Protective activity of gallic acid against glyoxal -induced renal fibrosis in experimental rats

Abstract

This study was designed to evaluate the protective activity of gallic acid (GA) against glyoxal (GO) an advanced glycation intermediate-induced renal fibrosis in experimental rats. Glyoxal (i.p) at a dose of 15 mg/Kg body weight/day for 4 weeks induces renal fibrosis. GA was administered orally (100 mg/Kg body weight/day) along with GO for 4 weeks. The anti-fibrotic activity of GA was analyzed by measuring the collagen synthesis and deposition in renal tissues using mRNA expression analysis and Masson trichrome staining (MTS), respectively. The nephroprotective potential of GA was assessed by quantifying the markers of kidney damage such as serum blood-urea-nitrogen (BUN), creatinine (CR) and alkaline phosphatase (AP). Moreover, basement membrane damage in renal tissues was analysed by periodic acid Schiff's (PAS) staining. GA co-treatment markedly suppressed the GO-induced elevation in mRNA expression of collagenIand III, MMP-2, MMP-9 and NOX (p < 0.05, respectively) genes as compared with GO alone infused rats. In addition, GA co-treatment significantly attenuated the GO -induced elevation in serum markers such as BUN, CR and AP levels (p < 0.05, respectively). Furthermore, GA co-treatment restored back the decreased renal super oxide dismutase (SOD) activity (p < 0.05) thereby assuage the reactive oxygen species (ROS) generation, and maintained the normal architecture of glomerulus. The present study clearly indicates that GO -induces renal fibrosis by enhancing GO/receptor of advanced glycation end product (RAGE) induced ROS generation and GA effectively counteracted GO-induced renal fibrosis by its ROS quenching and anti-glycation activity.

Keywords: AGE intermediate; Fibrosis; Gallic acid (GA); Glycation; Glyoxal (GO); Renal hypertrophy.

Fig. 1

Kidney sections from the control and experimental group animals were stained with H & E; Masson trichrome and Periodic Schiff’s stain (n = 6/group). H&E stain (a–d). Panel (a) normal control; Panel (b) GO infused rats; Panel (c) GO + GA treated rats; Panel (d) GA control animals. Masson’s trichrome to visualize collagen deposition (e–h). Panel (e) normal control; Panel (f) GO infused rats shows increase blue staining reflects interstitial fibrosis, panel (g) gallic acid treated renal sections GO + GA shows mild increase in the blue stain in the tubules. Panel (h) GA control rats. PAS staining (i–l) visualize basement membrane damage. Panel (i) normal control; Panel (j) GO alone infused rats; Panel (k) GO + GA group animals; Panel (l) GA control animals, renal sections were shown at 40X magnification. Scale bar- 50 μm.

Fig. 2

(A) Shows the total collagen content in the kidney tissue of experimental animals. Increased in the collagen content in GO group animals compared with the control group and there was a significant decrease in the collagen levels of GO + GA group animals as compared with the GO group. No significant change was observed in the GA group compared with the control group animals. (B and C) represents an increased mRNA expression of Collagen I and Collagen III in GO group animals and there was a significant decrease in the mRNA expression levels of GO + GA group animals. GAPDH was used as internal control. Results were expressed as Mean ± SEM (n = 6/group). Significance is indicated as *p < 0.05; **p < 0.01 and NS- non significant.

Fig. 3

Zymogram analysis of MMP-2 and -9 activity showing activity of MMP-2 and MMP-9 in the tissue extracts. Gels were stained with 0.25% Coommassie brilliant blue and destained until the clear lytic bands were visible. Results were expressed as Mean ± SEM (n = 6/group). Significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.01 and NS- non significant. (B) mRNA expression of MMP-2 and -9 in control and experimental group animals. GAPDH was used as internal control. Results were expressed as Mean ± SEM (n = 6/group). Significance is indicated as *p < 0.05; **p < 0.01 and NS- non significant. (C) mRNA expression levels of NOX-4 in control and experimental group animals. GAPDH was used as internal control. Results were expressed as Mean ± SEM (n = 6/group). Significance is indicated as *p < 0.05; **p < 0.01 and NS- non significant. (D) The figure shows the spectroflourimetric analysis of ROS generation in control, GO, GO + GA and GA treated tissue extract. Results were expressed as Mean ± SEM (n = 6/group). Significance is indicated as *p < 0.05; ***p < 0.001 and NS- non significant.

Fig. 4

Immunostaining analysis of kidney sections from the control and experimental group animals (n = 6/group). Panel (a–d) shows MMP-2, Panel (e–h) shows MMP-9, Panel (i–l) shows RAGE, Panel (m–p) shows SOD expressions in the kidney sections of control and experimental group of rats respectively. An increased MMP-2 and -9 expression is evidenced in (panel b and f, respectively) in GO treated rat renal sections. An increased RAGE and decreased SOD expression is evidenced in (panel j and n, respectively) in GO treated rat renal sections. GA treatment neutralizes GO induced MMPs (panel c and g, respectively) RAGE (panel k) and SOD (panel n) expressions in the renal tissue sections. Kidney tissue sections were observed at 40X Magnification.

Fig. 5

Immunoblotting analysis of MMP-2, MMP-9 and RAGE. β-actin was used as internal control. Where, Lane C-control; Lane GO – Glyoxal (GO) alone infused rats; Lane GO + GA - Gallic acid (GA) along with GO group animals; Lane GA - GA control animals. Results were expressed as Mean ± SEM (n = 6/group). Significance is indicated as *p < 0.05; **p < 0.01 and NS- non significant.

Fig. 6

(A) indicates the released Malondialdehyde-an indicator of LPO is expressed as nmoles/mg protein. (B) Activity is expressed as 50% inhibition of epinephrine auto-oxidation for SOD. (C) Native polyacrylamide gel activity assay for SOD activity in kidney tissue extract of experimental group animals. Where, Lane C-control; Lane GO – Glyoxal (GO) alone infused rats; Lane GO + GA - Gallic acid (GA) along with GO group animals; Lane GA - GA control animals. Results were expressed as Mean ± SEM (n = 6/group). Significance is indicated as *p < 0.05; **p < 0.01 and NS- non significant.