Karen M. Lochhead, Richard A. Zager Kidney International

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Fluorinated anesthetic exposure “activates” the renal cortical sphingomyelinase cascade Karen M. Lochhead, Richard A. Zager Kidney International Volume 54, Issue 2, Pages 373-381 (August 1998) DOI: 10.1046/j.1523-1755.1998.00022.x Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 1 Renal cortical ceramide concentrations in normal mouse kidneys and in kidneys obtained from mice after six hours of isoflurane (ISO; A; P < 0.0003), desflurane (DES; B; P < 0.0003), or pentobarbital (Pent; C; P = NS) anesthesia. Isoflurane and desflurane, but not pentobarbital, caused significant increases in ceramide concentrations. Kidney International 1998 54, 373-381DOI: (10.1046/j.1523-1755.1998.00022.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 2 Autoradiograph, obtained during ceramide assay. Lanes 1 and 2 are from control mice, and lanes 3 and 4 are from isoflurane treated mice. The ceramide-132P bands (Cer) appear denser in the isoflurane samples (with equivalent phospholipid phosphate loading for all samples). Note that the ceramide-1-32P band appears as a doublet (due to differing acyl group lengths (C16/C18 and C24 fatty acids). The whole band was cut and counted together to quantitate total ceramide levels. PA = phosphatidic acid. Kidney International 1998 54, 373-381DOI: (10.1046/j.1523-1755.1998.00022.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 3 Sphingomyelinase activity in cortical tissue extracts obtained from isoflurane anesethetized (ISO) and control mice. There were no significant differences in either acidic (A) or neutral (B) enzyme activities, indicating that the isoflurane-induced ceramide increments were not a result of increased enzyme levels. Kidney International 1998 54, 373-381DOI: (10.1046/j.1523-1755.1998.00022.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 4 Effects of isoflurane additions (0.5 and 1.0mm) on sphingomyelinase activity in cell free extracts. Both doses of isoflurane induced an approximate 20 to 25% increase in acidic sphingomyelinase-mediated 14C-sphingomyelin breakdown. However, neither isoflurane dosage stimulated neutral sphingomyelinase activity (indicating the relative specificity of the acidic sphingomyelinase result). Kidney International 1998 54, 373-381DOI: (10.1046/j.1523-1755.1998.00022.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 5 Ceremide in proximal tubular segments (A) and in cultured tubular (HK-2) cells (B). (A) Ceramide concentrations in isolated PTS incubated for one hour under control oxygenated conditions or in the presence of 1mm isoflurane. Isoflurane induced significant ceramide increments. (B) When HK-2 cells were incubated with isoflurane for four hours, a 33% increase in ceramide concentrations resulted. This result was not impacted by ceramide synthase inhibition (fumonisin B1; FB1), indicating that it was not secondary to de novo ceramide synthesis. Kidney International 1998 54, 373-381DOI: (10.1046/j.1523-1755.1998.00022.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 6 Western blot demonstrating heat shock protein 72 (HSP 72) expression in cortical tissue extracts. Lane 1, myohemoglobinuria-induced positive control; lanes 2 and 3, six hours of isoflurane anesthesia; lanes 4 and 5, six hours of isoflurane + 18hours of recovery; lanes 6 and 7, normal mouse kidneys. Isoflurane did not noticeably increase HSP 72 levels above normal levels. Kidney International 1998 54, 373-381DOI: (10.1046/j.1523-1755.1998.00022.x) Copyright © 1998 International Society of Nephrology Terms and Conditions