Emerging evidence suggests that transforming growth factor-␤ (TGF-␤) is an important mediator of diabetic nephropathy. We showed previously that short-term treatment with a neutralizing monoclonal anti-TGF-␤ antibody (␣T) in streptozotocin-diabetic mice prevents early changes of renal hypertrophy and increased matrix mRNA. To establish that overactivity of the renal TGF-␤ system mediates the functional and structural changes of the more advanced stages of nephropathy, we tested whether chronic administration of ␣T prevents renal insufficiency and glomerulosclerosis in the db͞db mouse, a model of type 2 diabetes that develops overt nephropathy. Diabetic db͞db mice and nondiabetic db͞m littermates were treated intraperitoneally with ␣T or control IgG, 300 g three times per week for 8 wk. Treatment with ␣T, but not with IgG, significantly decreased the plasma TGF-␤1 concentration without decreasing the plasma glucose concentration. The IgG-treated db͞db mice developed albuminuria, renal insufficiency, and glomerular mesangial matrix expansion associated with increased renal mRNAs encoding ␣1(IV) collagen and fibronectin. On the other hand, treatment with ␣T completely prevented the increase in plasma creatinine concentration, the decrease in urinary creatinine clearance, and the expansion of mesangial matrix in db͞db mice. The increase in renal matrix mRNAs was substantially attenuated, but the excretion of urinary albumin factored for creatinine clearance was not significantly affected by ␣T treatment. We conclude that chronic inhibition of the biologic actions of TGF-␤ with a neutralizing monoclonal antibody in db͞db mice prevents the glomerulosclerosis and renal insufficiency resulting from type 2 diabetes.

Pirfenidone is an oral antifibrotic agent that benefits diabetic nephropathy in animal models, but whether it is effective for human diabetic nephropathy is unknown. We conducted a randomized, double-blind, placebocontrolled study in 77 subjects with diabetic nephropathy who had elevated albuminuria and reduced estimated GFR (eGFR) (20 to 75 ml/min per 1.73 m 2 ). The prespecified primary outcome was a change in eGFR after 1 year of therapy. We randomly assigned 26 subjects to placebo, 26 to pirfenidone at 1200 mg/d, and 25 to pirfenidone at 2400 mg/d. Among the 52 subjects who completed the study, the mean eGFR increased in the pirfenidone 1200-mg/d group (ϩ3.3 Ϯ 8.5 ml/min per 1.73 m 2 ) whereas the mean eGFR decreased in the placebo group (Ϫ2.2 Ϯ 4.8 ml/min per 1.73 m 2 ; P ϭ 0.026 versus pirfenidone at 1200 mg/d). The dropout rate was high (11 of 25) in the pirfenidone 2400-mg/d group, and the change in eGFR was not significantly different from placebo (Ϫ1.9 Ϯ 6.7 ml/min per 1.73 m 2 ). Of the 77 subjects, 4 initiated hemodialysis in the placebo group, 1 in the pirfenidone 2400-mg/d group, and none in the pirfenidone 1200-mg/d group during the study (P ϭ 0.25). Baseline levels of plasma biomarkers of inflammation and fibrosis significantly correlated with baseline eGFR but did not predict response to therapy. In conclusion, these results suggest that pirfenidone is a promising agent for individuals with overt diabetic nephropathy.

Diabetic nephropathy is a common complication in patients with either type I or type II diabetes. The pathogenesis of diabetic nephropathy is thought to involve both metabolic and vascular factors leading to chronic accumulation of glomerular mesangial matrix. In this context, both transforming growth factor-beta (TGF-beta) and endothelin may contribute to these processes. To determine if diabetic patients demonstrate increased renal production of TGF-beta and endothelin, aortic, renal vein, and urinary levels of these factors were measured in 14 type II diabetic patients and 11 nondiabetic patients who were undergoing elective cardiac catheterization. Renal blood flow was measured in all patients to calculate net mass balance across the kidney. Diabetic patients demonstrated net renal production of immunoreactive TGF-beta1 (830 +/- 429 ng/min [mean +/- SE]), whereas nondiabetic patients demonstrated net renal extraction of circulating TGF-beta1 (-3479 +/- 1010 ng/min, P < 0.001). Urinary levels of bioassayable TGF-beta were also significantly increased in diabetic patients compared with nondiabetic patients (2.435 +/- 0.385 vs. 0.569 +/- 0.190 ng/mg creatinine, respectively; P < 0.001). Renal production of immunoreactive endothelin was not significantly increased in diabetic patients. In summary, type II diabetes is associated with enhanced net renal production of TGF-beta1, whereas nondiabetic patients exhibit net renal extraction of circulating TGF-beta1. Increased renal TGF-beta production may be an important manifestation of diabetic kidney disease.

Although several interventions slow the progression of diabetic nephropathy, current therapies do not halt progression completely. Recent preclinical studies suggested that pirfenidone (PFD) prevents fibrosis in various diseases, but the mechanisms underlying its antifibrotic action are incompletely understood. Here, we evaluated the role of PFD in regulation of the extracellular matrix. In mouse mesangial cells, PFD decreased TGF-␤ promoter activity, reduced TGF-␤ protein secretion, and inhibited TGF-␤-induced Smad2-phosphorylation, 3TP-lux promoter activity, and generation of reactive oxygen species. To explore the therapeutic potential of PFD, we administered PFD to 17-wk-old db/db mice for 4 wk. PFD treatment significantly reduced mesangial matrix expansion and expression of renal matrix genes but did not affect albuminuria. Using liquid chromatography with subsequent electrospray ionization tandem mass spectrometry, we identified 21 proteins unique to PFD-treated diabetic kidneys. Analysis of gene ontology and protein-protein interactions of these proteins suggested that PFD may regulate RNA processing. Immunoblotting demonstrated that PFD promotes dosage-dependent dephosphorylation of eukaryotic initiation factor, potentially inhibiting translation of mRNA. In conclusion, PFD is renoprotective in diabetic kidney disease and may exert its antifibrotic effects, in part, via inhibiting RNA processing.