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Divergent effects of low versus high dose anti-TGF-β antibody in puromycin aminonucleoside nephropathy in rats.BackgroundTransforming growth factor-β (TGF-β) modulates immune/inflammatory cells, promotes extracellular matrix (ECM) accumulation, and is increased in fibrotic organs. Here we report the effects of administering a puromycin aminonucleoside nephropathy (PAN)-specific TGF-β neutralizing antibody on glomerulosclerosis in vivo.MethodsAdult male Sprague-Dawley rats underwent uninephrectomy (Nx) followed by intraperitoneal PAN at weeks 2, 6, 7 and 8. Rats were treated with either high (5mg/kg body weight) (N = 9) or low (0.5mg/kg body weight) (N = 7) dose TGF-β antibody intraperitoneally three times weekly until sacrifice at week 10. A PAN untreated control group (N = 7) was dosed with an isotype specific, null antibody. The nephrectomy samples were studied as normal kidney control (NL) (N = 5). Rats undergoing left kidney Nx (N = 5) only were also included as age-matched control. Renal function and morphology were assessed, and molecular studies performed.ResultsSystolic blood pressure was increased in parallel over time in all groups (at 10weeks, control 137 ± 10mm Hg; high 129 ± 4mm Hg; low 137 ± 3mm Hg) (P = NS). Both TGF-β antibody treatments decreased renal cortex mRNA expressions similarly for TGF-β1, TGF-β2, and collagen III (TGF-β1, control 0.36 ± 0.02mm Hg; high 0.19 ± 0.01mm Hg; low 0.19 ± 0.02mm Hg; P < 0.01 low and high vs. control; TGF-β2, control 0.38 ± 0.03mm Hg; high 0.19 ± 0.02mm Hg; low 0.20 ± 0.03mm Hg; P < 0.01 low and high vs. control; and collagen III, control 0.33 ± 0.01mm Hg; high 0.14 ± 0.01mm Hg; low 0.19 ± 0.01mm Hg; P < 0.01 low and high vs. control; P < 0.05 low vs. high, data expressed as mRNA normalized density units vs. 18S RNA). However, only low dose TGF-β antibody improved renal function and sclerosis measured by serum creatinine and creatinine clearance (serum creatinine, control 2.3 ± 0.5mg/dL; high 2.5 ± 0.5mg/dL; low 0.8 ± 0.1mg/dL; P < 0.05 low vs. control and high; creatinine clearance, control 0.44 ± 0.11mL/min; high 0.70 ± 0.26mL/min; low 1.34 ± 0.30mL/min; P < 0.05 low vs. control, P = NS vs. high). In parallel, sclerosis index (0 to 4+ scale) was improved in low dose (control 2.67 ± 0.27; high 2.37 ± 0.30; low 1.78 ± 0.24; P < 0.05 low vs. control). This improved function and structure was linked to decreased glomerular infiltrating macrophages (0 to 4+ score, control 2.3 ± 0.2; high 1.8 ± 0.4; low 0.8 ± 0.1; P < 0.01 low vs. control; P < 0.05 low vs. high; P = NS high vs. control). Further, plasminogen activator inhibitor-1 (PAI-1) mRNA expression in renal cortex was attenuated after low dose TGF-β antibody treatment compared to control and high dose group (PAI-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio, NL 0.18 ± 0.003; control 0.45 ± 0.03; high 0.40 ± 0.04; low 0.23 ± 0.01; P < 0.05 low vs. control and high). Matrix metalloproteinase-9 (MMP-9) activity was maintained at higher levels in kidneys of the low dose TGF-β antibody-treated group.ConclusionThese results show an in vivo dose-response with an agent that blocks the biologic activity of TGF-β. Higher dose of TGF-β antibody was without beneficial effect, suggesting that TGF-β–mediated effects on PAI-1 and macrophage influx are bimodal and closely regulated. Given that both antibody doses reduced the expression of TGF-β isoforms and collagen III production, but only low dose ameliorated histologic sclerosis, it appears that pharmacologic effects of anti-TGF-β antibody on matrix synthesis and degradation are not equivalent.

Antibody to transforming growth factor-β ameliorates tubular apoptosis in unilateral ureteral obstruction. Background Unilateral ureteral obstruction (UUO) is characterized by progressive renal atrophy, renal interstitial fibrosis, an increase in renal transforming growth factor-β (TGF-β), and renal tubular apoptosis. The present study was undertaken to determine the effect of a monoclonal antibody to TGF-β (1D11) in UUO. Methods Mechanical stretch was applied to tubular epithelial cells (NRK-52E) by a computer-assisted system. Three doses of 1D11 (either 0.5, 2, or 4 mg/rat) were administered to rats one day prior to UUO and every two days thereafter, and kidneys were harvested at day 13. Fibrosis was assessed by measuring tissue hydroxyproline and mRNA for collagen and fibronectin. Apoptosis was assessed with the terminal deoxy transferase uridine triphosphate nick end-labeling assay. TGF-β levels were determined by bioassay. Western blot and immunostaining were used to identify proliferating cell nuclear antigen (PCNA), p53, bcl-2, and inducible nitric oxide synthase (iNOS). Results Stretch significantly induced apoptosis in NRK-52E cells, which was accompanied by an increased release of TGF-β; 1D11 (10 μg/mL) totally inhibited stretch-induced apoptosis. Control obstructed kidney contained 20-fold higher TGF-β as compared with its unobstructed kidney; 1D11 neutralized tissue TGF-β of the obstructed kidney. Control obstructed kidney exhibited significantly more fibrosis and tubular apoptosis than its unobstructed counterpart, which was blunted by 1D11. In contrast, 1D11 significantly increased tubular proliferation. p53 immunostaining was localized to renal tubular nuclei of control obstructed kidney and was diminished by 1D11. In contrast, bcl-2 was up-regulated in the 1D11-treated obstructed kidney. Total NOS activity and iNOS activity of the obstructed kidney were increased by 1D11 treatment. Conclusion The present study strongly suggests that an antibody to TGF-β is a promising agent to prevent renal tubular fibrosis and apoptosis in UUO.