1. The effects of fisetin on eGFR, plasma BUN, creatinine, and urinary albumin/creatinine ratio in rats subjected to renal ischemia reperfusion injury.
The effect of fisetin on the renal physiological function and corresponding renal markers were measured and the results are given in figure 1. According to figure 1a, IR induced a fall in the eGFR from the sham group (S- 15.91 ml/min, IR- 0.35 ml/min). The plasma level of BUN (S- 18.34 mg/dl, IR- 111.83 mg/dl), creatinine (S- 0.69 mg/dl, IR-2.20 mg/dl) and urinary albumin/creatinine ratio (Al/Cr-S-0.46, IR-6.09) were significantly declined from the normal range in IR group (Figure 1b-1d).
Fisetin pre-treatment prior to IR protocol protected the kidney from damage and improved the glomerular function significantly (p<0.05) from the IR (IR- 0.35 ml/min, F_IR- 9.03 ml/min). The injury markers exhibited a marked reduction in their level in fisetin pretreated renal tissues from the IR group (F-IR: Cr- 0.92 mg/dl, BUN- 81.03 mg/dl, Al/Cr-2.16).
The renal injury markers in the blood exhibited a marked reduction in the level in fisetin pretreated renal tissues from the IR group (F-IR: Cr- 0.92 mg/dl vs 2.20 mg/dl, BUN- 81.03 mg/dl vs 111.83 mg/dl, Al/Cr-2.16 vs 6.09 from IR group).
2. Effect of fisetin on renal injury in rats subjected to renal ischemia reperfusion.
Histopathological analysis of the renal tissue from different experimental groups reveals normal architecture in the sham group, which was significantly altered in the IR tissue. Compared with the sham (Figure 1e), the kidneys from the IR group rats (Figure 1f) showed severe glomerular congestion, tubular dilatation, tubular degeneration with vacuolization in proximal tubules and cast formation. The pathological changes in the renal tissues from IR groups were minimal with prior treatment of fisetin (Figure 1h) confirming the protective effect of fisetin. Accordingly, the characteristic histological changes observed in F_IR were decrease in renal congestion and tubular dilatation and minimal tubular cast formation indicated a reduced injury (Figure 1i).
3. Fisetin attenuates the IR induced apoptosis in rats subjected to renal ischemia reperfusion injury.
Apoptotic renal injury was assessed by measuring the caspase 3 activities and visualizing the fragmented DNA in agarose gel (Figure 1j and 1k). Caspase 3 activity was high in IR tissue by 27% from the sham tissue. Fragmented DNA was visible in the electrogram of IR tissue which was absent in the sham group. However, compared with the IR, fisetin pre-treated IR renal tissue exhibited significantly low apoptotic injury evident by the reduced fragmentation and decline in caspase 3 activity by 19% (Figure 1k).
4. Fisetin reduced the oxidative stress associated with renal ischemia reperfusion injury.
Figure 2 shows the renal lipid peroxidation level and antioxidant enzyme activities in the tissues of different experimental groups. Upon reperfusion, the renal tissues exhibited increased concentration of TBARS (in nM MDA/mg tissue, S- 2.00, IR- 3.41) and correspondingly decreased level of GSH/GSSG (S- 0.43, IR- 0.27). The antioxidant enzymes like SOD (in U/mg protein, S-5.8, IR-4.56), catalase (mU/mg protein, S-12.8, IR- 6.03) and GPx (µM GSH/mg protein, S- 0.94, IR- 0.65) were declined in their activities. Preconditioning the animal with fisetin showed a significant decline in the level of TBARS (IR- 3.41, F_IR- 2.74) and improved the endogenous antioxidant GSH/GSSG level (IR- 0.27, F_IR- 0.34) compared with the IR group. The antioxidant enzyme activities (SOD, catalase, GPx, and GR) were improved in F_IR (Figure 2c–f) group than IR.
Figure 3 shows the lipid peroxidation and antioxidant status in the renal mitochondria. Lipid peroxidation measured in terms of TBARS was high in IR group (11% higher than in the homogenate). The corresponding decline in reduced glutathione (GSH) was prominent in IR tissue (31% higher than in homogenate). Similar patterns of changes were observed in the antioxidant enzymes of mitochondrial fraction from IR tissue. Fisetin pretreatment significantly reduced TBARS (42%) and increased GSH/GSSG (18%) level in the mitochondria from the IR group animals. Activities of antioxidant enzymes like catalase (63%), SOD (35%), and GPx (20%) were also improved significantly from the IR group.
5. Fisetin improved the mitochondrial bio-energetic functions in rats subjected to ischemia reperfusion injury.
The quality of mitochondria in the renal tissue is a critical factor that regulate renal function. The different mitochondria functions like bioenergetics (Figure 4) and its regulatory events such as copy number (Figure 5), mitophagy, mitofusion, and mitofission (Figure 6) were evaluated to assess the renal mitochondrial quality.
Bioenergetic potential of renal mitochondria was measured via ETC enzyme activities like NQR, SQR, QCR and COX along with ATP level (Figure 4). Animals subjected to IR exhibited low enzyme activities in NQR, SQR, QCR and COX by 41%, 43%, 50%, and 82% respectively from the sham. ATP level in IR tissue was significantly low (18%) from the sham. However, Fisetin treatment in tissue subjected to IR improved the mitochondrial ETC enzyme activities of NQR (25%), SQR (25%), QCR (39%), COX (71%), and ATP level (22%) from the IR control (Figure 4a-d and 7a).
To assess if the mitochondrial copy number plays a significant role in the reduced functional activity, we evaluated the mitochondrial copy number in the rat renal tissues from all the experimental groups and noted a significant (p<0.05) reduction in the copy number in IR treated animal which was improved by the pre-treatment of the animals with fisetin (Figure 5a).
Further analysis of the expression level of PGC-1α (Figure 5b), which plays a critical role in the regulation of mitochondrial biogenesis and respiratory function (bioenergetics) showed 62 % (p<0.05) decline in IR kidney. Fisetin pretreatment was found to improve the PGC-1α expression by 63% from the IR group confirming the mitochondrial biogenetic potential of the drug. However, fisetin found to exert minimum impact on the mitochondrial transcription regulation factor A (Tfam) and mitochondrial DNA polymerase γ (POLG) which regulates the mitochondrial encoded gene expressions, where 13 mitochondrial encoded genes influenced the activity of bio-energetic enzymes (Figure 5c- 5d).
6. Fisetin improved renal mitochondrial dynamics and mitophagy in rats subjected to ischemia reperfusion injury.
The damage to mitochondria associated with IR injury can disrupt the balance of the dynamic processes controlling mitochondrial fusion and fission. In order to analyze the same we estimated the gene expression of mitochondrial fission and fusion genes in different experimental groups and the results are given in figure 6. We observed an augmented expression of mitochondrial fission genes Mff, Fis 1 and Dmn 1 by 1.66, 1.17 and 2.23 folds respectively in the IR treated groups compared with sham (Figure 6a- 6c). Treatment with fisetin prior the IR reduced the mitochondrial fission (Dmn 1 expressed in Fold change: IR-2.23 F_IR-1.21, Fis 1: IR- 1.17 F_IR-0.35, Mff: IR-1.66 F_IR-1.14). Furthermore the mRNA expression of Mfn1 gene, which codes for the principal GTP-dependent membrane tethering protein for mitochondrial fusion exhibited a significant (p<0.05) increase in mitchondrial fusion gene expression due to in IR (S- 1.00, IR- 1.57) group animals. However, mfn1 expression was further increased in F_IR group (IR- 1.57, F_IR- 1.92).
To determine the effect of fisetin on mitophagy -an important event that maintain the mitochondrial quality was evaluated by measuring the gene expression of the key regulatory molecules such as Pink, Parkin and Optn (Figure 6f- 6h). Among the three genes, Pink and Parkin showed significant elevation (3.7 fold) in the gene expression in response to IR induction in the sham control. On the other hand, compared to sham all the three genes showed significantly higher expression in F_IR. Two out of three genes were significantly expressed in F_IR group compared to the IR control group (Parkin: IR- 1.25, F_IR- 1.98 and Optn: IR- 1.00, F_IR- 1.28). In general, fisetin pretreatment prior to IR challenged improved the mitochondrial function and maintained the mitochondrial homeostasis and quality.
7. Fisetin improved the mitochondrial ATP producing capacity, and attenuated swelling and mPTP opening in rats subjected to ischemia reperfusion injury.
In order to assess if alterations in the electron transport chain affected the ATP machinery in renal tissue we estimated the ATP producing capacity of isolated mitochondria in different energised conditions (Glutamate- Malate/Succinate) and non-energised conditions. After incubating the mitochondria in the respective respiratory buffers for 10 minutes, ATP content was measured by using ATPlite kit and the results are presented in Figure 7 (a- c). The IR significantly compromised the ATP producing capacity of the isolated renal mitochondria in both energized and non-energized conditions. However, isolated renal mitochondria from the fisetin pretreatment group retain the ATP producing capacity significantly. We also noted another interesting observation where the ATP level showed a striking increase in the fisetin control group than the sham control.
The corresponding mitochondrial membrane potential, that determine the oxidative phosphorylation, in different energized medium did not show significant changes across the experimental groups (Figure 7d- 7f). However, mitochondrial swelling was high in IR group in both energized and non-energized conditions, which was improved substantially in fisetin treated IR group (Figure 7g- 7i).
7. Fisetin’s protective ability towards renal IR injury is independent of PI3K/Akt signalling pathway
In order to assess the role of PI3K/Akt pathway in the renal protection mediated by fisetin, we used an inhibitor based study. The study results presented in figure 8 shows that the IR induced renal injury was found to be intensified in presence of Wortmannin, a PI3K inhibitor confirming the role of PI3K in renal IR protection. However, the protective ability of fisetin towards IR was retained even in the presence of Wortmannin indicated that the fisetin mediated renal protection towards renal IR injury is independent of PI3K/Akt signalling pathway.