Volume 79, Issue 6, Pages 613-623 (March 2011) Increased susceptibility to acute kidney injury due to endoplasmic reticulum stress in mice lacking tumor necrosis factor-α and its receptor 1  Lianghu Huang, Ruihua Zhang, Jin Wu, Jian Chen, Fabrizio Grosjean, Lisa H. Satlin, Janet D. Klein, Jeffrey M. Sands, Gary E. Striker, Jianming Tan, Feng Zheng  Kidney International  Volume 79, Issue 6, Pages 613-623 (March 2011) DOI: 10.1038/ki.2010.469 Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 1 Tunicamycin-induced ER stress kidney injury. (a) Representative gel of western blot shows that the levels of GRP78 and phosphorylated eIF2α are increased in mouse kidney 24h after tunicamycin (tuni) injection. (b) RT-PCR. Spliced XBP-1 is present in tunicamycin-treated mouse kidney. (c) Normal renal histology of control mouse. (d) Renal histology of mouse killed 72h after tunicamycin injection. Tubular vacuolation (light green arrow) and nuclear changes (dark green arrow) are prominent. (e) Cleaved PARP is present only in tunicamycin-treated mouse kidney. (f) Appearance of apoptotic cell death in the kidney from tunicamycin-treated mouse. TUNEL staining was performed. Arrow points to apoptotic cell. eIF2α, eukaryotic translation initiation factor 2α; ER, endoplasmic reticulum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GRP78, glucose regulatory protein 78; PARP, poly ADP ribose polymerase; RT-PCR, reverse transcriptase-PCR; TUNEL, TdT-mediated dUTP nick end labeling; XBP-1, X-box binding protein 1. Kidney International 2011 79, 613-623DOI: (10.1038/ki.2010.469) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 2 Proximal tubular cells are susceptible to endoplasmic reticulum (ER) stress injury. (a) Staining for alkaline phosphatase (AKP), a marker for proximal tubule in normal kidney. Positive tubules are abundant in renal cortex. (b) The number of AKP-positive tubules is decreased in mouse kidney 72h after tunicamycin injection. (c) AKP activity in urine from control mice and mice injected with tunicamycin (tuni) (n=6 per group). The activity was determined by an enzymatic reaction and was corrected for creatinine levels. **P<0.01 vs control. (d) Double staining for AKP and apoptosis in the kidney collected from the mouse 72h after tunicamycin injection. Arrow points to apoptotic cell. Note that apoptotic cells are localized in AKP-positive tubules. (e) Double staining for aquaporin 2 (a marker for collecting duct) and apoptosis. Kidney was harvested from the mouse 72h after tunicamycin injection. Arrow points to apoptotic cell or aquaporin 2-positive tubule. Note that apoptotic cells are not localized in aquaporin 2-positive tubules. (f) Cultured proximal tubular cells (right) and distal tubular cells (left) were treated with 50ng of tunicamycin. Apoptotic cells were quantitated by Annexin V staining and flow cytometry. A total of 31.5% of proximal tubular cells died of apoptosis. (g) Dose-dependent increase in apoptotic cell death occurred in proximal tubular cells when cells were exposed to tunicamycin. Distal tubular cells (Distal) were much more resistant to tunicamycin-induced cell death when compared with proximal tubular cells (Proximal). **P<0.01 vs distal tubular cells treated with the same dose of tunicamycin. Kidney International 2011 79, 613-623DOI: (10.1038/ki.2010.469) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 3 Tumor necrosis factor α (TNFα)−/− mice are susceptible to acute endoplasmic reticulum (ER) stress kidney injury. Both TNFα−/− and wild-type mice were injected with tunicamycin (1μg/g) and killed 72h later (n=6 per group). (a) Wild-type mouse kidney shows moderate tubular injury. (b) Extensive tubular injury in TNFα−/− mouse kidney. (c) Percentage of injured area in renal cortex. **P<0.01 vs wild-type mice. (d,e) Although the number of alkaline phosphatase (AKP)-positive proximal tubules is reduced in wild-type mouse kidney (d), the loss was more prominent in TNFα−/− mice (e). (f) Urine excretion of AKP. **P<0.01 vs wild-type mice. (g,h) Double staining for apoptosis (arrows) and aquaporin 2 in kidneys from wild-type (g) and TNFα−/− mice (h). Note that apoptotic cells were visibly increased in TNFα−/− kidney. (i) More apoptotic cell counts in kidneys of tunicamycin-treated TNFα−/− mice. **P<0.01 vs wild-type mice. Kidney International 2011 79, 613-623DOI: (10.1038/ki.2010.469) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 4 Lipid accumulation in kidney of TNFα−/− mouse with acute ER stress kidney injury. Oil red O staining of kidneys from wild-type (a) and tumor necrosis factor α (TNFα)−/− (b) mice 48h after tunicamycin injection. Extensive staining is found in TNFα−/− kidney. Kidney International 2011 79, 613-623DOI: (10.1038/ki.2010.469) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 5 Reconstitution of TNFα−/− mice with tumor necrosis factor α (TNFα) substantially reduced the severity of acute endoplasmic reticulum (ER) stress kidney injury. Recombinant murine TNFα (200ng/mouse) was given intraperitoneally to TNFα−/− mice 24h before tunicamycin injection. (a,b) Severe renal injury in TNFα−/− mice (a) was nearly completely prevented by TNFα (b). (c) Percentage of injured area in renal cortex. **P<0.01 vs TNFα −/− mice. (d,e) Alkaline phosphatase (AKP)-positive tubules were largely lost in tunicamycin-treated TNFα−/− mice (d). Giving back TNFα (e) prevented the loss. (f) Increased urinary AKP in tunicamycin-treated TNFα−/− mice were reduced by TNFα. **P<0.01 vs TNFα−/− mice. (g,h) Double staining for apoptosis (arrows) and aquaporin 2. The kidney from tunicamycin-treated TNFα−/− mouse (g) shows extensive apoptosis. Putting back TNFα (h) 24h before the injury largely prevented the apoptosis in TNFα−/− mice. (i) Number of apoptotic cells was counted in kidney sections from tunicamycin-treated TNFα−/− and TNFα-reconstituted TNFα−/− mice. **P<0.01 vs TNFα−/− mice. Kidney International 2011 79, 613-623DOI: (10.1038/ki.2010.469) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 6 Severe acute endoplasmic reticulum (ER) stress kidney injury in tumor necrosis factor receptor-1 (TNFR1)−/− and TNFR1R2−/− mice. TNFR1−/−, TNFR2−/−, and TNFR1R2−/− mice were given tunicamycin and killed 72h later. (a) Blood urea nitrogen (BUN) levels were elevated in TNFR1−/− mice, but not in wild-type and TNFR2−/− mice. **P<0.01 vs wild-type mice. (b) Urinary alkaline phosphatase (AKP) activity. **P<0.01 vs wild-type mice. (c,d,e,f) Representative renal histology of wild-type (c), TNFR1−/− (d), TNFR2−/− (e), and TNFR1R2−/− (f) mice with tunicamycin-induced injury. Extensive vacuolation and nuclear change are seen in TNFR1−/− and TNFR1R2−/− kidneys. (g) Measurement of injured area in renal cortex. **P<0.01 vs wild type mice with tunicamycin injury. (h) Apoptotic cell number in kidney. *P<0.05, **P<0.01 vs wild-type mice with tunicamycin injury. Kidney International 2011 79, 613-623DOI: (10.1038/ki.2010.469) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 7 Lipid accumulation in kidneys of TNFR1−/− and TNFR2−/− mice with acute ER stress kidney injury. Oil red O staining of kidneys from (a) wild-type, (b) tumor necrosis factor receptor-1 (TNFR1)−/−, (c) TNFR2−/−, and (d) TNFR1R2−/− mice 48h after tunicamycin injection. Extensive staining is found in TNFR1−/− and TNFR1R2−/− kidneys. Kidney International 2011 79, 613-623DOI: (10.1038/ki.2010.469) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 8 mRNA levels of GRP78, GRP94, spliced XBP-1, EDEM, and CHOP. RNA was extracted from kidneys of control and tunicamycin-treated mice. mRNA levels were determined by real-time PCR. The levels in wild-type control were arbitrarily defined as 1. (a) GRP78 mRNA. (b) GRP94 mRNA. #P<0.05, ##P<0.01 vs wild-type mice treated with tunicamycin. (c) Spliced XBP-1 mRNA. (d) EDEM mRNA. (e) CHOP mRNA. ##P<0.01 vs wild-type mice treated with tunicamycin. CHOP, C/EBP homologous protein; EDEM, ER degradation enhancing α-mannosidase I-like protein; GRP78, glucose regulatory protein 78; XBP-1, X-box binding protein 1. Kidney International 2011 79, 613-623DOI: (10.1038/ki.2010.469) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 9 Differences in renal GRP94 and phosphorylated eIF2α levels in tunicamycin-treated wild-type, TNFα−/−, TNFα-reconstituted TNFα−/−, TNFR1−/−, TNFR2−/−, and TNFR1R2−/− mice. Renal proteins were collected from mice 48h after tunicamycin injection (n=6 per group). (a) Representative western blot gels show that although GRP78 levels were equally elevated in the kidneys of all mice treated with tunicamycin, GRP94 levels were more increased in the kidneys of TNFα−/−, TNFR1−/−, and TNFR1R2−/− mice. (b) Quantitatively assessment of the intensity of GRP94 western blot bands from the kidneys of wild-type (WT) control, tunicamycin-treated WT mice (WT+tuni), and tunicamycin-treated TNFα−/−, TNFα-reconstituted TNFα−/−, TNFR1−/−, TNFR2−/−, and TNFR1R2−/− mice. The levels in WT control were arbitrarily defined as 1. **P<0.01, vs WT control; #P<0.05, ##P<0.01 vs WT mice treated with tunicamycin (WT+tuni). (c,d) CHOP protein levels. Tunicamycin treatment increased CHOP levels in all groups. The increase was more prominent in most of groups except for WT. **P<0.01 vs WT control; #P<0.05, ##P<0.01 vs WT mice treated with tunicamycin (WT+tuni). (e,f) The levels of phosphorylated eIF2α were significantly increased in WT mice kidneys after tunicamycin treatment. The increase was not visible in TNFα−/− mice kidneys but was obvious in the kidneys from TNFα−/− mice reconstituted with TNFα 24h before tunicamycin challenge. TNFR1−/− and TNFR1R2−/− mice kidneys also showed less increase in eIF2α phosphorylation in response to tunicamycin. **P<0.01 vs WT control; #P<0.05 vs WT mice treated with tunicamycin (WT+tuni). CHOP, C/EBP homologous protein; eIF2α, eukaryotic translation initiation factor 2α; GRP94, glucose regulatory protein 94; TNFα, tumor necrosis factor α; TNFR1, TNF receptor-1. Kidney International 2011 79, 613-623DOI: (10.1038/ki.2010.469) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 10 Decreased eIF2α phosphorylation contributes to the susceptibility of TNFR1−/− proximal tubular cells to ER stress injury. (a) Tumor necrosis factor α (TNFα; 10ng/ml) causes an increase in eIF2α phosphorylation in cultured proximal tubular cells. (b) Proximal tubular cells isolated from TNF receptor-1 (TNFR1)−/− mice were more susceptible to tunicamycin-induced cell death. **P<0.01 vs proximal tubular cells isolated from wild-type (WT) or TNFR2−/− mice treated with the same dosage of tunicamycin. (c) Tunicamycin (1μg/ml) induced an increase in eIF2α phosphorylation in proximal tubular cells from WT and TNFR2−/− mice, but not in proximal tubular cells from TNFR1−/− mice. (d) Treatment of proximal tubular cells from TNFR1−/− mice with salubrinal causes an increase in eIF2α phosphorylation. (e) Salubrinal substantially reduced the susceptibility of TNFR1−/− proximal tubular cells to tunicamycin-induced cell death. **P<0.01 vs salubrinal-treated cells at the same dosage. Kidney International 2011 79, 613-623DOI: (10.1038/ki.2010.469) Copyright © 2011 International Society of Nephrology Terms and Conditions