STAT-3 signaling role in an experimental model of nephropathy induced by doxorubicin.

The focal segmental glomerulosclerosis (FSGS) is one of the most frequent glomerulopathy in the world, being considered a significative public health problem worldwide. The disease is characterized by glomerular loss mainly due to inflammation process and collagen fibers deposition. STAT-3 is a transcription factor associated with cell differentiation, migration and proliferation and in renal cells it has been related with fibrosis, acting on the progression of the lesion. Considering this perspective, the present study evaluated the involvement of STAT-3 molecule in an experimental model of FSGS induced by Doxorubicin (DOX). DOX mimics primary FSGS by causing both glomerular and tubular lesions and the inhibition of the STAT3 pathway leads to a decrease in fibrosis and attenuation of kidney damage. We described here a novel FSGS experimental model in a strain of genetically heterogeneous mice which resembles the reality of FSGS patients. DOX-injected mice presented elevated indices of albuminuria and glycosuria, that were significantly reduced in animals treated with a STAT-3 inhibitor (STATTIC), in addition with a decrease of some inflammatory molecules. Moreover, we detected that SOCS-3 (a regulator of STAT family) was up-regulated only in STATTIC-treated mice. Finally, histopathological analyzes showed that DOX-treated group had a significant increase in a tubulointerstitial fibrosis and tubular necrosis, which were not identified in both control and STATTIC groups. Thus, our results indicate that STAT-3 pathway possess an important role in experimental FSGS induced by DOX and may be an important molecule to be further investigated.

Chronic VEGF blockade worsens glomerular injury in the remnant kidney model.

VEGF inhibition can promote renal vascular and parenchymal injury, causing proteinuria, hypertension and thrombotic microangiopathy. The mechanisms underlying these side effects are unclear. We investigated the renal effects of the administration, during 45 days, of sunitinib (Su), a VEGF receptor inhibitor, to rats with 5/6 renal ablation (Nx). Adult male Munich-Wistar rats were distributed among groups S+V, sham-operated rats receiving vehicle only; S+Su, S rats given Su, 4 mg/kg/day; Nx+V, Nx rats receiving V; and Nx+Su, Nx rats receiving Su. Su caused no change in Group S. Seven and 45 days after renal ablation, renal cortical interstitium was expanded, in association with rarefaction of peritubular capillaries. Su did not worsen hypertension, proteinuria or interstitial expansion, nor did it affect capillary rarefaction, suggesting little angiogenic activity in this model. Nx animals exhibited glomerulosclerosis (GS), which was aggravated by Su. This effect could not be explained by podocyte damage, nor could it be ascribed to tuft hypertrophy or hyperplasia. GS may have derived from organization of capillary microthrombi, frequently observed in Group Nx+Su. Treatment with Su did not reduce the fractional glomerular endothelial area, suggesting functional rather than structural cell injury. Chronic VEGF inhibition has little effect on normal rats, but can affect glomerular endothelium when renal damage is already present.

Invariant natural killer T cell agonist modulates experimental focal and segmental glomerulosclerosis.

A growing body of evidence demonstrates a correlation between Th2 cytokines and the development of focal and segmental glomerulosclerosis (FSGS). Therefore, we hypothesized that GSL-1, a monoglycosylceramide from Sphingomonas ssp. with pro-Th1 activity on invariant Natural Killer T (iNKT) lymphocytes, could counterbalance the Th2 profile and modulate glomerulosclerosis. Using an adriamycin(ADM)-based model of FSGS, we found that BALB/c mice presented albuminuria and glomerular degeneration in association with a Th2-like pro-fibrogenic profile; these mice also expressed a combination of inflammatory cytokines, such as IL-4, IL-1α, IL-1ß, IL-17, TNF-α, and chemokines, such as RANTES and eotaxin. In addition, we observed a decrease in the mRNA levels of GD3 synthase, the enzyme responsible for GD3 metabolism, a glycolipid associated with podocyte physiology. GSL-1 treatment inhibited ADM-induced renal dysfunction and preserved kidney architecture, a phenomenon associated with the induction of a Th1-like response, increased levels of GD3 synthase transcripts and inhibition of pro-fibrotic transcripts and inflammatory cytokines. TGF-ß analysis revealed increased levels of circulating protein and tissue transcripts in both ADM- and GSL-1-treated mice, suggesting that TGF-ß could be associated with both FSGS pathology and iNKT-mediated immunosuppression; therefore, we analyzed the kidney expression of phosphorylated SMAD2/3 and SMAD7 proteins, molecules associated with the deleterious and protective effects of TGF-ß, respectively. We found high levels of phosphoSMAD2/3 in ADM mice in contrast to the GSL-1 treated group in which SMAD7 expression increased. These data suggest that GSL-1 treatment modulates the downstream signaling of TGF-ß through a renoprotective pathway. Finally, GSL-1 treatment at day 4, a period when proteinuria was already established, was still able to improve renal function, preserve renal structure and inhibit fibrogenic transcripts. In conclusion, our work demonstrates that the iNKT agonist GSL-1 modulates the pathogenesis of ADM-induced glomerulosclerosis and may provide an alternative approach to disease management.

Podocyte Wnt/ß-catenin pathway is activated by integrin-linked kinase in clinical and experimental focal segmental glomerulosclerosis.

BACKGROUND: Changes in podocyte phenotype and function are characteristic of proteinuric glomerular diseases. Integrin-linked kinase (ILK) functions as a common downstream effector in proteinuric diseases. In addition, ILK was shown to interact with the Wnt signaling pathway. Here, we investigated ILK expression as well as its involvement with the Wnt signaling pathway in renal biopsies of patients with primary focal segmental glomerulosclerosis (FSGS), and in a correspondent in vivo model of podocyte lesion. METHODS: Biopsies from 37 patients with primary FSGS were evaluated by immunohistochemistry for ILK, phosphorylated GSK-3ß (pGSK-3ß) and ß-catenin expression. As experimental model, male Wistar rats received 5 injections of puromycin aminonucleoside (PAN) at 2-week intervals, and their kidneys were evaluated for ILK, P-cadherin and pAkt expression as well as ß-catenin and LEF-1 colocalization. RESULTS: Patients presented de novo ILK expression and pGSK-3ß in podocytes. In animals, there was an increase in gene and protein expression of ILK, mainly detected in the podocytes, as well as increased protein expression of pAkt compared with controls. ß-Catenin translocated to the nuclei of podocytes in animals and patients. ß-Catenin colocalized with LEF-1 in the nuclei of podocytes of animals. Gene expression of ß-catenin and P-cadherin in PAN rats was lower compared with controls. CONCLUSIONS: Our findings suggest that activation of ILK activated the Wnt signaling pathway in damaged podocytes. This phenomenon could have an important role in development and/or progression of clinical and experimental FSGS.

Bradykinin receptor 1 activation exacerbates experimental focal and segmental glomerulosclerosis.

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. The bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and α-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling.

The renal clearance of dextran sulfate decreases in puromycin aminonucleoside-induced glomerulosclerosis: a puzzle observation.

BACKGROUND: Puromycin aminonucleoside-induced nephrosis is characterized by increased renal excretion of plasma proteins. We employed this experimental model to study the urinary clearance of dextran sulfate. METHODS: The dextran sulfate eliminated by the urine was determined using a metachromatic assay. Polysaccharide fragments were analyzed by chromatographic and electrophoretic procedures. Disaccharide composition of the glomerular heparan sulfate was assessed using digestion with specific lyases. RESULTS: In normal rats dextran sulfate is partially degraded to lower molecular weight fragments and only then eliminated by the urine. Surprisingly, in puromycin aminonucleoside-induced glomerulosclerosis the molecular size of the fragments of dextran sulfate found in the urine is the same or even lower than in control animals in spite of the marked proteinuria. Furthermore, urinary excretion of dextran sulfate decreases in the experimentally induced nephrosis. This observation cannot be totally attributed to a reduced number of physiologically active nephrons since the glomerular filtration rate decreases approximately 32% after puromycin aminonucleoside administration while the urinary excretion of 8 kDa-dextran sulfate decreases 3-fold. The glomerular heparan sulfate shows reduced sulfation when compared with normal animals. Possibly puromycin aminonucleoside decreases the activity of kidney endoglycosidases, which reduce the molecular size of the sulfated polysaccharide, leading to a decrease in its renal clearance. Reduced sulfation of the glomerular heparan sulfate in the puromycin aminonucleoside-induced nephrosis does not alter the size of the dextran sulfate eliminated by the kidney, as suggested for protein. CONCLUSIONS: Each pathological process induces a particular modification in the kidney, which in turn can affect the renal selectivity to specific macromolecules in different ways.

Life-threatening thrombocytopenia and nephrotic syndrome due to focal segmental glomerulosclerosis associated with pegylated interferon alpha-2b and ribavirin treatment for hepatitis C.

BACKGROUND: Interferon-alpha (IFN-alpha)and ribavirin combination therapy for chronic infection with hepatitis C virus produces a number of well-described side effects. Combination therapy with pegylated interferon (PEG-IFN) yields an adverse event profile similar to that observed with the standard IFN, although the frequency of certain adverse events may vary according to the preparation. CASE REPORT: We report the case of a 44-year-old man who was treated with ribavirin and PEG-IFN-alpha-2b for chronic hepatitis C and developed two rare side effects simultaneously on the 16th week of therapy: severe thrombocytopenia and nephrotic syndrome due to focal segmental glomerulosclerosis. The antiviral treatment was immediately interrupted and the patient received immunosuppressive therapy. He promptly recovered from the thrombocytopenia and partially and slowly from the nephrotic syndrome. To our knowledge, this is the first case reported of the development of such complications at the same time.

Low molecular weight heparin in the treatment of puromycin-induced nephrosis.

Heparin may have a beneficial effect in proteinuric renal diseases, where negative charges of the glomerular capillary membrane are compromised. We evaluated the role of low molecular weight heparin (LMWH - 3000 Da) in puromycin aminonucleoside (PAN)-induced focal and segmental glomerulosclerosis in male Wistar rats: Controls (C) n=7, LMWH-treated group, n=9, subcutaneously (SC), 6 mg/kg every day. The PAN group (n=7) received 7 doses on weeks 0, 1, 2, 4, 6, 8, 10 (SC - 2mg/100g), and a group PAN+LMWH (n=6). After 12 weeks, cholesterol and triglycerides were higher in nephrotic groups, as well as proteinuria and urinary IgG. Kidney weight, glomerular volume, and glomerular sclerosis index were higher in the PAN-treated groups. Glomerular capillary length density (L(Vcap)) and glomerular capillary surface density (S(Vcap)) were lower in the PAN group, and mesangial fractional volume was higher. Fibronectin immunostaining was more intense in the PAN group, and collagens I and III were absent in the studied glomeruli. Thus, LMWH prevented mesangial expansion and capillaries changes, showing antiproliferative properties, despite worsening glomerular permeability changes in the PAN model. In conclusion, LMWH interferes in the complications of PAN model, but not through inhibition of the proteinuria.

Mycophenolate mofetil prevents salt-sensitive hypertension resulting from nitric oxide synthesis inhibition.

Recent studies have suggested that subtle microvascular and tubulointerstitial injury in the kidney can cause salt-sensitive hypertension. To test this hypothesis, we determined whether the mild renal disease induced by transient blockade of nitric oxide (NO) synthesis would result in salt-sensitive hypertension and whether prevention of the renal injury by coadministration of the immunosuppressive agent mycophenolate mofetil (MMF) would block the development of salt sensitivity. N(omega)-nitro-L-arginine-methyl ester (L-NAME; 70 mg/100 ml in the drinking water) was administered for 3 wk to rats with or without MMF (30 mg x kg(-1) x day(-1) by gastric gavage), followed by a 1-wk "washout" period in which the MMF was continued, which was followed in turn by placement on a high-salt (4% NaCl) diet for an additional 4 wk. Renal histology was examined at 3 and 8 wk, and blood pressure was measured serially. L-NAME treatment resulted in acute hypertension and the development of mild renal injury. During the washout period, blood pressure returned to normal, only to return to the hypertensive range on exposure of the animals to a high-salt diet. MMF treatment prevented the development of hypertension in response to a high-salt diet. This correlated with the ability of MMF to inhibit specific aspects of the renal injury, including the development of segmental glomerulosclerosis, the infiltration of T cells and ANG II-positive cells, and the thickening of afferent arterioles.

Reduction of urine volume ameliorates adriamycin-induced nephropathy.

1. Adriamycin, a commonly used antineoplastic antibiotic, induces glomerular lesions in rats, resulting in persistent proteinuria and glomerulosclerosis. 2. The effect of urine volume on the progression of adriamycin-induced nephropathy was studied in 70 male Wistar rats (180-200 g) observed for 30 weeks and separated into 4 groups: healthy control group (HCG, N = 10) inoculated i.v. with 1 ml of saline, and nephrotic groups inoculated iv with a single dose of adriamycin of 3 mg/kg body weight. The nephrotic rats were separated into 3 groups (N = 20): nephrotic control group (NCG) receiving only adriamycin; dehydrated nephrotic group (DNG) water deprived for 36 h within each 48-h period, and furosemide nephrotic group (FNG) treated with 12 mg/dl furosemide, and 0.9 g/dl NaCl in the drinking water. 3. The 30-week survival rates of the DNG (100%) and HCG (100%) were significantly higher than those of the NCG (85%) and FNG (55%). 4. The proteinuria observed in the HCG (range, 7.38 +/- 0.7 to 13.6 +/- 1.27 mg/24 h) was significantly lower than that observed for all the nephrotic groups throughout the experiment. The DNG presented significantly less proteinuria (range, 42.71 +/- 6.83 to 140.10 +/- 19.22 mg/24 h) than the NCG (range, 35.32 +/- 7.64 to 250.00 +/- 25.91 mg/24 h) from week 10 on. There was no significant difference between the mean 24-h proteinuria of the NCG (range, 35.32 +/- 7.64 to 250.00 +/- 25.91 mg/24 h) and the FNG (range, 35.82 +/- 7.91 to 221.54 +/- 26.74). 5. The mean frequency of damaged glomeruli was 0.3% +/- 0.3 for HCG, 42% +/- 6% for CNG, 40.8% +/- 8% for DNG, and 47% +/- 14% for FNG. The median value of the tubulointerstitial lesion, evaluated by a semiquantitative method, was 0 in HCG, 10 in CNG, 8.5 in DNG and 9.5 in FNG (P < 0.05 for all groups compared to HCG). 6. The data indicate that reduction of urine volume has a protective effect on adriamycin-induced nephropathy.

Reduction of urine volume ameliorates adriamycin-induced nephropathy

1. Adriamycin, a commonly used antineoplastic antibiotic, induces glomerular lesions in rats, resulting in persistent proteinuria and glomerulosclerosis. 2. The effect of urine volume on the progression of adriamycin-induced nephropathy was studied in 70 male Wistar rats (180-200 g) observed for 30 weeks and separated into 4 groups: healthy control group (HCG, N = 10) inoculated i.v. with 1 ml of saline, and nephrotic groups inoculated iv with a single dose of adriamycin of 3 mg/kg body weight. The nephrotic rats were separated into 3 groups (N = 20): nephrotic control group (NCG) receiving only adriamycin; dehydrated nephrotic group (DNG) water deprived for 36 h within each 48-h period, and furosemide nephrotic group (FNG) treated with 12 mg/dl furosemide, and 0.9 g/dl NaCl in the drinking water. 3. The 30-week survival rates of the DNG (100 per cent ) and HCG (100 per cent ) were significantly higher than those of the NCG (85 per cent ) and FNG (55 per cent ). 4. The proteinuria observed in the HCG (range, 7.38 +/- 0.7 to 13.6 +/- 1.27 mg/24 h) was significantly lower than that observed for all the nephrotic groups throughout the experiment. The DNG presented significantly less proteinuria (range, 42.71 +/- 6.83 to 140.10 +/- 19.22 mg/24 h) than the NCG (range, 35.32 +/- 7.64 to 250.00 +/- 25.91 mg/24 h) from week 10 on. There was no significant difference between the mean 24-h proteinuria of the NCG (range, 35.32 +/- 7.64 to 250.00 +/- 25.91 mg/24 h) and the FNG (range, 35.82 +/- 7.91 to 221.54 +/- 26.74). 5. The mean frequency of damaged glomeruli was 0.3 per cent +/- 0.3 for HCG, 42 per cent +/- 6 per cent for CNG, 40.8 per cent +/- 8 per cent for DNG, and 47 per cent +/- 14 per cent for FNG. The median value of the tubulointerstitial lesion, evaluated by a semiquantitative method, was 0 in HCG, 10 in CNG, 8.5 in DNG and 9.5 in FNG (P < 0.05 for all groups compared to HCG). 6. The data indicate that reduction of urine volume has a protective effect on adriamycin-induced nephropathy