Volume 85, Issue 1, Pages 62-71 (January 2014) The heat-shock protein-70–induced renoprotective effect is partially mediated by CD4+CD25+Foxp3+ regulatory T cells in ischemia/reperfusion-induced acute kidney injury  Myung-Gyu Kim, Eun Jung Cho, Jae Won Lee, Yoon Sook Ko, Hee Young Lee, Sang-Kyung Jo, Won Yong Cho, Hyoung Kyu Kim  Kidney International  Volume 85, Issue 1, Pages 62-71 (January 2014) DOI: 10.1038/ki.2013.277 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Effect of heat preconditioning (HP) on splenic CD4+CD25+ cells. Splenocytes were obtained from HP mice (41±0.5°C for 15min) or control mice at 24h, and the numbers of regulatory T cells (Tregs) were compared; n=5–7 per group. *P<0.05 compared with control mice. Kidney International 2014 85, 62-71DOI: (10.1038/ki.2013.277) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Proliferative response upon mitogenic stimulus. Different numbers (5 × 105∼0.625 × 105) of spleen mononuclear cells (MNCs) from heat-preconditioned (HP) or control mice were cultured with or without anti-mouse CD3/CD28 antibodies (Abs) for 72h. Neg, negative. Following incubation with bromodeoxyuridine (BrdU) for 18h, the amount of BrdU incorporation was measured; n=4 per group, triplicates. *P<0.05 compared with control mice. Kidney International 2014 85, 62-71DOI: (10.1038/ki.2013.277) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Effect of heat preconditioning (HP) on T-cell function. Isolated CD3+ cells (1 × 107) from control or HP mice were adoptively transferred to nu/nu mice, and 32min of bilateral renal pedicle clamping was done. Mice were killed at 24h after reperfusion, and kidney function, histology, and neutrophil infiltration were compared. (a) Purity of isolated CD3+ cells was 95.8%. (b) Serum creatinine. For comparison, ischemia/reperfusion (I/R) injury was performed in wild-type (WT) mice. (c) The nu/nu mice, histology (periodic acid–Schiff (PAS) stain), original magnification × 100, n=4–6 per group, scale bar=200μm. (d) The nu/nu mice, neutrophil infiltration (Gr-1 immunohistochemistry), original magnification × 200, n=4–6 per group, scale bar=100μm. #P<0.05 compared with sham in control mice, *P<0.05 compared with IR in nu/nu mice, and **P<0.05 compared with T cellscontrol+I/R. Kidney International 2014 85, 62-71DOI: (10.1038/ki.2013.277) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Effect of depletion (dep) and adoptive transfer (adop transf) of regulatory T cells (Tregs) on the renoprotective effect by heat preconditioning (HP). Anti-mouse CD25 monoclonal antibodies (mAbs; PC61) were intraperitoneally administered on day −5, and HP was performed on day −1 before ischemia/reperfusion (I/R) injury. Depletion of CD4+CD25+ cells was confirmed by flow cytometric analysis of splenocytes. Purified CD4+CD25+ Tregs (1 × 106) were adoptively transferred on day −3. Mice were killed on day 1 after I/R and serum creatinine was measured. (a) Serum creatinine at 24h. (b) Semiquantitative tubular injury score. (c) Periodic acid–Schiff (PAS)–stained kidney tissue sections. Original magnification × 100, n=4−6 per group, scale bar=200μm. *P<0.05 compared with sham, **P<0.05 compared with I/R, #P<0.05 compared with HP+I/R, and ##P<0.05 compared with Tregdep+ HP+I/R. Kidney International 2014 85, 62-71DOI: (10.1038/ki.2013.277) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Infiltrating regulatory T cells (Tregs) increased significantly in heat-preconditioned (HP) ischemic kidneys. Renal pedicles were clamped for 32min in HP or control mice and killed at 24h after ischemia/reperfusion (I/R) injury. HP was performed by maintaining the core temperature of mice at 41±0.5°C for 15min. (a) Flow cytometric analysis of kidney CD4+Foxp3+ cells. The number of Tregs (per g kidney) was expressed as fold difference compared with sham. (b) Kidney Foxp3 mRNA expression determined as fold difference compared with sham mice; n=4–7 per group. *P<0.05 compared with sham or I/R. Kidney International 2014 85, 62-71DOI: (10.1038/ki.2013.277) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Heat preconditioning (HP) induced heat-shock protein-70 (HSP70) expression in immune cells in spleen. HP was performed by maintaining the core temperature of mice at 41±0.5°C for 15min, and mice were killed 24h later. (a) HSP70 immunochemistry in kidney, liver, and lung, original magnification × 200, n=3, scale bar=100μm; spleen, original magnification × 40, scale bar=500μm. (b) Western blot analysis of HSP70 in kidney, liver, lung, and immune cells in spleen. Relative fold difference in densitometry value compared with respective tissue control from sham animal is provided. (c) Double immunofluorescence of HSP70 with CD4, F4/80, and CD11c. Original magnification × 200, n=3, scale bar=100μm. (d) Flow cytometric detection of HSP70 in CD11c+ cells. Splenocytes were stained with CD11c-allophycocyanin (APC) antibody (Ab) and subsequently stained with intracellular HSP70–fluorescein isothiocyanate (FITC) Ab following fixation and permeabilization. Kidney International 2014 85, 62-71DOI: (10.1038/ki.2013.277) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 Effect of quercetin pretreatment on spleen heat-shock protein-70 (HSP70) expression, number of splenic CD4+CD25+ regulatory T cells (Tregs), and T-cell proliferation. Heat preconditioning (HP) was performed by maintaining the core temperature of mice at 41±0.5°C for 15min, and mice were killed 24h later. Quercetin (100mg/kg) was administered intraperitoneally 3h before HP. (a) Western blot of HSP70 in immune cells in spleen. (b) Flow cytometric analysis of CD4+CD25+ Tregs. (c) 5 × 105 spleen mononuclear cells (MNCs) from control (C), quercetin-treated, HP, or quercetin (Q)+HP mice were cultured with anti-mouse CD3/CD28 antibodies (Abs) for 72h, and after incubation with bromodeoxyuridine (BrdU) for 18h, the amount of BrdU incorporation was measured; n=4–5 per each group. *P<0.05 compared with sham and #P<0.05 compared with HP group. Kidney International 2014 85, 62-71DOI: (10.1038/ki.2013.277) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 8 Effect of transfer of regulatory T cells (Tregs) on quercetin-induced loss of renoprotective effect of heat preconditioning (HP) in ischemia/reperfusion (I/R) injury. HP was performed by maintaining the core temperature of mice at 41±0.5°C for 15min and I/R injury was performed 24h later. Quercetin (Q) was intraperitoneally administered 3h before HP. In Q+Tregs+HP+I/R group, purified Tregs (1 × 106) were intravenously (i.v.) administered right after HP. Then, 32min of bilateral renal pedicle clamping was done and mice were killed at 24h after reperfusion; n=4–6 per group, serum creatinine at 24h. *P<0.05 compared with HP+I/R and **P<0.05 compared with Q+HP+I/R. Kidney International 2014 85, 62-71DOI: (10.1038/ki.2013.277) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 9 Adoptive transfer of T cells from heat-preconditioned (HP) heat-shock protein-70 knockout (HSP70 KO) mice reconstitute postischemic injury in nu/nu mice. HP was performed by maintaining the core temperature of mice at 41±0.5°C for 15min. CD3+ T cells from control, HP, and HP HSP70 KO mice were isolated by MACS, adoptively transferred, and their function to reconstitute postischemic injury in nu/nu mice was compared; n=4 per group, *P< 0.05 compared with ischemia/reperfusion (I/R) injury, **P<0.05 compared with T cellcontrol+I/R, and #P<0.05 compared with T cellHP+I/R. Kidney International 2014 85, 62-71DOI: (10.1038/ki.2013.277) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 10 Pharmacologic induction of heat-shock protein-70 (HSP70) in cultured splenocytes was associated with expansion of regulatory T cells (Tregs). Splenocytes (1 × 105) from control mice were incubated with 1, 10, and 100μmol/l geranylgeranylacetone (GGA) for 18h. (a) Western blot for HSP70. (b) Percentages of CD4+CD25+ Tregs were compared; n=3 per group. *P<0.05 compared with no GGA. Kidney International 2014 85, 62-71DOI: (10.1038/ki.2013.277) Copyright © 2014 International Society of Nephrology Terms and Conditions