Protective effect of metalloporphyrins against cisplatin-induced kidney injury in mice

Abstract

Oxidative and nitrative stress is a well-known phenomenon in cisplatin-induced nephrotoxicity. The purpose of this work is to study the role of two metalloporphyrins (FeTMPyP and MnTBAP), water soluble complexes, in cisplatin-induced renal damage and their ability to scavenge peroxynitrite. In cisplatin-induced nephropathy study in mice, renal nitrative stress was evident by the increase in protein nitration. Cisplatin-induced nephrotoxicity was also evident by the histological damage from the loss of the proximal tubular brush border, blebbing of apical membranes, tubular epithelial cell detachment from the basement membrane, or intra-luminal aggregation of cells and proteins and by the increase in blood urea nitrogen and serum creatinine. Cisplatin-induced apoptosis and cell death as shown by Caspase 3 assessments, TUNEL staining and DNA fragmentation Cisplatin-induced nitrative stress, apoptosis and nephrotoxicity were attenuated by both metalloporphyrins. Heme oxygenase (HO-1) also plays a critical role in metalloporphyrin-mediated protection of cisplatin-induced nephrotoxicity. It is evident that nitrative stress plays a critical role in cisplatin-induced nephrotoxicity in mice. Our data suggest that peroxynitrite is involved, at least in part, in cisplatin-induced nephrotoxicity and protein nitration and cisplatin-induced nephrotoxicity can be prevented with the use of metalloporphyrins.

Figure 1. Effects of metalloporphyrins on Cisplatin induced renal dysfunction in mice.

Cisplatin-induced renal dysfunction was measured by the levels of BUN and creatinine. A. BUN and Creatinine levels were measured at 72 h after cisplatin administration. Cisplatin administration resulted in severe kidney injuries which were attenuated by FeTMPyP and MnTBAP treatments. B. BUN and Creatinine levels were measured at 72 h after cisplatin administration. FeTMPyP and MnTBAP were administered 12 hour after cisplatin treatment. Results are mean ± S.E.M. n = 4–5/group. *p<0.05 versus vehicle; and ^#p<0.05 versus cisplatin.

Figure 2. Effects of metalloporphyrins on Cisplatin induced kidney tubular damage in mice.

Histological examination (Panel A) and quantification (Panel B) revealed necrosis, protein cast, vacuolation, and desquamation of epithelial cells in the renal tubules of the cisplatin-treated group. Cisplatin administration resulted in severe tubular damage. Cisplatin induced damages were attenuated by FeTMPyP and MnTBAP treatments (n = 4–5/each group; p<0.01). Results are mean ± S.E.M. n = 4–5/group. *p<0.05 versus vehicle; and ^#p<0.05 versus cisplatin.

Figure 3. Effects of metalloporphyrins on Cisplatin induced nitrative stress in mice.

Panel A: Histological examination revealed significant protein nitration in the renal tubules of the cisplatin-treated group. FeTMPyP and MnTBAP treatments lead to reduced protein nitration similar to vehicle level (n = 4–5/each group; p<0.01). Panel B: Quantitative measurement of protein nitration also demonstrated 3.9 fold increased by cisplatin and the protein nitration is decreased to 1.5 and 1.4 fold for FeTMPyP and MnTBAP treated groups respectively. Results are mean ± S.E.M. n = 4–5/group. *p<0.05 versus vehicle; and ^#p<0.05 versus cisplatin.

Figure 4. Effects of metalloporphyrins on Cisplatin induced early apoptosis marker Caspase 3.

Histological examination (Panel A) demonstrated cisplatin induced cleaved caspase 3 staining in kidney and the caspase 3 staining were significantly attenuated with pretreatments of FeTMPyP or MnTBAP. Panel B: caspase 3 activity were measured and similar trend was observed. Results are mean ± S.E.M. n = 4–5/group. *p<0.05 versus vehicle; and ^#p<0.05 versus cisplatin.

Figure 5. Effects of metalloporphyrins on Cisplatin induced tubular apoptosis.

TUNEL-positive apoptotic cell numbers were also increased in cisplatin-treated mice and attenuated by FeTMPyP or MnTBAP treatments.

Figure 6. Effects of metalloporphyrins on Cisplatin induced DNA fragmentation.

Cisplatin induced significant DNA fragmentation and the effect was attenuated by FeTMpyP or MnTBAP treatments. Results are mean ± S.E.M. n = 4/group. *p<0.05 versus vehicle; and ^#p<0.05 versus cisplatin.

Figure 7. Effects of metalloporphyrins on Cisplatin induced HO-1 mRNA.

Metallophorphyrins induced HO-1 mRNA by itself ($ p<0.05 vs vehicle). Cisplatin induced significant HO-1 mRNA level and the induction was attenuated by FeTMpyP or MnTBAP treatments. Results are mean ± S.E.M. n = 4/group. *p<0.05 versus vehicle; and ^#p<0.05 versus cisplatin.

Figure 8. Schematic diagram of protection mechanism of metalloporphyrins in Cisplatin induced kidney injury.

Metalloporphyrins induce HO-1 resulting in antioxidant defense and autophagy during cisplatin induced kidney injury. Metalloporphyrins also neutralize superoxide or scavenge peroxynitrite generated during cisplatin exposure. All combitorial effects leads to reduced inflammation and cell death, thus protecting against cisplatin induced kidney injury.