Stephen Shafizadeh

Fatty Acid Synthase Blockade Protects Steatotic Livers from Warm Ischemia Reperfusion Injury and Transplantation

Cerulenin has been shown to reduce body weight and hepatic steatosis in murine models of obesity by inhibiting fatty acid synthase (FAS). We have shown that attenuating intrahepatocyte lipid content diminished the sensitivity of ob/ob mice to ischemia/reperfusion injury and improved survival after liver transplantation. The mechanism of action is by inhibition of fatty acid metabolism by downregulating PPARα, as well as mitochondrial uncoupling protein 2 (UCP2), with a concomitant increase in ATP. A short treatment course of cerulenin prior to I/R injury is ideal for protection of steatotic livers. Cerulenin opens the potential for expanding the use of steatotic livers in transplantation.

Development of an unbiased method for the estimation of liver steatosis

Abstract:  Background:  Steatosis significantly contributes to an organs transplantability. Livers with >30% fat content have a 25% chance of developing primary non‐function (PNF). The current practice of evaluating a hematoxylin and eosin (H&E) stained donor biopsy by visual interpretation is subjective. We hypothesized that H&E staining of frozen sections fails to accurately estimate the degree of steatosis present within a given liver biopsy. To address this problem of evaluating steatosis in prospective donor organs, we developed a fast, user friendly computer methodology to objectively assess fat content based on the differential quantification of color pixels in Oil Red O (ORO) stained liver biopsies.

Anti‐Endotoxin Monoclonal Antibodies are Protective against Hepatic Ischemia/Reperfusion Injury in Steatotic Mice

Steatotic mice are particularly susceptible to hepatic ischemia/reperfusion injury compared with their lean littermates. We have previously demonstrated that livers of mice having a spontaneous mutation in the leptin gene (ob/ob), resulting in global obesity and liver steatosis, are ATP depleted, are endotoxin sensitive, and do not survive (I/R) injury. We hypothesize that administration of an anti‐LPS monoclonal antibody (mAb) prior to initiation of I/R would be protective from that insult. Steatotic mice (ob/ob) were subjected to 15 min of ischemia via complete porta‐hepatis occlusion and varying lengths of reperfusion with or without pre‐treatment with an anti‐LPS mAb. There was 14–31% survival of isotype matched control mAb treated ob/ob mice after 15 min of ischemia and 24 h of reperfusion. In contrast, 75–83% of ob/ob mice pre‐treated with an anti‐LPS mAb prior to initiation of I/R survived both ischemia and 24 h of reperfusion. Furthermore, there was a decrease in ALT and circulating endotoxin levels when treated with an anti‐LPS mAb compared with control antibodies. Attenuation of the endotoxin load with anti‐LPS mAb, prior to initiation of I/R, was cytoprotective and improved survival. Consequently, these studies might offer a solution to the problems associated with using steatotic livers in clinical transplantation.

Decrease of intracellular ATP content downregulated UCP2 expression in mouse hepatocytes

Mitochondrial uncoupling protein 2 (UCP2) plays an important role in regulating energy metabolism. We previously reported that UCP2 expression in steatotic livers is increased which leads to diminished hepatic ATP stores and renders steatotic hepatocytes vulnerable to ischemic damage. In this study, reagents that inhibit the production of ATP were used to mimic an ischemic state in the liver in order to investigate the effects of decreased intracellular ATP levels on UCP2 expression in a murine hepatocyte cell line (HEP6-16). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), an oxidative phosphorylation uncoupler, was found to decrease intracellular ATP levels in a dose- and time-dependent manner. Relatively high concentrations of FCCP from 8 to 80 microM were required to reduce the intracellular concentration of ATP. The inhibitory effect of FCCP on intracellular ATP was significantly potentiated by 2-deoxy-D-glucose, an inhibitor of glycolysis that when administered alone had no negative effect on cellular ATP levels in mouse hepatocytes. Decreased intracellular ATP levels were accompanied by lower UCP2 mRNA expression. Upon removal of FCCP and/or 2-deoxy-D-glucose and reculture with normal medium, ATP and UCP2 mRNA levels returned to normal within a few hours. Mitochondrial membrane potential in HEP6-16 cells was dissipated by 80 microM FCCP but not 8 microM FCCP, suggesting that the downregulation of UCP2 expression by FCCP was not related to mitochondrial potential changes. Consequently, the in vitro manipulation of ATP stores is consistent with the in vivo observations associated with ischemia/reperfusion injury.

Chylous ascites secondary to laparoscopic donor nephrectomy.

Live donor renal transplantation offers many significant advantages over cadaveric donor transplantation. Yet living donation continues to be underused, accounting for less than 30% of all donor renal transplants. In an attempt to remove the disincentives to live donation, Ratner et al. developed laparoscopic donor nephrectomy (LDN). LDN is gaining acceptance in the transplant community. The overriding concern must always be the safety and welfare of the donor. To this end, potential complications of LDN must be identified and discussed. We present a patient who developed the complication of chylous ascites from LDN. To improve the laparoscopic technique further, a discussion of its successes and complications needs to be encouraged. To this end, we present chylous ascites as a potential complication after LDN. We also offer suggestions to minimize the likelihood of this complication.

Further improvements in laparoscopic donor nephrectomy: decreased pain and accelerated recovery

Fear of postoperative pain is a disincentive to living donor kidney transplantation. Laparoscopic donor nephrectomy (LDN) was developed in part to dispel this disincentive. The dramatic increase in the number of laparoscopic donor nephrectomies performed at our institution has been in part due to the reduction in postoperative pain as compared to traditional, open donor nephrectomy. We sought to further diminish the pain associated with this surgical technique. The purpose of this study was to compare the efficacy of three different postoperative pain management regimens after LDN. All living kidney donors performed laparoscopically (n=43) between September 1998 and April 2000 were included for analysis. Primary endpoints included postoperative narcotic requirements and length of stay. Narcotic usage was converted to morphine equivalents (ME) for comparison purposes. Patients received one of three pain control regimens (group I: oral and intravenous narcotics; group II: oral and intravenous narcotics and the On‐Q™ pump delivering a continuous infusion of subfascial bupivicaine 0.5%; and group III: oral and intravenous narcotics and subfascial bupivicaine 0.5% injection). Postoperative intravenous and oral narcotic use as measured in morphine equivalents was significantly less in group III versus groups I and II (group III: 28.7 ME versus group I: 40.2 ME, group II: 44.8 ME; P<0.05). Postoperative length of stay was also shorter for group III (1.8 days) versus group I (2.5 days) and group II (2.9 days). LDN has been shown to be a viable alternative to traditional open donor nephrectomy for living kidney donation. We observed that the use of combined oral and intravenous narcotics alone is associated with greater postoperative narcotic use and increased length of stay compared to either a combined oral and intravenous narcotics plus continuous or single injection subfascial administration of bupivicaine. The progressive modification of our analgesic regimen has resulted in decreased postoperative oral and intravenous narcotic use and a reduction in the length of stay. We recommend subfascial infiltration with bupivicaine to the three laparoscopic sites and the pfannenstiel incision at the conclusion of the procedure to reduce postoperative pain. We believe this improvement in postoperative pain management will continue to make LDN even more appealing to the potential living kidney donor.