Endoplasmic reticulum stress is activated in post-ischemic kidneys to promote chronic kidney disease

Abstract

Background: Acute kidney injury (AKI) may lead to the development of chronic kidney disease (CKD), i.e. AKI-CKD transition, but the underlying mechanism remains largely unclear. Endoplasmic reticulum (ER) stress is characterized by the accumulation of unfolded or misfolded proteins in ER resulting in a cellular stress response. The role of ER stress in AKI-CKD transition remains unknown.

Methods: In this study, we examined ER stress in the mouse model of AKI-CKD transition after unilateral renal ischemia-reperfusion injury (uIR). To determine the role of ER stress in AKI-CKD transition, we tested the effects of two chemical chaperones: Tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA).

Findings: uIR led to the induction of ER stress in kidneys, as indicated by increased expression of UPR molecules CHOP (C/EBP homologous protein) and BiP(binding immunoglobulin protein; also called GRP78-78 kDa glucose-regulated protein). Given at 3 days after uIR, both TUDCA and 4-PBA blocked ER stress in post-ischemic kidneys. Notably, both chemicals promoted renal recovery and suppressed tubulointerstitial injury as manifested by the reduction of tubular atrophy, renal fibrosis and myofibroblast activation. Inhibition of ER stress further attenuated renal tubular epithelial cell apoptosis, inflammation and autophagy in post-ischemic kidneys.

Interpretation: These findings suggest that ER stress contributes critically to the development of chronic kidney pathologies and CKD following AKI, and inhibition of ER stress may represent a potential therapeutic strategy to impede AKI-CKD transition.

Keywords: AKI-CKD transition; Apoptosis; Autophagy; ER stress; Fibrosis; Renal ischemia-reperfusion.

Fig. 1

Unilateral renal ischemia-reperfusion induces chronic kidney problems. C57BL/6 mice (male, 8–10 week old) were subjected to sham operation or 30 min of unilateral ischemia of left kidney followed by reperfusion. For biochemical and histological analyses, kidney tissues were collected at 2, 7 or 14 days after renal ischemia. To determine the function of post-ischemic left kidney, right nephrectomy was performed one day before animal sacrifice and blood samples were then collected at animal sacrifice to measure serum creatinine. (A) Post-ischemic kidney weight/body weight (mg/g). (B) Contralateral kidney weight/body weight (mg/g). (C) Serum creatinine(mg/dL). (D) Representative histology images of hematoxylin-eosin staining. Bar = 200 μm. (E) Pathological score of tubular atrophy. (F) Representative immunoblots of collagen 1, α-SMA, vimentin, and GAPDH. GAPDH was used as a loading control. (G) Quantitative analysis of immunoblot of collagen 1, α-SMA, and vimentin. For Densitometric analysis, the protein level of sham group was arbitrarily set as 1, and the signals of other conditions were normalized with the sham control group to indicate their protein fold changes. (H) Representative images of Masson trichrome staining. Bar = 200 μm. (I) Quantitative analysis of Masson trichrome staining. Data are expressed as mean ± SEM. n = 4. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Fig. 2

ER stress is induced and sustained in renal tubules in post-IRI kidney. Mice were subjected to uIR without contralateral nephrectomy or sham-operation as described in Fig. 1. (A) Representative images of immunohistochemical staining of BiP. Bar = 200 μm. (B) Quantitative analysis of BiP staining. (C) Representative immunoblots of p-PERK, PERK, BiP, CHOP, and GAPDH. GAPDH was used as a loading control. (D) Quantitative analysis of immunoblots of p-PERK, BiP and CHOP. For Densitometric analysis, the protein level of sham group was arbitrarily set as 1, and the signals of other conditions were normalized with the sham control group to indicate their protein fold changes. (E) Representative images of immunohistochemical staining of CHOP. Arrows was used to indicate a few positive activated CHOP proteins. Bar = 200 μm. (F) Quantitative analysis of CHOP staining. Data are expressed as mean ± SEM. n = 4 mice. *p < 0.05;**p < 0.01;***p < 0.001; ns, not significant.

Fig. 3

4-PBA and TUDCA suppress post-IRI ER stress in kidney tubules. C57Bl/6 mice (male, 8–10 week old) were subjected to 30 min of unilateral ischemia of left kidney followed by reperfusion for 7 or 14 days. 4-PBA at 20 mg/kg/day, TUDCA at 250 mg/kg/day, or normal saline (NS) was given from day 3 after uIR. Then kidney tissues were collected for biochemical and histological analyses. (A) Representative images of immunohistochemical staining of BiP. Bar = 200 μm. (B) Quantitative analysis of BiP staining. (C) Representative immunoblots of p-PERK, PERK, BiP, CHOP, and GAPDH. GAPDH was used as a loading control. (D) Quantitative analysis of immunoblot signals of p-PERK, BiP and CHOP. For densitometric analysis, the protein level of sham group was arbitrarily set as 1, and the signals of other conditions were normalized with the sham control group to indicate their protein fold changes. (E) Representative images of immunohistochemical staining of CHOP. Arrows point to positive CHOP staining. Bar = 200 μm. (F) Quantitative analysis of CHOP staining. Data are expressed as mean ± SEM. n = 4. *p < 0.05;**p < 0.01;***p < 0.001; ns, not significant.

Fig. 4

4-PBA and TUDCA given after IRI attenuates renal tubular atrophy, renal dysfunction and renal fibrosis. C57BL/6 mice (male, 8–10 week old) were subjected to 30 min of unilateral ischemia followed by reperfusion. 4-PBA at 20 mg/kg/day, TUDCA at 250 mg/kg/day, or normal saline (NS) was given from day 3 after uIR. For biochemical and histological analyses, kidney tissues were collected at day 7 or day 14. To determine the function of post-ischemic left kidney, right nephrectomy was performed one day before animal sacrifice and blood samples were then collected at animal sacrifice to measure serum creatinine. (A) Ischemic kidney weight/body weight (mg/g). (B) Contralateral kidney weight/body weight (mg/g). (C) Serum creatinine(mg/dL). (D) Representative renal images of hematoxylin-eosin staining. Bar = 200 μm. (E) Pathological score of tubular atrophy. (F) Representative immunoblots of collagen 1, α-SMA, vimentin and GAPDH. GAPDH was used as a loading control. (G) Quantitative analysis of immunoblot of collagen 1 and α-SMA. For densitometric analysis, the protein level of sham group was arbitrarily set as 1, and the signals of other conditions were normalized with the sham control group to indicate their protein fold changes. (H) Representative images of Masson trichrome staining. Bar = 200 μm. (I) Quantitative analysis of Masson trichrome staining. Data are expressed as mean ± SEM. n = 4. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Fig. 5

4-PBA and TUDCA given after IRI alleviates renal tubular cell apoptosis. C57Bl/6 mice were subjected to 30 min of left uIR or sham-operation. 4-PBA at 20 mg/kg/day, TUDCA at 250 mg/kg/day, or normal saline (NS) was given from day 3 after uIR. Kidney tissues were collected for biochemical and histological analyses. (A) Representative images of TUNEL staining. Bar = 200 μm. (B) Quantitative analysis of TUNEL staining positive cells. (C) Representative immunoblots of cleaved caspase 12, cleaved caspase 3, and GAPDH (loading control). (D) Quantitative analysis of immunoblots of cleaved caspase 12 and cleaved caspase 3. For densitometric analysis, the protein level of sham group was arbitrarily set as 1, and the signals of other conditions were normalized with the sham control group to indicate their protein fold changes. Data are expressed as mean ± SEM. n = 4. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Fig. 6

4-PBA and TUDCA given after IRI reduces renal interstitial inflammation. C57Bl/6 mice were subjected to 30 min of left uIR or sham-operation. 4-PBA at 20 mg/kg/day, TUDCA at 250 mg/kg/day, or normal saline (NS) was given from day 3 after uIR to collect kidney tissues for analyses. (A) Representative images of immunohistochemical staining of F4/80 to show macrophages. Arrows were used to indicate a few positive macrophages. Bar = 200 μm. (B) Quantitative analysis of macrophages. (C-E) Real-time PCR analysis of MCP-1, IL-6, TNF-α and ACTB (internal control). Data are expressed as mean ± SEM. n = 4. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Fig. 7

4-PBA and TUDCA given after IRI reduces renal tubular autophagy. Mice were subjected to 30 min of left uIR or sham-operation. 4-PBA at 20 mg/kg/day, TUDCA at 250 mg/kg/day, or normal saline (NS) was given from day 3 after uIR to collect kidney tissues for analyses. (A) Representative images of immunohistochemical staining of LC3. Arrows was used to indicate a few positive LC3 puncta or dots. Bar = 200 μm. (B) Quantitative analysis of LC3 dots. (C) Representative immunoblot images of LC3B and GAPDH. GAPDH was used as a loading control. (D) Quantitative analysis of immunoblot of LC3B-II. For Densitometric analysis, the protein level of sham group was arbitrarily set as 1, and the signals of other conditions were normalized with the sham control group to indicate their protein fold changes. Data are expressed as mean ± SEM. n = 4 mice. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Supplementary Fig. 1

(A) C57BL/6 mice (male, 8-10 week old) were subjected to sham operation or 30 minutes of unilateral ischemia of left kidney followed by reperfusion for 2, 7, or 14 days. (B) 4-PBA at 20 mg/kg/day, TUDCA at 250mg/kg/day, or normal saline (NS) was given from day 3 after uIR. Kidney tissues were collected at day 7 and 14 for PAS staining. Bar=50 μm.