The role of nitric oxide in obstructive nephropathy.

PURPOSE: Ureteral obstruction leads to tubulointerstitial fibrosis and loss of renal function. Nitric oxide production ameliorates fibrosis due to obstructive uropathy. However, nitric oxide is produced by 3 isoforms of the enzyme, nitric oxide synthase. We evaluated the role of inducible nitric oxide synthase in obstructive uropathy using nitric oxide synthase knockout mice, and determined whether the administration of L-arginine to promote nitric oxide synthesis by alternative nitric oxide synthase isoforms modulates renal fibrosis in these animals. MATERIALS AND METHODS: Complete unilateral ureteral obstruction was created in wild-type C57 and inducible nitric oxide synthase knockout mice. Control animals of each strain underwent sham surgery. Throughout the experiment mice had free access to untreated tap water or water supplemented with 10 gm./l. L-arginine. Animals were sacrificed 1 and 2 weeks, respectively, after creation of unilateral ureteral obstruction. We obtained serum as well as bladder and obstructed renal pelvic urine, and determined the nitrite level in each fluid. Renal cortical thickness was measured in the normal and obstructed kidneys. The degree of tubulointerstitial fibrosis was evaluated by trichrome staining and type I collagen deposition in kidney tissue specimens. RESULTS: Nitrite was significantly decreased in the serum, bladder and renal pelvic urine of inducible nitric oxide synthase knockout mice with unilateral ureteral obstruction compared with that in wild-type C57 mice at 1 and 2 weeks (p<0.05). In knockout mice with unilateral ureteral obstruction 1 week in duration that drank tap or L-arginine supplemented water nitrite in serum and each urine sample was higher than in sham operated knockout controls. The level returned to baseline after 2 weeks of obstruction (p<0.05). After 2 weeks of obstruction there was significantly greater cortical thinning in knockout than in C57 mice (p<0.05). Moreover, knockout mice given L-arginine supplemented water for 2 weeks had even greater cortical thinning than after 1 week or than mice given tap water for 1 to 2 weeks (p<0.05). Decreased renal cortical thickness in knockout mice after 2 weeks of obstruction was associated with less intense trichrome staining and a virtual absence of type I collagen deposition compared with findings in the wild-type C57 strain. CONCLUSIONS: Inducible nitric oxide synthase knockout mice with unilateral ureteral obstruction have significantly lower nitrite in serum and urine than wild-type C57 mice. Knockout mice also have more severe renal cortical thinning than C57 animals after creation of unilateral ureteral obstruction. Providing L-arginine supplemented water to inducible nitric oxide synthase knockout mice exacerbates the loss of cortical thickness. Alterations in cortical thinning that we observed in knockout mice were associated with decreased tubulointerstitial fibrosis and a decreased net renal extracellular matrix accumulation. These data indicate that endothelial or neuronal nitric oxide synthase may be more important than inducible nitric oxide synthase for modulating renal fibrosis in obstructive uropathy.

Vitamin E treatment of focal segmental glomerulosclerosis: results of an open-label study.

Experimental data indicate that excessive production of reactive oxygen molecules contributes to progressive renal injury and that treatment with antioxidants attenuates this damage. Therefore, we investigated whether vitamin E supplementation could ameliorate renal disease and reduce proteinuria in children with a variety of kidney disorders. Vitamin E, 200 IU twice daily, was administered orally to 11 children with focal segmental glomerulosclerosis (FSGS) (group A) and 9 patients with miscellaneous kidney diseases (group B) [Henoch-Schönlein purpura nephritis (n=3), urinary tract anomalies (n=2), non-specific immune complex glomerulonephritis (n=2), IgA nephropathy (n=1), and reflux nephropathy (n=1)]. The duration of vitamin E treatment, when no other therapy was introduced, was 2.9+/-0.4 months. Proteinuria was determined by measuring the protein:creatinine ratio (mg/mg) in an early morning urine specimen. In children with FSGS, administration of vitamin E lowered the protein:creatinine ratio in 10 of 11 patients from 9. 7+/-5.1 to 4.1+/-1.1 (P<0.005). In contrast, among children with miscellaneous renal diseases, vitamin E had no beneficial impact on urinary protein excretion-protein:creatinine ratio 2.5+/-1.0 pre versus 2.4+/-1.2 post antioxidant. Vitamin E supplementation had no effect on glomerular filtration rate, serum albumin, or cholesterol concentration in either group of patients. These findings suggest that reactive oxygen molecules may play a more-prominent role in causing renal injury in patients with FSGS than in other kidney disorders. Antioxidant therapy may be a useful adjunct in the treatment of children with FSGS and proteinuria that is refractory to standard medical management.

Influence of alpha-tocopherol over the time course of experimental IgA nephropathy.

The present study investigated the pathogenesis and the time course of kidney injury in experimental IgA nephropathy. In order to determine an appropriate period in the course of experimental IgA nephropathy to study renal injury and repair, we examined proteinuria and IgA deposition in the renal mesangium after 4, 8, and 16 weeks of mucosal challenge by bovine gamma globulins (BGG) provided in the drinking water. The hallmark of IgA deposition in the mesangium was present after 4 weeks and 8 weeks of BGG inoculation, but by 16 weeks, the mesangial IgA deposition had resolved. In addition, we confirmed our previous report on the beneficial effects of alpha-tocopherol in reducing proteinuria in IgA nephropathy at 8 weeks, and extended this observation to investigate the effects of dietary supplementation of alpha-tocopherol at both 4 weeks and 16 weeks. Proteinuria resolved spontaneously at 16 weeks. There is oxidative stress, as suggested by the elevation in plasma and renal malondialdehyde content, and increased fibrogenic cytokine message, as suggested by elevated transforming growth factor beta1 mRNA. These increases were clearly blunted by alpha-tocopherol at both 4 weeks and 8 weeks. Treatment with alpha-tocopherol was associated with a significant reduction in the severity of proteinuria. Thus, our data suggest that the period between 4 and 8 weeks of BGG vaccination could be relevant in designing an appropriate model to study the molecular biology of the pathogenesis of renal injury and the effects of treatment. The 16-week model may be useful in exploring gene expression involved with spontaneous resolution.

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.

Effects of fish oil and alpha-tocopherol in immunoglobulin A nephropathy in the rat.

Alpha-tocopherol and fish oil have been reported to modulate the progression of IgA nephropathy in animals and humans. Because fish oil has been reported to exacerbate renal disease in subtotal nephrectomized rats, we investigated the effects of fish oil, with and without alpha-tocopherol, on the course of IgA nephropathy. Experimental IgA nephropathy was induced in male Sprague-Dawley rats, weighing 170-200 g, by oral and i.v. immunization with bovine gamma-globulin for 8 wk. IgA nephropathy was evidenced by hematuria, proteinuria, and IgA deposition in the mesangium. Standard rodent chow, containing 30 IU of alpha-tocopherol/kg of diet, was given to the control and IgA nephropathy rats. Fish oil (20% wt/wt), stripped of alpha-tocopherol preservative, was given to control and a second group of IgA nephropathy rats. Alternatively, corn oil or fish oil was supplemented with alpha-tocopherol at 100 IU/kg of diet and given to the third and fourth groups of IgA nephropathy rats. All animals were killed at 8 wk. Urinary protein excretion, plasma and kidney alpha-tocopherol concentrations, as well as glomerular planar area, and kidney transforming growth factor-beta1 mRNA were analyzed. As determined by reductions in proteinuria, glomerular planar area, and TGF-beta1 mRNA, fish oil with alpha-tocopherol ameliorated the renal injury induced by bovine gamma-globulin, whereas fish oil without alpha-tocopherol did not. Our findings support the importance of alpha-tocopherol, more so than fish oil, in mitigating the injury and promoting repair in experimental IgA nephropathy.

Vitamin E ameliorates renal injury in an experimental model of immunoglobulin A nephropathy.

IgA nephropathy is one of the most common forms of glomerular disease. Nearly 25% of affected patients progress to end-stage renal disease over a 20-25-y follow-up period. IgA-containing immune complexes stimulate oxygen-free radical production by mesangial cells in vitro. The excessive oxidant stress may mediate glomerular injury in this disorder. Therefore, we studied whether dietary supplementation with the antioxidant agent, vitamin E, attenuates renal disease in an experimental model of incipient IgA nephropathy with mild kidney inflammation. IgA nephropathy was induced in male Lewis rats by oral immunization with 0.1% bovine gamma-globulin (BGG)-containing drinking water for 8 wk. At the completion of this period, animals received BGG, 1 mg/dose i.v., on three successive days. Experimental rats (n = 10) received a specially formulated diet containing 100 IU of vitamin E/kg of chow, whereas control animals (n = 10) were fed chow containing 30 IU of vitamin/kg of chow. The BGG immunization regimen induced mesangial IgA deposition in all rats. Vitamin E supplementation resulted in a nearly 5-fold increase in the serum vitamin E concentration. Vitamin E-treated rats gained more weight and had a lower incidence of hematuria, 20% versus 80% (p < 0.03). Moreover, proteinuria was decreased by 50%, and reduced renal plasma flow was restored to normal, compared with untreated rats with IgA nephropathy. Glomerular hypertrophy occurred in animals with IgA nephropathy, but less so in those receiving vitamin E supplementation. Renal cortical malondialdehyde content was reduced from 1.55 +/- 0.10 to 1.22 +/- 0.09 nmol/mg of protein (p < 0.01) in rats fed the vitamin E-enriched diet. Finally, renal transforming growth factor-beta 1 gene expression was reduced by 34% in rats with IgA nephropathy receiving vitamin E treatment (p < 0.05). We conclude that experimental IgA nephropathy is associated with increased renal oxidant injury. Dietary treatment with the antioxidant agent, vitamin E, attenuated renal functional and structural changes in this experimental glomerulopathy. These studies support the importance of clinical trials for the evaluation of the efficacy of antioxidant therapy in patients with IgA nephropathy.

Taurine: A therapeutic agent in experimental kidney disease.

Taurine is an abundant free amino acid in the plasma and cytosol. The kidney plays a pivotal role in maintaining taurine balance. Immunohistochemical studies reveal a unique localization pattern of the amino acid along the nephron. Taurine acts as an antioxidant in a variety ofin vitro andin vivo systems. It prevents lipid peroxidation of glomerular mesangial cells and renal tubular epithelial cells exposed to high glucose or hypoxic culture conditions. Dietary taurine supplementation ameliorates experimental renal disease including models of refractory nephrotic syndrome and diabetic nephropathy. The beneficial effects of taurine are mediated by its antioxidant action. It does not attenuate ischemic or nephrotoxic acute renal failure or chronic renal failure due to sub-total ablation of kidney mass. Additional work is required to fully explain the scope and mechanism of action of taurine as a renoprotective agent in experimental kidney disease. Clinical trials are warranted to determine the usefulness of this amino acid as an adjunctive treatment of progressive glomerular disease and diabetic nephropathy.

Taurine ameliorates chronic streptozocin-induced diabetic nephropathy in rats.

We examined the effect of two endogenous antioxidant agents, taurine and vitamin E, on renal function in experimental diabetes. Male Sprague-Dawley rats, rendered diabetic with streptozocin (STZ), were assigned to one of the following groups: 1) untreated; 2) insulin treatment with 6 U Ultralente insulin/day in two doses; 3) taurine supplementation by 1% taurine in drinking water; and 4) vitamin E supplementation at 100 IU vitamin E/kg chow. Animals were kept for 52 wk. The survival rate was similar (70-90%) in all groups except vitamin E-treated animals, of which 84% died by 6 mo. At 52 wk, glomerular filtration rate was elevated in untreated and taurine-treated STZ rats compared with normal or insulin-treated diabetic rats. Taurine supplementation reduced total proteinuria and albuminuria by nearly 50%. This treatment also prevented glomerular hypertrophy, preserved immunohistochemical staining for type IV collagen in glomeruli, and diminished glomerulosclerosis and tubulointerstitial fibrosis in diabetic animals. The changes in renal function and structure in taurine-treated diabetic rats were associated with normalization of renal cortical malondialdehyde content, lowering of serum free Fe2+ concentration, and decreased formation of the advanced glycooxidation products, pentosidine, and fluorescence in skin collagen. Administration of the vitamin E-enriched diet exacerbated the nephropathy in STZ-diabetic rats. In addition, vitamin E supplementation increased serum free Fe2+ concentration, enhanced renal lipid peroxidation, and accelerated the accumulation of advanced glycosylation end products (AGEs) in skin collagen. We conclude that administration of taurine, but not vitamin E, to rats with STZ-diabetes ameliorates diabetic nephropathy. The beneficial effect of taurine is related to reduced renal oxidant injury with decreased lipid peroxidation and less accumulation of AGEs within the kidney.

Dietary vitamin E supplementation ameliorates renal injury in chronic puromycin aminonucleoside nephropathy.

Chronic puromycin aminonucleoside nephropathy (PAN) is an experimental analog of focal segmental glomerulosclerosis. Progressive renal damage in this model is partly mediated by excessive production of oxidant species. Whether dietary supplementation with vitamin E, an endogenous lipophilic antioxidant, ameliorates the severity of chronic PAN was tested. PAN was induced by seven serial injections of the glomerular epithelial cell toxin puromycin aminonucleoside, 2 mg/100 g body wt per dose, over a 12-wk period. Experimental animals (N = 8) received vitamin E-enriched chow (100 IU/kg), whereas control PAN rats (N = 10) were fed standard rodent diet containing vitamin E (30 IU/kg of chow). The administration of vitamin E had no effect on somatic growth or blood pressure; however, rats with PAN fed the vitamin E-enriched diet had an increased hematocrit. In addition, the experimental diet resulted in a 50% reduction in urinary total protein and albumin excretion and stabilization of the serum albumin, cholesterol, and triglyceride concentrations (P < 0.01). The inulin clearance was 69% higher in the vitamin E-supplemented animals (P < 0.001). Tubular function, namely, phosphate reabsorption and beta 2-microglobulin excretion, was improved in rats with chronic PAN treated with the vitamin E-enriched diet. There was a significant decrease in glomerulosclerosis and glomerular planar area, and tubulointerstitial scarring was diminished in vitamin E-treated animals with chronic PAN (P < 0.01). These beneficial effects on renal structure and function were associated with reduced malondialdehyde content in the kidney and liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Taurine attenuates renal disease in chronic puromycin aminonucleoside nephropathy.

Repeated administration of low doses of puromycin aminonucleoside (PAMN) to rats induces a proteinuric renal disease that resembles focal segmental glomerulosclerosis (FSGS). Reactive oxygen molecules may be involved in the progressive course of this nephropathy. Therefore we evaluated whether taurine, an endogenous antioxidant, could limit the extent of renal injury. Sprague-Dawley rats received low-dose injections of PAMN, 2 mg/100 g body wt, over a 12-wk period. Two groups were studied: 1) controls given tap water (n = 23), and 2) an experimental group that drank 1% taurine-supplemented water (n = 22). Taurine-treated nephrotic rats had a reduction in albuminuria, as assessed by the urinary albumin-to-creatinine ratio (26 +/- 4 vs. 44 +/- 4, P less than 0.0001). After 12 wk, creatinine clearance was 0.33 +/- 0.03 (experimental) vs. 0.17 +/- 0.03 ml.min-1.100 g body wt-1 (control) (P less than 0.001), and inulin clearance (n = 6 pairs) was 0.26 +/- 0.04 (experimental) vs. 0.13 +/- 0.02 ml.min-1.100 g body wt-1 (control) (P less than 0.025). Administration of taurine reduced the percentage of segmentally sclerosed glomeruli (9.8 +/- 1.7 vs. 16.2 +/- 1.8%, P less than 0.02) and the tubulointerstitial injury score (1.36 +/- 0.19 vs. 2.61 +/- 0.25, P less than 0.0025) in experimental vs. control rats. Taurine treatment normalized the elevated renal cortical malondialdehyde level in rats with PAMN nephropathy (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Parenteral calcitriol for treatment of severe renal osteodystrophy in children with chronic renal insufficiency.

Renal osteodystrophy is a common and incapacitating complication of chronic renal failure in children. Standard therapy with oral calcium supplements, phosphate binders, and vitamin D preparations is often inadequate to control progressive bone disease. We report the use of parenteral calcitriol therapy in two children, aged 2 and 15 years, respectively, with chronic renal failure. This treatment effectively suppressed secondary hyperparathyroidism in both patients, causing a nearly 50% reduction in circulating parathyroid hormone level and a parallel decline in serum alkaline phosphatase activity. In the younger patient, therapy was associated with healing of subperiosteal bone resorption and accelerated growth velocity. These findings indicate that parenteral administration of calcitriol may be an effective treatment option in some patients with refractory renal osteodystrophy and secondary hyperparathyroidism.