Volume 68, Issue 5, Pages 2029-2041 (November 2005) Disturbed Ca2+-signaling by chloroacetaldehyde: A possible cause for chronic ifosfamide nephrotoxicity  Andreas Benesic, Gerald Schwerdt, Sigrid Mildenberger, Ruth Freudinger, Nader Gordjani, Michael Gekle  Kidney International  Volume 68, Issue 5, Pages 2029-2041 (November 2005) DOI: 10.1111/j.1523-1755.2005.00657.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Apical distribution of γ-glutamyl-transpeptidase (γGT) in human renal proximal tubule cells (RPTEC) (N = 6 to 12) changes with passage number. Human embryonic kidney 293 (HEK-293) cells (passage 42) were used as negative control cells with no proximal tubular phenotype (N = 6). Kidney International 2005 68, 2029-2041DOI: (10.1111/j.1523-1755.2005.00657.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Effect of chloroacetaldehyde (CAA). (A) Original tracing of the effect of CAA on intracellular Ca2+ in human renal proximal tubule cells (RPTECs). (B) CAA increased baseline Ca2+ in RPTEC dose-dependently with a maximal effect at 15 μmol/L. *P < 0.05 vs. control. Kidney International 2005 68, 2029-2041DOI: (10.1111/j.1523-1755.2005.00657.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 Effect of lowering extracellular Ca2+ below 10 μmol/L. (A) There was a decrease of [Ca2+]i in human renal proximal tubule cells (RPTEC) and abolished the effect of chloroacetaldehyde (CAA). (B) Store depletion with 100 nmol/L thapsigargin (TG) still produced a significant Ca2+ response under these conditions. Kidney International 2005 68, 2029-2041DOI: (10.1111/j.1523-1755.2005.00657.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Addition of 50 μmol/L Mn2+ led to a decrease in fura-2 fluorescence by excitation with 365nm (isosbestic wavelength). Chloroacetaldehyde (CAA) did not influence the time course of Mn2+ entry. High extracellular K+ (20 mmol/L) was used as negative, 1 μmol/L ionomycin (iono) as positive control. Kidney International 2005 68, 2029-2041DOI: (10.1111/j.1523-1755.2005.00657.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 Measured and calculated Ca2+ peak and plateau concentrations after store-depletion by 100 nmol/L thapsigargin (control N = 50; chloroacetaldehyde (CAA) N = 44). Inset, Time constant of Ca2+ extrusion under control conditions and CAA exposure. The Ca2+ peak as well as the area under the curve (data not shown) were slightly but not significantly increased in CAA-exposed cells. Kidney International 2005 68, 2029-2041DOI: (10.1111/j.1523-1755.2005.00657.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 6 Original tracings of [Ca2+]i in human renal proximal tubule cells (RPTECs). Na+ removal switched the Na+/Ca2+ exchanger to Ca2+ entry mode, leading to an increase of [Ca2+]i. By readdition of extracellular sodium, [Ca2+]i decreased rapidly due to Ca2+ exit mode of the exchanger. Under control conditions repeated Na+ removal left the slope unaltered (A). Chloroacetaldehyde (CAA) exposure reduced Na+-dependent Ca2+ extrusion in RPTECs (B). (C) Δ[Ca2+]i (nmol * L-1 * second-1) vs. [Ca2+]i (nmol/L) for control and CAA-exposed cells. CAA reduced Na+-dependent Ca2+ extrusion resulting in a right shift of the concentration-response curve for Na+/Ca2+ exchange. *P < 0.05 vs. control. Kidney International 2005 68, 2029-2041DOI: (10.1111/j.1523-1755.2005.00657.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 7 Reverse transcription-polymerase chain reaction (RT-PCR) and PCR of human renal proximal tubule cells (RPTECs) mRNA-preparations revealed Na+/Ca2+ exchanger mRNA expression in human renal proximal tubule cells (RPTECs). Abbreviations are: M, DNA molecular weight marker; -, H2O as negative control; +, RPTEC genomic DNA as positive control; 1 and 2, mRNA preparations of RPTEC; +RT, RT-PCR, 35 cycles; -RT, = RT-PCR, 35 cycles after denaturation of RT by incubating the Mastermix 4 minutes at 94°C. Representative of three independent experiments. Kidney International 2005 68, 2029-2041DOI: (10.1111/j.1523-1755.2005.00657.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 8 Effects of modulators of protein kinase C (PKC) and protein kinase A (PKA) on Na+/Ca2+ exchange (NCX) activity in human renal proximal tubule cells (RPTECs). Inhibition of PKC by 100 nmol/L bisindolylmaleimide I (BIM) stimulated activation of PKC by 100 nmol/L phorbol 12-myristate 13-acetate (PMA) decreased NCX. Activation of PKA by 10 μmol/L forskolin or 100 μmol/L dibutyryl-cyclic adenosine monophosphate (cAMP) reduced Na+-dependent Ca2+ extrusion, whereas inhibition of PKA by 500 nmol/L H-89 showed no effect. Modulation of PKA activity in both directions blunted the effect of chloroacetaldehyde (CAA) on NCX. n.s. is not significant. *P < 0.05 vs. control) Kidney International 2005 68, 2029-2041DOI: (10.1111/j.1523-1755.2005.00657.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 9 Long-term incubation (72 hours) with different chloroacetaldehyde (CAA) concentrations. Concentrations ≥ 15 μmol/L led to a significant decrease of renal proximal tubule cell (RPTEC) protein content. Kidney International 2005 68, 2029-2041DOI: (10.1111/j.1523-1755.2005.00657.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 10 Effect of chloroacetaldehyde (CAA) incubations. (A) Forty-eight hours' incubation with 15 and 150 μmol/L CAA induced a reduction of cell membrane integrity, shown by increased uptake of trypan blue. *P < 0.05 vs. control. (B and C) Morphologic changes in renal proximal tubule cells (RPTECs) after 48 hours' incubation with 15 μmol/L CAA. (D) Caspase-3 activity was not increased after 24 hours' incubation with 15 μmol/L CAA, indicating a subordinate role of apoptosis in CAA-induced cell death. (E and F) Confirming the caspase-3 data, 4′,6-diamino-2-phenylindol (DAPI) staining showed no increased DNA condensation after 48 hours' incubation with 15 μmol/L CAA compared to controls. Representative of three independent experiments. Kidney International 2005 68, 2029-2041DOI: (10.1111/j.1523-1755.2005.00657.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 11 (A) Chelation of intracellular free Ca2+ by 1,2-bis(2-aminophenoxy)ethane-N,N,N′N, ′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) (50 μmol/L) completely abolished the effect of 15 μmol/L chloroacetaldehyde (CAA) on renal proximal tubule cell (RPTEC) protein content after 48 hours of incubation. The toxicity of 150 μmol/L CAA remained unchanged by BAPTA-AM. (B) The decrease in cell number caused by 15 μmol/L CAA was also inhibited by 50 μmol/L BAPTA-AM. Kidney International 2005 68, 2029-2041DOI: (10.1111/j.1523-1755.2005.00657.x) Copyright © 2005 International Society of Nephrology Terms and Conditions