MnTBAP Therapy Attenuates Renal Fibrosis in Mice with 5/6 Nephrectomy

Jing Yu, Song Mao, Yue Zhang, Wei Gong, Zhanjun Jia, Songming Huang, Aihua Zhang, Jing Yu, Song Mao, Yue Zhang, Wei Gong, Zhanjun Jia, Songming Huang, Aihua Zhang

Abstract

Renal fibrosis is a common pathological feature of all kinds of chronic kidney diseases (CKDs) with uncertain mechanisms. Accumulating evidence demonstrated an important role of oxidative stress in the pathogenesis of CKD. Here we hypothesized that MnTBAP (manganese (III) tetrakis (4-benzoic acid)porphyrin chloride), a cell-permeable mimic of superoxide dismutase (SOD), may protect against the fibrotic response in CKD by antagonizing oxidative stress. To verify this hypothesis, we performed experiments in tubular epithelial cells and mice with 5/6 nephrectomy (Nx). In mouse tubular epithelial cells, TGF-β1 induced a significant transition to fibrotic phenotype in line with a remarkable mitochondrial dysfunction, which was markedly improved by MnTBAP (1.14 μM) pretreatment. In remnant kidneys of 5/6 Nx mice, tubulointerstitial fibrosis occurred in parallel with mitochondrial abnormality in renal tubular cells. Administration of MnTBAP significantly attenuated the deposition of extracellular matrix as evidenced by the blocked expressions of fibronectin, collagen I, and collagen III. Masson staining also displayed an ameliorated accumulation of collagenous matrix in MnTBAP-treated mice. Moreover, MnTBAP also significantly improved the severity of proteinuria without altering CKD-related hypertension. Collectively, MnTBAP therapy served as a promising strategy in preventing renal fibrosis in CKDs possibly via antagonizing mitochondrial-derived oxidative stress and subsequent protection of mitochondrial function.

Figures

Figure 1
Figure 1
TGF-β1 altered cellular phenotype in mPTCs. (a) Western blots of E-cadherin, α-SMA, and vimentin. (b–d) Densitometric analysis of Western blots. (e–g) qRT-PCR analysis of E-cadherin (e), α-SMA (f), and vimentin (g). All values are means ± SD (n = 5 in each group). ∗p < 0.05 versus control group.
Figure 2
Figure 2
TGF-β1 induced mitochondrial dysfunction in mPTCs. (a) Analysis of mitochondrial membrane potential by JC-1 fluorescence dye in mPTCs treated with TGF-β1. (b) qRT-PCR analysis of mtDNA copy number in mPTCs treated with TGF-β1. (c) Analysis of ROS by DCF fluorescence in mPTCs treated with TGF-β1. (d) Electron microscopy analysis of mitochondrial morphology. All values are means ± SD (n = 5 in each group). ∗p < 0.05 versus control group.
Figure 3
Figure 3
Effects of MnTBAP treatment on protein expressions of E-cadherin, α-SMA, and vimentin in mPTCs following TGF-β1 treatment. (a) Western blots of E-cadherin, α-SMA, and vimentin. (b–d) Densitometric analysis of Western blots. All values are means ± SD (n = 5 in each group).  ∗p < 0.05 versus control group.   #p < 0.05 versus TGF-β1 group.
Figure 4
Figure 4
Effects of MnTBAP treatment on mRNA expressions of E-cadherin, α-SMA, and vimentin in mPTCs following TGF-β1 treatment. (a–c) qRT-PCR analysis of E-cadherin (a), α-SMA (b), and vimentin (c). All values are means ± SD (n = 5 in each group).  ∗p < 0.05 versus control group.   #p < 0.05 versus TGF-β1 group.
Figure 5
Figure 5
MnTBAP attenuated mitochondrial abnormality induced by TGF-β1 in mPTCs. (a) qRT-PCR analysis of mtDNA copy number in mPTCs. (b) Flow cytometry analysis of ROS production by DCF fluorescence in mPTCs. All values are means ± SD (n = 5 in each group).  ∗p < 0.05 versus control group.   #p < 0.05 versus TGF-β1 group.
Figure 6
Figure 6
Kidney mass reduction by 5/6 Nx induced renal fibrosis and mitochondrial abnormality. (a) Masson staining. (b) Electron microscopy analysis of mitochondrial morphology in tubular cells. (c) qRT-PCR analysis of mtDNA copy number and TFAM (d) in remnant kidneys. All values are means ± SD (n = 5 in each group).  ∗p < 0.05 versus control group.
Figure 7
Figure 7
MnTBAP therapy attenuated renal fibrosis in 5/6 Nx mice. (a) Masson staining. (b–d) qRT-PCR analysis of fibronectin (b), collagen I (c), and collagen III (d) in 5/6 Nx mice. All values are means ± SD (n = 5 in each group).  ∗p < 0.05 versus control group.   #p < 0.05 versus 5/6 Nx group.
Figure 8
Figure 8
MnTBAP therapy reduced protein expressions of fibronectin and collagen III in remnant kidneys. (a) Western blots of fibronectin and collagen III. (b) Densitometric analysis of Western blots. All values are means ± SD (n = 5 in each group).  ∗p < 0.05 versus control group.   #p < 0.05 versus 5/6 Nx group.
Figure 9
Figure 9
Effects of MnTBAP treatment on proteinuria and blood pressure in 5/6 Nx mice. (a) Urinary albumin excretion. (b) Systolic blood pressure. All values are means ± SD (n = 5 in each group).  ∗p < 0.05 versus control group.   #p < 0.05 versus 5/6 Nx group.

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Source: PubMed

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