Blockade of endogenous tissue kallikrein aggravates renal injury by enhancing oxidative stress and inhibiting matrix degradation

Abstract

Levels of tissue kallikrein (TK) are significantly lower in the urine of patients with kidney failure, and TK expression is specifically diminished in rat kidney after recovery from ischemia-reperfusion injury. In this study, we investigated the functional consequence of blocking endogenous TK activity in a rat model of chronic kidney disease. Inhibition of endogenous TK levels for 10 days by neutralizing TK antibody injection in DOCA-salt rats caused a significant increase in blood urea nitrogen and urinary protein levels, and a decrease in creatinine clearance. Kidney sections from anti-TK antibody-treated rats displayed a marked rise in tubular dilation and protein cast accumulation as well as glomerular sclerosis and size. TK blockade also increased inflammatory cell infiltration, myofibroblast and collagen accumulation, and collagen fraction volume. Elevated renal inflammation and fibrosis by anti-TK antibody were associated with increased expression of tumor necrosis factor-alpha, intercellular adhesion molecule-1, tissue inhibitor of metalloproteinase-2 (TIMP-2), and plasminogen activator inhibitor-1 (PAI-1). Moreover, the detrimental effect of TK blockade resulted in reduced nitric oxide (NO) levels as well as increased serum lipid peroxidation, renal NADH oxidase activity, and superoxide formation. In cultured proximal tubular cells, TK inhibited angiotensin II-induced superoxide production and NADH oxidase activity via NO formation. In addition, TK markedly increased matrix metalloproteinase-2 activity with a parallel reduction of TIMP-2 and PAI-1 synthesis. These findings indicate that endogenous TK has the propensity to preserve kidney structure and function in rats with chronic renal disease by inhibiting oxidative stress and activating matrix degradation pathways.

Fig. 1.

Blockade of endogenous tissue kallikrein (TK) by neutralizing anti-TK antibody worsens renal injury in DOCA-salt rats, as demonstrated by exacerbated tubular dilation, brush-border loss, protein cast formation, glomerulosclerosis, and glomerular size. A: representative images of periodic acid Schiff (PAS) and silver staining in kidney sections. Original magnification is ×200. B: semiquantitative glomerular sclerotic score. C: semiquantitative analysis of glomerular size. Values are expressed as means ± SE (n = 6). *P < 0.05 vs. sham. †P < 0.05 vs. DOCA/IgG.

Fig. 2.

Blockade of endogenous TK by neutralizing anti-TK antibody promotes the accumulation of monocytes/macrophages and elevates tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) expression in the kidney of DOCA-salt rats. A: representative images of cells positive for ED-1, a marker of monocytes/macrophages, in the renal cortex. Original magnification is ×200. B: quantification of monocytes/macrophages in the renal cortex. TNF-α (C) and ICAM-1 (D) mRNA levels determined by quantitative RT-PCR. Values are expressed as means ± SE (n = 6). *P < 0.05 and ‡P < 0.01 vs. sham. †P < 0.05 vs. DOCA/IgG.

Fig. 3.

Blockade of endogenous TK by neutralizing anti-TK antibody exacerbates DOCA-salt-induced renal fibrosis and profibrotic gene expression. A: representative images of histochemical staining with Sirius red, which stains collagen fibers red, and immunohistochemical staining for α-smooth muscle actin (α-SMA), a marker of myofibroblasts. Original magnification is ×200. B: quantification of collagen fraction volume. C: quantification of α-SMA-positive cells. TIMP-2 (D) and PAI-1 (E) mRNA levels in the kidney determined by quantitative RT-PCR. Values are expressed as means ± SE (n = 6). *P < 0.05 vs. sham. †P < 0.05 vs. DOCA/IgG.

Fig. 4.

TK reduces ANG II-induced reactive oxygen species (ROS) generation and NADH oxidase activity in human proximal tubular cells (HKCs) via nitric oxide (NO) formation. A: representative images of ANG II-induced DCF fluorescence. DCF fluorescence results from the oxidation of 2′,7′-dichlorofluorescein diacetate (DCF-DA) by H₂O₂. Original magnification is ×400. B: quantification of ROS production as determined by DCF-DA fluorescence. C: NADH oxidase activity in HKCs as determined by chemiluminescence assay. Control, no treatment; rlu, relative light units. Values are expressed as means ± SE (n = 4). *P < 0.05 vs. control. †P < 0.05 vs. ANG II.

Fig. 5.

TK exerts anti-fibrotic actions in HKCs stimulated with transforming growth factor-β (TGF-β). Gelatin zymography (A) and densitometric analysis (B) of matrix metalloproteinase-2 (MMP-2) activity of HKC-conditioned media. Western blot (C) and quantitative RT-PCR analysis (D) of tissue inhibitor of metalloproteinase-2 (TIMP-2) expression. Western blot (E) and quantitative RT-PCR analysis (F) of plasminogen activator inhibitor-1 (PAI-1) expression. Control, no treatment. Values are expressed as means ± SE (n = 4). *P < 0.05 and ‡P < 0.01 vs. control. †P < 0.01 and §P < 0.05 vs. TGF-β.