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Nitrative Stress-Related Autophagic Insufficiency Participates in Hyperhomocysteinemia-Induced Renal Aging - PubMed

  • ️Wed Jan 01 2020

Nitrative Stress-Related Autophagic Insufficiency Participates in Hyperhomocysteinemia-Induced Renal Aging

Shangyue Zhang et al. Oxid Med Cell Longev. 2020.

Abstract

The kidneys are important organs that are susceptible to aging. Hyperhomocysteinemia (HHcy) is a risk factor for nephropathy and is associated with chronic nephritis, purpuric nephritis, and nephrotic syndrome. Numerous studies have shown that elevated serum homocysteine levels can damage the kidneys; however, the underlying mechanism of HHcy on kidney damage remains unclear. In this study, we make use of a diet-induced HHcy rat model and in vitro cell culture to explore the role of autophagy in HHcy-induced renal aging and further explored the underlying mechanism. We demonstrated that HHcy led to the development of renal aging. Promoted kidney aging and autophagic insufficiency were involved in HHcy-induced renal aging. HHcy decreased the expression of transcription factor EB (TFEB), the key transcription factor of autophagy-related genes in renal tissue. Further experiments showed that nitrative stress levels were increased in the kidney of HHcy rats. Interestingly, pretreatment with the peroxynitrite (ONOO-) scavenger FeTMPyP not only reduced the Hcy-induced nitrative stress in vitro but also partially attenuated the decrease in TFEB in both protein and mRNA levels. Moreover, our results indicated that HHcy reduced TFEB expression and inhibited TFEB-mediated autophagy activation by elevating nitrative stress. In conclusion, this study showed an important role of autophagic insufficiency in HHcy-induced renal aging, in which downregulation of TFEB plays a major role. Furthermore, downexpression of TFEB was associated with increased nitrative stress in HHcy. This study provides a novel insight into the mechanism and therapeutic strategy for renal aging.

Copyright © 2020 Shangyue Zhang et al.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1

HHcy led to the development of renal aging. (a) Representative Western blot showing the increased expression of aging-related proteins in the kidneys of the HHcy rats (b–d). Quantification of the protein levels of p53, p21, and p16, respectively (∗P < 0.05, ∗∗P < 0.01vs. Control, n = 4-5). (e) Serum level of the aging-related inflammatory cytokine IL-6 (∗P < 0.05, ∗∗P < 0.01vs. Control, n = 6-10). (f) Serum level of urea increased in both HHcy and natural aged mice (∗P < 0.05vs. Control, n = 6-10). (g) Masson staining of fibrotic lesions in kidney tissue. (h) Sirius red staining of fibrotic lesions in kidney tissue. Control: control group; HHcy: hyperhomocysteinemia group; IL-6: interleukin-6. Data were expressed as mean ± SEM.

Figure 2
Figure 2

Autophagic insufficiency was involved in HHcy-induced renal aging. (a) Representative Western blot showing the protein expression levels of autophagy in HHcy. Graphs representing (b, c) LC3 by quantitative analysis. (∗P < 0.05, ∗∗P < 0.01, n = 4-5). (d) Graph representing quantitative analysis showing the mRNA level of LC3 in HHcy (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001; HHcy vs. Control; n = 5-6). (e) Representative Western blot showing the protein expression levels of aging and autophagy in MPC-5 treated with rapamycin. Graphs representing (f) p53, (g) p16, and (h) LC3 by quantitative analysis (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, Hcy vs. Control, rapamycin vs. Hcy, n = 3-4). (i) Representative Western blot showing the protein expression levels of aging and autophagy in MPC-5 infected with Atg5 adenovirus. Graphs representing (j) p53, (k) p21, and (l) LC3 by quantitative analysis (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, Hcy vs. Control, Atg5 adenovirus vs. Hcy, n = 3-4). Control: control group; Hcy: homocysteinemia group; Ad-Atg5: Atg5 adenovirus. Data were expressed as mean ± SEM.

Figure 3
Figure 3

HHcy decreased the expression of TFEB in renal tissue. (a–f) The protein expression of TFEB in kidney extracts of HHcy rats and MPC-5 cells measured by Western blot. (a) Representative Western blot showing the protein expression of TFEB in HHcy. Graphs representing (b) TFEB by quantitative analysis (∗P < 0.05, HHcy vs. Control, n = 4-5). (c) Representative Western blot showing the protein expression of TFEB in MPC-5 treated with rapamycin. Graphs representing (d) quantitative analysis (∗P < 0.05, Hcy vs. Control, rapamycin vs. Hcy, n = 3-4). (e) Representative Western blot showing the protein expression of TFEB in MPC-5 infected with Atg5 adenovirus. Graphs representing (f) (∗P < 0.05, Hcy vs. Control, Atg5 adenovirus vs. Hcy, n = 3-4). (g) Graphs representing quantitative analysis showing the mRNA level of TFEB in MPC-5 treated with Hcy for 6, 12, and 24 h (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, Hcy vs. Control, n = 3-4). Control: control group; Hcy: homocysteinemia group; Ad-Atg5: Atg5 adenovirus. Data were expressed as mean ± SEM.

Figure 4
Figure 4

Nitrative stress participated in Hcy-induced TFEB downregulation. (a–c) Representative Western blot and immunohistochemistry detecting 3-NT contents in renal tissue of HHcy rats, respectively. (∗P < 0.05, HHcy vs. Control, n = 4-5). (d) Representative immunoprecipitation showing the contents of nitrated TFEB. Graphs representing (e) quantitative analysis (ns, P > 0.05, Hcy vs. Control, FeTMPyP vs. Hcy, n = 6). (f) Representative Western blot showing the protein expression of TFEB and 3-NT in MPC-5 treated with FeTMPyP. Graphs representing (g) quantitative analysis (∗P < 0.05, Hcy vs. Control, FeTMPyP vs. Hcy, n = 3-4). (h–j) Graphs representing quantitative analysis showing the mRNA level of TFEB in MPC-5 treated with FeTMPyP for 6, 12, and 24 h (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, Hcy vs. Control, FeTMPyP vs. Hcy, n = 3-4). Control: control group; Hcy: homocysteinemia group. Data were expressed as mean ± SEM.

Figure 5
Figure 5

Correlation between renal function, autophagy, and Hcy level in human. (a, b) Correlation between serum urea, creatinine, and Hcy level, respectively (urea: r2 = 0.23, P < 0.0001; creatinine: r2 = 0.078, P < 0.05; n = 80). (c) Correlation between urea and Hcy (Beclin-1: 0-2.0 ng/mL, n = 32, r = −0.4087, P < 0.05). (d) Correlation between urea and Hcy (Beclin-1: 2.0-6.0 ng/mL, n = 41, r = 0.3197, P < 0.05). (e) Correlation between creatinine and Hcy (Beclin-1: 0-1.6 ng/mL, n = 22, r = −0.4524, P < 0.05). (f) Correlation between creatinine and Hcy (Beclin-1: 1.6-7.0 ng/mL, n = 48, r = 0.2895, P < 0.05). Crea: creatinine.

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