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. 2021 Jul 26:12:697751.
doi: 10.3389/fimmu.2021.697751. eCollection 2021.

Long-Term Protection of CHBP Against Combinational Renal Injury Induced by Both Ischemia-Reperfusion and Cyclosporine A in Mice

Yufang Zhang  1 , Yuanyuan Wu  1   2 , Wei Wang  2 , Feng Liu  2 , Yiwen Zhang  2 , Cheng Yang  3 , Aifen Liu  1 , Jing Wu  2 , Tongyu Zhu  3 , Michael L Nicholson  4   5 , Yaping Fan  2 , Bin Yang  1   2   4
Affiliations

Long-Term Protection of CHBP Against Combinational Renal Injury Induced by Both Ischemia-Reperfusion and Cyclosporine A in Mice

Yufang Zhang et al. Front Immunol. .

Abstract

Renal ischemia-reperfusion (IR) injury and cyclosporine A (CsA) nephrotoxicity affect allograft function and survival. The prolonged effects and underlying mechanisms of erythropoietin derived cyclic helix B peptide (CHBP) and/or caspase-3 small interfering RNA (CASP-3siRNA) were investigated in mouse kidneys, as well as kidney epithelial cells (TCMK-1), subjected to transplant-related injuries. Bilateral renal pedicles were clamped for 30 min followed by reperfusion for 2 and 8 weeks, with/without 35 mg/kg CsA gavage daily and/or 24 nmol/kg CHBP intraperitoneal injection every 3 days. The ratio of urinary albumin to creatinine was raised by IR injury, further increased by CsA and lowered by CHBP at 2, 4, 6 and 8 weeks, whereas the level of SCr was not significantly affected. Similar change trends were revealed in tubulointerstitial damage and fibrosis, HMGB1 and active CASP-3 protein. Increased apoptotic cells in IR kidneys were decreased by CsA and CHBP at 2 and/or 8 weeks. p70 S6 kinase and mTOR were reduced by CsA with/without CHBP at 2 weeks, so were S6 ribosomal protein and GSK-3β at 8 weeks, with reduced CASP-3 at both time points. CASP-3 was further decreased by CHBP in IR or IR + CsA kidneys at 2 or 8 weeks. Furthermore, in TCMK-1 cells CsA induced apoptosis was decreased by CHBP and/or CASP-3siRNA treatment. Taken together, CHBP predominantly protects kidneys against IR injury at 2 weeks and/or CsA nephrotoxicity at 8 weeks, with different underlying mechanisms. Urinary albumin/creatinine is a good biomarker in monitoring the progression of transplant-related injuries. CsA divergently affects apoptosis in kidneys and cultured kidney epithelial cells, in which CHBP and/or CASP-3siRNA reduces inflammation and apoptosis.

Keywords: CASP-3; apoptosis; cyclic helix B peptide; cyclosporine A; ischemia–reperfusion injury.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic picture of in vivo study design and urinary albumin/creatinine. (A) Bilateral renal pedicles were clamped for 30 min followed by reperfusion for 2 and 8 weeks. Approximately 35 mg/kg CsA gavage daily, and/or 24 nmol/kg CHBP intraperitoneal injection every 3 days. Urine samples were collected every 2 weeks, blood samples were drawn and animals were sacrificed at 2 and 8 weeks. (B–E) The ratio of urinary albumin/creatinine was shown in the IR group with/without CsA treatment at 2, 4, 6 and 8 weeks. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (F) The dynamic profile of urinary albumin/creatinine at all-time points. † P < 0.05, †† P ≤ 0.01, †††† P ≤ 0.0001 versus IR; # P < 0.05, ### P ≤ 0.0005 versus IR + CsA and $ P < 0.05 versus the control. Data were expressed as the mean ± SD of each group (n ≥ 3).
Figure 2
Figure 2
The semi-quantitative score of TID in H&E staining sections. (A, C) Tubular dilation, epithelial cell vacuolation, interstitial expansion with edema or inflammation, and cells or cell debris in tubular lumens were mainly seen in IR kidneys. (B) CHBP treatment improved the TID compared with IR and IR + CsA groups at 2 weeks. (D) The score was further increased in the IR + CsA group but reversed by CHBP at 8 weeks. Data were expressed as the mean ± SD of each group (n ≥ 3). *P ≤ 0.05; **P ≤ 0.01.
Figure 3
Figure 3
The score of interstitial fibrosis in Masson’s trichrome stained sections. (A, C) Masson’s trichrome staining revealed the interstitial fibrosis. (B, D) Interstitial fibrosis was increased by IR compared with the control, but improved by CHBP at both time points. (D) The score of fibrosis was significantly decreased by CHBP in the CsA treated group at 8 weeks. Data were expressed as the mean ± SD of each group (n ≥ 3). **P ≤ 0.01.
Figure 4
Figure 4
Apoptotic cells detected by labelling fragmented DNAs. (A–J) Apoptosis were mainly shown in tubular and interstitial areas. (B1–D1, G1–I1) Enlarged pictures were from (B–D) and (G–I). (K, L) The number of apoptotic cells was greatly increased by IR, but decreased by CsA at 2 and 8 weeks. CHBP treatment decreased apoptotic cells in the IR kidneys at 8 weeks. Data were expressed as the mean ± SD of each group (n ≥ 3). *P ≤ 0.05; **P ≤ 0.01.
Figure 5
Figure 5
CASP-3 mRNA and protein, and HMGB1 protein expression. (A, E) The relative expression of caspase 3 mRNA was increased by IR at both time points, and further increased by CsA at 2 weeks, but decreased by CHBP in the CsA group at 8 weeks. (B–D, F, G) The level of 17 kD active CASP-3 protein was increased by IR and reversed by CHBP at 2 weeks, and increased by CsA at 8 weeks and also reversed by CHBP. (H–J) The expression of HMGB1 protein in 2 and 8 weeks was detected by western blotting. The level of HMGB1 was significantly decreased by CHBP in the IR group at 2 weeks, increased by CsA compared with the IR group and reversed by CHBP at 8 weeks. Data were expressed as the mean ± SD (n ≥ 3). The volume density of western blots was corrected by against 42 kD β-actin as a loading control. *P ≤ 0.05; **P ≤ 0.01.
Figure 6
Figure 6
Simultaneous detection of 18 proteins by slide-based antibody array. (A, B) The expression of 18 proteins in the kidneys revealed by slide-based antibody array. (C–E) CsA decreased p70 S6 kinase, mTOR and caspase-3 at 2 weeks. (F–H) S6RP was decreased by CsA in IR kidneys, but reversed by CHBP. CsA decreased GSK-3β and caspase-3 at 8 weeks in IR kidneys with/without CHBP treatment. (E–H) Caspase-3 was decreased by CHBP in CsA treated IR kidneys at 2 weeks and in IR kidneys at 8 weeks. Data were expressed as the mean ± SD (n ≥ 3). The volume density was corrected by against the control. *P ≤ 0.05; **P ≤ 0.01.
Figure 7
Figure 7
Schematic picture of in vitro study design and effects of CsA, CHBP and CASP-3siRNA on TCMK-1 cells. (A) TCMK-1 cells were transfected with CASP-3siRNA 4–6 h before treated by CsA with/without CHBP for 24 h. (B) The level of CASP-3 mRNA was gradually increased by 2.5–20 μg/ml CsA. (C) The percentage of early apoptotic cells was significantly increased by CsA by 20–40 μg/ml and peaked at 20 μg/ml. (D, E) Early apoptotic cells was gradually reduced by 2.5–40 ng/ml CHBP, and reached statistical significance at 20–40 ng/ml. (F) The expression of CASP-3 mRNA was reduced by 10–30 nM/ml CASP-3siRNA compared with the control treated by 20 μg/ml CsA or with NCsiRNA, but the suppression was maximized at 30 nM/ml with 64.85% reduction. (G) CASP-3 mRNA were significantly increased by 20 μg/ml CsA, but reversed by 20 ng/ml CHBP with/without 30 nM CASP-3siRNA. (H, I) Early apoptotic cells was significantly increased by 20 μg/ml CsA, but reversed by CHBP with/without CASP-3siRNA or NCsiRNA. Data were expressed as the mean ± SD, (n = 3). *P < 0.05, **P < 0.01.
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