Renal tubule necrosis and apoptosis modulation by A1 adenosine receptor expression  H.T. Lee, M. Kim, M. Jan, R.B. Penn, C.W. Emala  Kidney International  Volume 71, Issue 12, Pages 1249-1261 (June 2007) DOI: 10.1038/sj.ki.5002227 Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 1 Overexpression of the A1AR-GFP in LLC-PK1 cells produces cytoprotection against necrosis induced with H2O2. (a) Dose- (LDH release was examined at 2h) and (b) time-dependent cytoprotection against H2O2-induced necrosis (n=6). *P<0.05 compared to GFP LLC-PK1 cells. Kidney International 2007 71, 1249-1261DOI: (10.1038/sj.ki.5002227) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 2 Overexpression of the A1AR-GFP in LLC-PK1 cells produces cytoprotection against apoptosis induced with TNF-α plus cycloheximide. (a) (upper panel) Representative immunoblot image of fragmentation of PARP or caspase 3 induced by TNF-α plus cycloheximide as compared to vehicle (V) which was attenuated in A1AR-GFP-overexpressing LLC-PK1 cells compared to control cells (GFP-overexpressing LLC-PK1 cells). (lower panel) Relative band intensities from immunoblots of fragmented PARP or caspase 3 (n=6) (*P<0.05 compared to GFP-LLC-PK1 cells treated with TNF-α plus cycloheximide (CXM)). (b) Representative gel image of internucleosomal DNA fragmentation induced by TNF-α plus cycloheximide illustrating less DNA fragmentation in A1AR-GFP-overexpressing LLC-PK1 cells. (c) (upper panel) Representative immunoblot image illustrating the translocation of cytochrome c from mitochondrial to cytosolic fractions induced by TNF-α plus cycloheximide and attenuation in A1AR-GFP-overexpressing LLC-PK1 cells. (lower panel) Relative band intensities from immunoblots of cytosolic cytochrome c (n=6) (*P<0.05 compared to vehicle-treated GFP LLC-PK1 cells). Kidney International 2007 71, 1249-1261DOI: (10.1038/sj.ki.5002227) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 3 A1AR antagonist attenuate protection from necrosis and apoptosis in A1AR-LLC-PK1 cells. (a) Endogenous protection against H2O2 (500μM, 2 or 4h)-induced necrosis (as percent LDH release) in A1AR-GFP LLC-PK1 cells is blocked by 48h pretreatment with the irreversible A1AR antagonist FSCPX (20μM) (n=6) (*P<0.05 compared to A1AR-GFP LLC-PK1 cells treated with FSCPX). (b) Representative (of six) gel image of DNA laddering illustrating the loss of endogenous protection against TNF-α plus cycloheximide (CXM)-induced apoptosis in A1AR-GFP-overexpressing LLC-PK1 cells by chronic exposure (48h) to FSCPX (20μM). (c) Increased resistance to apoptosis A1AR-GFP LLC-PK1 cells is blocked by 48h pretreatment with 20μM FSCPX (n=4). Top: representative blots from four independent experiments performed. Densitometric quantifications of unfragmented PARP (middle) and caspase 3 (bottom) from GFP- and A1AR-GFP-overexpressing LLC-PK1 cells treated with vehicle, TNF-α plus cycloheximide or TNF-α plus cycloheximide plus FSCPX. *P<0.05 vs GFP-overexpressing cells treated with vehicle, #P<0.05 vs A1AR-GFP-overexpressing cells treated with vehicle. Kidney International 2007 71, 1249-1261DOI: (10.1038/sj.ki.5002227) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 4 Primary cultures of renal proximal tubule cells from A1KO mice are more susceptible to necrosis and apoptosis. Cells from A1AR KO mice showed increased necrosis in response to H2O2 in both a dose- (a) and time- (b) dependent manner compared to renal proximal tubule cells isolated from A1AR WT mice (n=6) (*P<0.05 compared to cells from A1AR KO mice). (c) Representative (of six) gel image of internucleosomal DNA fragmentation in primary cultures of proximal tubule cells isolated from A1AR WT or A1AR KO mice after treatment with vehicle (V) or TNF-α plus cycloheximide (CXM) illustrating reduced fragmentation in cells from A1AR WT compared to A1AR KO mice. Kidney International 2007 71, 1249-1261DOI: (10.1038/sj.ki.5002227) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 5 Basal levels of HSP27 mRNA and protein are increased in LLC-PK1 cells overexpressing the A1AR. (a) (upper panel) Representative gel image of RT-PCR results for mRNA encoding HSP27 in unstimulated LLC-PK1 cells overexpressing the A1AR-GFP vs GFP alone. GAPDH mRNA is shown to correct for RNA input in each sample (lower panel). Quantification of relative band intensities (as ratio of GAPDH content) of RT-PCR results for HSP27 in A1AR-GFP vs GFP LLC-PK1 overexpressing cells (n=6) (*P<0.05 compared to control GFP LLC-PK1 cells). (b) (upper panel) Representative gel image of immunoblot results for HSP27 protein in unstimulated LLC-PK1 cells overexpressing the A1AR-GFP vs GFP alone. (lower panel) Quantification of relative band intensities of immunoblot results for HSP27 in A1AR-GFP vs GFP LLC-PK1 overexpressing cells (n=6) (*P<0.05 compared to control GFP LLC-PK1 cells). Kidney International 2007 71, 1249-1261DOI: (10.1038/sj.ki.5002227) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 6 HSP27 mRNA and protein are increased by an A1AR agonist. (a) (upper panel) Representative gel image of RT-PCR results for mRNA encoding HSP27 in renal proximal tubule cells from LLC-PK1 cells stimulated with the A1AR agonist CCPA for indicated time points. (lower panel) Quantification of HSP27 mRNA (as ratio of GAPDH content) in A1AR-GFP- or GFP-overexpressing LLC-PK1 cells following 0–16h of 1μM CCPA treatment (n=5) (*P<0.05 compared to unstimulated GFP LLC-PK1 cells). (b) Representative gel image of immunoblot results for HSP27 in LLC-PK1 cells (upper panel) stimulated with the A1AR agonist CCPA for 0–16h. (lower panel) Quantification of relative band intensities of HSP27 protein in GFP LLC-PK1 cells following 0–16h of 1μM CCPA treatment (n=5) (*P<0.05 compared to baseline (CCPA 0h) GFP LLC-PK1 cells). Kidney International 2007 71, 1249-1261DOI: (10.1038/sj.ki.5002227) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 7 A1AR agonist increases phosphorylation of HSP27 protein. (upper panel) Representative (of eight) gel image of immunoblot of phosphorylated (phospho)-HSP27 in A1AR-GFP- and GFP-overexpressing LLC-PK1 cells following 0–20h exposure to the A1AR agonist CCPA (1μM). (lower panel) Relative immunoblot band intensities of phospho-HSP27 in A1AR-GFP- or GFP-overexpressing LLC-PK1 cells following 0–20h of 1μM CCPA (n=8) (*P<0.05 compared to unstimulated GFP LLC-PK1 cells; #P<0.05 compared to A1AR-GFP LLC-PK1 cells). Kidney International 2007 71, 1249-1261DOI: (10.1038/sj.ki.5002227) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 8 A1AR agonist induces increases in phosphorylation of p38 MAPK and MAPKAP2. (a) (upper panel) Representative (of four) gel image of immunoblot of phosphorylated (phospho)-MAPKAP2 (1μM CCPA for 30min) or phosphorylated (p) p38 MAPK (1μm CCPA for 0–8h) in GFP-overexpressing LLC-PK1 cells following exposure to the A1AR agonist CCPA (1μM). (lower panel) Relative immunoblot band intensities of phosphorylated p38 MAPK in GFP-overexpressing LLC-PK1 cells following 0–8h of 1μM CCPA (n=4) (*P<0.05 compared to baseline (0h CCPA)). (b) (upper panel) Representative (of three) gel image of immunoblot of phosphorylated (p) HSP27 in A1AR-GFP- or GFP-overexpressing LLC-PK1 cells following 30min exposure to 1μM CCPA following 30min pretreatment with inhibitors of MEK1 (50μM PD98059) (PD), PI3-kinase (100nM wortmannin) (Wort), p38 MAPK (20μM SB203580) (SB), or the A1AR antagonist (DPCPX) (10μM). (lower panel) Relative immunoblot band intensities of phosphorylated HSP27 in A1AR-GFP- or GFP-overexpressing LLC-PK1 cells following 30min of 1μM CCPA following 30min pretreatment with indicated inhibitors (n=3) (*P<0.05 compared to unstimulated GFP LLC-PK1 cells; #P<0.05 compared to 1μM CCPA-stimulated GFP LLC-PK1 cells; % P<0.05 compared to 1μM CCPA-stimulated A1AR-GFP LLC-PK1 cells). Kidney International 2007 71, 1249-1261DOI: (10.1038/sj.ki.5002227) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 9 Endogenous upregulation of HSP27 mRNA and protein in A1AR overexpressing LLC-PK1 cells are attenuated by an A1AR antogonist. (a) (upper panel) Representative (of six) gel image of RT-PCR results for mRNA encoding HSP27, HSP70, or GAPDH in A1AR-GFP LLC-PK1 overexpressing cells chronically (48h) treated with the irreversible A1AR antagonist FSCPX (2 or 20μM) illustrating attenuation of the endogenous upregulation of HSP27 mRNA expression without effects on HSP70 mRNA. (lower panel) Quantification of relative band intensities of RT-PCR results for HSP27 in A1AR-GFP or GFP LLC-PK1 overexpressing cells chronically exposed to 2 or 20μM with the irreversible A1AR antagonist FSCPX (n=6) (*P<0.05 compared to untreated A1AR-GFP LLC-PK1 cells; #P<0.05 compared to untreated GFP LLC-PK1 cells). (b) (upper panel) Representative (of six) gel image of immunoblot for HSP27 and HSP70 in A1AR-GFP LLC-PK1 overexpressing cells chronically (48h) treated with the irreversible A1AR antagonist FSCPX (2 or 20μM) illustrating attenuation of the endogenous upregulation of HSP27 protein expression without effects on HSP70 protein. (lower panel) Quantification of relative band intensities from immunoblots of HSP27 in A1AR-GFP or GFP LLC-PK1 overexpressing cells chronically exposed to 2 or 20μM with the irreversible A1AR antagonist FSCPX (n=6) (*P<0.05 compared to untreated A1AR-GFP LLC-PK1 cells; #P<0.05 compared to untreated GFP LLC-PK1 cells). Kidney International 2007 71, 1249-1261DOI: (10.1038/sj.ki.5002227) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 10 Endogenous protection against necrosis and apoptosis is attenuated by inhibition of HSP protein synthesis in LLC-PK1 cells overexpressing the A1AR or in proximal cells isolated from A1WT mice. (a) Endogenous protection against H2O2 (500μM, 2 or 4h)-induced necrosis (as percent LDH release) in A1AR-GFP LLC-PK1 cells is blocked by 16h pretreatment with a specific inhibitor of HSP (KNK-347) (50 or 100μM) (n=5) (*P<0.05 compared to GFP LLC-PK1 cells treated with H2O2 plus vehicle). (b) Increased resistance to apoptosis A1AR-GFP LLC-PK1 cells is blocked by 16h pretreatment with 100μM KNK-347 (n=4). Top: representative blots from four independent experiments performed. Densitometric quantifications of unfragmented PARP (middle) and caspase 3 (bottom) from GFP- and A1AR-GFP-overexpressing LLC-PK1 cells treated with vehicle, TNF-α plus cycloheximide or TNF-α plus cycloheximide plus KNK-437. *P<0.05 vs GFP-overexpressing cells treated with vehicle, #P<0.05 vs A1AR-GFP-overexpressing cells treated with TNF-α plus cycoheximide. (c) Reduced necrosis in response to 5mM H2O2 in proximal tubule cultures from A1WT mice is blunted in cells treated with KNK-437 (100μM for 16h). *P<0.01 vs A1WT tubules treated with vehicle. (d) Reduced apoptosis (detected with caspase 3 immunoblotting) in response to TNF-α plus cycloheximide in proximal tubule cultures from A1WT mice is no longer demonstrated in cells treated with KNK-437 (100μM for 16h). Representative of four separate experiments. Kidney International 2007 71, 1249-1261DOI: (10.1038/sj.ki.5002227) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 11 A1AR agonist increases phosphorylation of HSP in A1WT proximal tubule cells in vivo. (a) Representative (of four) gel image of immunoblot of phosphorylated (phospho)-HSP27 in renal proximal tubules from A1AR WT and A1AR KO mice treated for 0, 30, or 60min with the A1AR agonist CCPA (0.1mg/kg i.p.). (b) Relative immunoblot band intensities of phospho-HSP27 in isolated renal proximal tubule cells from A1AR WT and A1AR KO mice following 30 or 60min of 0.1mg/kg CCPA (n=4) (*P<0.05 compared to baseline (CCPA 0h) A1AR WT tubule cells; #P<0.05 compared to A1AR KO tubule cells). Kidney International 2007 71, 1249-1261DOI: (10.1038/sj.ki.5002227) Copyright © 2007 International Society of Nephrology Terms and Conditions