taurine staining of all structures, especially medullary tubules, in rats with streptozocin diabetes and puromycin aminonucleoside nephropathy. These changes were more pronounced in diabetic rats and were unrelated to renal medullary osmolality. The distribution of taurine within the kidney was unchanged in the models of acute and chronic renal failure. Alterations in the immunohistochemical localization of taurine correlated with the beneficial effect of this amino acid to preserve renal function in the rats with chronic diabetes and puromycin aminonucleoside nephropathy. These results suggest that taurine is preferentially localized in the medullary regions of the kidney, where it exerts a protective effect against renal injury in select disease states.

Taurine lowers blood pressure in the spontaneously hypertensive rat by a catecholamine independent mechanism.

Taurine, a sulfur aminoacid, has been studied for a role in blood pressure regulation since it functions as a generalized inhibitory neurotransmitter and is found in high concentrations in the myocardium. We reinvestigated the magnitude of the hypotensive effect of chronic taurine administration to the spontaneously hypertensive rat (SHR) and the role of catecholamines in such an action. The SHR received either a 1% taurine solution or tap water to drink for 16 weeks. Taurine treatment caused a significant persistent reduction in blood pressure by 4 weeks that was maximal at 16 weeks (146 +/- 6 [exp.] v 182 +/- 5 [control] mm Hg, P less than .01). While this taurine-induced decline in blood pressure in the SHR was not accompanied by alterations in plasma epinephrine levels, there was a steady 235% increment in the norepinephrine concentration from 231 +/- 31 pg/mL initially to 542 +/- 126 pg/mL at completion of the study, P less than .02. The reduction in blood pressure was associated with decreased proteinuria in the taurine-treated SHR (9.6 +/- 4 [exp.] v 21.5 +/- 7 [control] mg/24 h, P less than .02) and less cardiac and renal hypertrophy. We conclude that taurine administration results in a 20 to 25% reduction in blood pressure in the SHR. The mechanism of this hypotensive action requires further study but is independent of changes in plasma catecholamine levels. The vasodepressor effect of taurine leads to less hypertensive injury to the kidney and heart in the SHR.

Cerebral cell volume regulation during hypernatremia in developing rats.

Cell volume regulation is a vital biological function in all species. Maintenance of cerebral cell size in the face of osmotic stress is especially important because the brain is contained in the non-complaint skull. The developmental aspects of this adaptive process are not known. Therefore, we evaluated cerebral cell volume regulation during hypernatremia in pre-weaning and adult rats. Hypernatremia was induced by injections of 1 M NaCl for 48 h. Brain water, electrolyte, and organic osmolyte contents were measured in hypernatremia and sham injected littermate control rats at the following ages: 12, 18 and 20 days and adults. In normonatremic rats, there was a steady decline in brain water content during development that was paralleled by a gradual fall in the cerebral levels of Na+, K+, and all organic osmolytes. The change in brain water content correlated most closely with the decrease in cerebral taurine content. In the face of equivalent elevations in serum Na+ concentration, there was comparable brain cell shrinkage and similar increases in total cerebral electrolyte and organic osmolyte content in rats at all 4 ages studied. Taurine was the predominant organic osmolyte prior to weaning, constituting 16-49% of the increment in nonperturbing solute content in hypernatremic animals between 12-20 days of age; in contrast, taurine contributed only 10% to the cerebral organic osmolyte pool in adult rats. We conclude that the capacity of brain cells to accumulate inorganic electrolytes and organic osmolytes during adaptation to hypernatremia is adequately expressed in developing rats, aged 12 days or older. Moreover, we speculate that the immature animal behaves as if it has an elevated 'set point' to protect the higher brain water content that is present earlier in development.

Hyponatremia-induced brain edema in guinea pigs is reduced by treatment with the novel anion transport inhibitor L-644,711.

Cerebral edema in various disease states may result from astroglial swelling due to increased NaCl uptake mediated by enhanced Cl-HC03 exchange. We evaluated this mechanism in the pathogenesis of cerebral edema in acute hyponatremia by administering L-644,711, a fluorenyloxyacetate derivative that functions as an anion exchange inhibitor, to guinea pigs with severe reductions in serum Na+ concentration. Acute hyponatremia was induced for 54 hr by daily injections of arginine vasopressin (10 U/day) and 5% dextrose in water (7.5% body wt/day). Experimental animals received L-644,711, 20 mg/kg/day, while controls were given an equal volume of the diluent. This regimen lowered the serum Na from normal levels to 108 +/- 3 and 109 +/- 4 mM in experimental and control animals, respectively. Drug treatment resulted in less cerebral edema characterized by a reduction in brain total tissue water 432 +/- 4 vs 466 +/- 8 ml/100 g dry wt experimental vs control, P less than 0.005. This difference was composed mainly of less expansion of the intracellular water space, 287 +/- 11 vs 323 +/- 9 ml/100 g dry wt experimental vs control, p less than 0.005. The cerebral cortical Na+ +Cl content was reduced from 55.5 +/- 1.3 (control) to 39.5 +/- 1.1 mEq/100 g dry wt (experimental), p less than 0.01. These results indicate that treatment of guinea pigs with L-644,711 decreases brain NaCl content and attenuates cerebral edema during severe acute hyponatremia without normalizing the serum Na+ concentration.

The role of oxygen free radicals in the development of chronic renal failure.

This study examined whether there is increased production of oxygen free radicals during chronic renal failure. Rats subjected to 3/4 nephrectomy and sham operated controls were killed after 3 weeks. Lipid extracts of plasma and renal tissue were examined by HPLC and kidney specimens were also analyzed by EPR spectroscopy. The redox capacity of blood was assessed using nitroblue tetrazolium and plasma ascorbate levels were measured with HPLC. There was no detectable renal production of oxygen free radicals in rats with chronic renal failure. Kidney parenchymal content of other oxidants and the oxidant:reductant ratio were similar in control and uremic animals. The plasma redox capacity and ascorbate levels were elevated in uremic rats. We conclude that early in the course of chronic renal failure, there is not excessive production of oxygen free radicals. There is accumulation of reductants, primarily ascorbate, in the plasma of uremic animals.

Endogenous digoxin-like material in normotensive and hypertensive strains of rats.

We measured the concentration of endogenous digoxin-like materials (EDLM) in the serum of spontaneously hypertensive rats (SHR) and three normotensive rat strains at four stages during growth using a sensitive RIA. In the SHR, there was a significant peak in the EDLM level between 0.057-0.087 ngE/mL at 6 to 8 weeks of age, shortly after the onset of hypertension. The EDLM concentration returned to normal levels by 20 weeks of age. Sprague-Dawley and Wistar-Kyoto rats had EDLM levels below 0.050 ngE/mL at all time points studied. In contrast, Fischer 344 rats displayed persistently elevated serum EDLM concentrations that exceeded 0.124 ngE/mL from 3 to 20 weeks of life. We conclude that (1) there are significant interstrain differences in serum EDLM levels in rats; and (2) the SHR has a unique peak in serum EDLM levels at 6 to 8 weeks of age, indicating a possible role for the substance in the inception of hypertension.

The effect of hypertension and its treatment on cerebral cell volume regulation during hypernatremic dehydration.

Since hypertension may compromise the ability to withstand hypernatremic dehydration, we investigated the impact of two experimental models of hypertension and pharmacologic normalization of blood pressure on the tolerance to chronic hypernatremic dehydration. In DOCA-salt hypertensive animals and the spontaneously-hypertensive rat (SHR), there was increased mortality and cerebral cell shrinkage during hypernatremic dehydration, compared to control Sprague-Dawley or Wistar-Kyoto rats. These findings were paralleled by significant differences in the brain intracellular water compartment size (ml/100 g dry weight), i.e. 233 +/- 6, Sprague-Dawley vs 189 +/- 8, DOCA-salt, P less than 0.01; 246 +/- 3, Wistar-Kyoto vs 194 +/- 6, SHR, P less than 0.01. Normalization of the blood pressure in the SHR with captopril restored 48% of the cerebral cell volume regulatory capacity observed in normotensive Wistar-Kyoto rats. We conclude that sustained hypertension increases the risk of hypernatremic dehydration in select circumstances. Correction of the elevated blood pressure promotes partial recovery of normal cerebral cell volume regulation.

The role of polyols in cerebral cell volume regulation in hypernatremic and hyponatremic states.

To clarify the role of the sugar polyols, sorbitol and myo-inositol, in cerebral cell volume regulation, we studied the effect of sorbinil, an inhibitor of aldose and aldehyde reductase, on the size of the cerebral water compartments in rats with hypernatremia, hyponatremia and normonatremia. Experimental animals were pretreated with sorbinil, while comparison rats received the drug vehicle. Rats were made hypernatremic for 96 h by water deprivation and injections of hypertonic saline, while hyponatremia was provoked over 48 h by daily administration of 5% dextrose in water and vasopressin. Sorbinil treatment was continued throughout the hyper- and hyponatremic periods. The severity of hypernatremia and hyponatremia was similar in sorbinil-treated and corresponding vehicle-treated rats. Brain electrolyte content and the size of the cerebral intracellular water compartment were comparable in sorbinil-treated rats vs. controls under hypernatremic and hyponatremic conditions. Sorbinil reduced the cerebral sorbitol content by approximately 50%, irrespective of the serum Na+ concentration. In contrast, sorbinil had no effect on brain myo-inositol content which rose by 114% during chronic hypernatremia (P less than 0.0001). Cerebral levels of myo-inositol did not decline in hyponatremic rats. We conclude that (1) sorbitol is not an essential cerebral osmolyte; and (2) myo-inositol is involved in the maintenance of brain cell volume during severe hypernatremia but not under hyponatremic conditions.

Meprin activity in rats with experimental renal disease.

Activity of renal meprin, a membrane-bound proteinase in the proximal tubule brush border, was measured in normal rats and in two disease groups: chronic puromycin aminonucleoside nephropathy for 12 weeks and streptozocin-induced diabetes for 6 months. Enzyme activity in kidney homogenates was assayed using azocasein as substrate. The mean activity of mephrin was 3.22 +/- 0.34 U/g kidney weight in normal rats. In diabetic animals, enzyme activity was 8.58 +/- 2.11 U/g kidney weight, P < 0.01. In contrast, meprin activity was decreased in rats with puromycin-induced glomerulopathy, 2.13 +/- 0.17 U/g kidney weight, P < 0.01. These findings indicate that meprin activity is elevated in experimental diabetes. Diminished activity of this luminal membrane enzyme in puromycin aminonucleoside nephropathy may contribute to renal injury in this disease model associated with massive urinary protein excretion.

Anti-apoptotic effect of L-arginine in cultured rat mesangial cells.

Nitric oxide has been shown to be an important factor in the regulation of apoptosis in mesangial cells. The following studies were conducted to determine whether L-arginine, the metabolic precursor of nitric oxide synthesis, directly modulates programmed cell death in response to a variety of stimuli. Cultured rat mesangial cells were exposed for 24-48 h to one of the following four experimental conditions known to induce apoptosis: i) a cytokine combination consisting of interferon-gamma (50 U/ml), interleukin-1 (5 ng/ml), and LPS (10 microg/ml); ii) 0.75 mM hydrogen peroxide; iii) etoposide, 150 microg/ml; and iv) cis-platinum, 100 microg/ml. Addition of L-arginine resulted in a dose-dependent decrease in apoptosis in cytokine-treated RMC. This effect was demonstrable by gel electrophoresis, diphenylamine assay of DNA fragmentation, and an ELISA to detect DNA oligonucleosomes. The effect was not related to increased osmolality of the test media and was not reproduced by the addition of equimolar D-arginine. L-Arginine also reduced apoptosis and necrosis in RMC that were incubated with etoposide and cis-platinum. In contrast, the amino acid had no beneficial effect on RMC survival following exposure to hydrogen peroxide. These findings indicate that several components of the nitric oxide biosynthetic pathway including the precursor and the enzymatic product independently exert direct effects on apoptosis in cultured rat mesangial cells.

Growth of rat mesangial cells on oxidized extracellular matrix increases inducible nitric oxide synthase activity.

Biochemical modification of extracellular matrix (ECM) proteins can alter the function in overlying cells. We tested the hypothesis that metal-catalyzed oxidation of native ECM and individual matrix proteins modulates the activity of inducible nitric oxide synthase (iNOS) in cultured rat mesangial cells (RMC). Oxidized modification of native ECM resulted in a 32% increase in iNOS activity (P<0.01) without influencing the response to supplemental L-arginine or to the addition of the iNOS inhibitor, L-NAME. Immunoblot analysis indicated that enhanced iNOS activity was not associated with a parallel rise in the cytosolic content of iNOS. Synthesis of type IV collagen was unaffected by growth of RMC on oxidized native ECM. Oxidation of three normal constituents of the mesangial matrix - type IV collagen, laminin, and fibronectin - also stimulated iNOS activity in overlying RMC by 18-32% (P<0.05). Growth of RMC on oxidized type I collagen or Vitrogel had no effect on NO production. We conclude that oxidized modification of the mesangial matrix promotes increased iNOS activity and NO production by mesangial cells. Further work is required to determine whether this response limits glomerular injury or promotes damage to the mesangium in oxygen free radical-mediated diseases such as chronic renal failure, atherosclerosis and diabetes.

Circulating factor in patients with recurrent focal segmental glomerulosclerosis postrenal transplantation inhibits expression of inducible nitric oxide synthase and nitric oxide production by cultured rat mesangial cells.

BACKGROUND: Primary focal segmental glomerulosclerosis (FSGS) recurs in nearly 30% of patients who progress to end-stage renal disease and then receive a kidney transplant. A circulating plasma factor has been isolated from these patients that increases glomerular permeability to albumin in vitro. Because of the pivotal role of the mesangial cell in the accumulation of extracellular matrix (ECM) material within the glomerulus and the modulation of matrix protein synthesis by nitric oxide (NO), we examined the effect of the FSGS factor on inducible nitric oxide synthase (iNOS) expression and NO production by cultured rat mesangial cells (RMC). METHODS: RMC were incubated with the supernatant following 70% ammonium sulfate precipitation of serum from patients with recurrent FSGS. RESULTS: Addition of the FSGS factor to cultured RMC led to a significant inhibition of nitrite accumulation, an index of NO synthesis. There was a parallel decline in iNOS gene and protein expression. Sera obtained from control patients or those with minimal change nephrotic syndrome or diabetic nephropathy that was processed in the same manner as FSGS samples had no effect NO synthesis or iNOS activity. The inhibitory effect of the FSGS factor on NO production persisted despite addition of indomethacin (0.1-1 mumol/L) or cyclosporine (25 micrograms/mL) to test media. CONCLUSIONS: These data indicate that the FSGS factor independently alters two aspects of glomerular function--permselectivity and matrix protein synthesis--by distinct mechanisms. FSGS factor-induced disturbances in iNOS gene and protein expression and NO production by mesangial cells may antagonize the antifibrotic effect of NO within the mesangium and contribute to progressive glomerulosclerosis in patients with primary FSGS.