Heme, Heme Degradation Products and Systems Biology of Sepsis-associated Organ Failure
Acronym: HHDP
Principal Investigator: Prof. Dr. Michael Bauer
Team: Dr. rer. med. Marcel Kramer, Peter Recknagel, Bianka Wissuwa, Beate Szafranski, Monique Riedel
Research Area: D Sepsis related Organ Failure
Project Number: D1.2
Duration: 01.08.2010 bis 31.07.2015
Module: In-house Professorship Group
The Problem
Hepatic dysfunction characterized by jaundice is traditionally viewed as a late feature of sepsis and portends poor outcome. The project aims to unravel molecular mechanisms of liver failure as well as its interaction with the heme degradation pathway.
Results so far
We hypothesized that changes in liver function occur early yet pass undetected by standard laboratory tests. Using long-term rat models of faecal peritonitis, predicted nonsurvivors displayed downregulation of genes encoding for proteins involved in phase I and II metabolism, and hepatobiliary transport. Simultaneously, both HMOX-1 and PI3K signaling were induced as early as six hours. Functional changes at fifteen hours included hepatocellular accumulation of bilirubin, bile acids and xenobiotics, with disturbed bile acid conjugation and drug metabolism. A comparable pattern of unconjugated and conjugated bile acids was also observed in plasma of patients fulfilling ACCP/SCCM criteria for severe sepsis on the day of diagnosis. In experimental animals cholestasis was preceded by loss of microvilli and internalization of the multidrug resistance-associated protein (Mrp2)-dependent transport machinery at the canalicular pole. Inhibitors of PI3K partially prevented cytokine-induced loss of villi and Mrp2 in cultured HepG2 cells. Notably, mice lacking the PI3Kγ gene were protected against cholestasis and impaired bile acid conjugation. Liver dysfunction is thus an early event in sepsis, in which PI3K signaling plays a crucial role. All aspects of hepatic biotransformation are affected with severity relating to subsequent prognosis. Detected changes significantly precede conventional markers and are reᴀected by early alterations in plasma conjugated and unconjugated bile acids. These observations carry important implications for diagnosis, monitoring and pharmacotherapy in the critically ill.
To find genetic polymorphisms associated with human sepsis we screened genes involved in the heme degradation pathway in patients with severe human sepsis. We found a single nucleotide polymorphism rs2071746 and a GT-dinucleotide repeat in the promoter region of HMOX1, but no polymorphism in genes encoding biliverdin reductases (BLVRA and BLVRB) associated with the outcome of severe human sepsis. The functional impact of polymorphisms in the HMOX1 locus on sepsis outcome is far from being understood, but the polymorphisms potentially correlate with HO-1 expression. A correlation of the GT-dinucleotide repeat with HO-1 mRNA and protein levels has already been shown. Therefore, we analyzed the HMOX1 gene structure in more detail by sequencing HMOX1 transcripts. We were able to detect various alternatively spliced transcripts including a novel first exon in the 5` untranslated region (UTR) extending the current gene model. The evaluation of the alternative 5’-UTR splicing in the context of polymorphisms associated with sepsis outcome was done by a minigene approach. We found a dependency of splice-isoform frequencies and GT-dinucleotide repeat length. Since alternative 5’-UTR splicing is known to be involved in translation regulation we are currently validating the novel 5’-UTR transcripts in Luciferase reporter assays. This may provide a link between the genetic features of HMOX1, regulation of HO-1 activity and outcome.
Publications
Gonnert FA, Kunisch E, Gajda M, Lambeck S, Weber M, Claus RA, Bauer M, Kinne RW: Hepatic Fibrosis in a Long-Term Murine Model of Sepsis. Shock 2012 Apr, 37(4):399-407.
Gonnert FA, Recknagel P, Seidel M, Jbeily N, Dahlke K, Bockmeyer CL, Winning J, Lösche W, Claus RA, Bauer M: Characteristics of clinical sepsis reflected in a reliable and reproducible rodent sepsis model. J Surg Res 2011 Sep, 170(1):e123-34.
Kleiman A, Huebner S, Rodriguez Parkitna JM, Neumann A, Hofer S, Weigand MA, Bauer M, Schmid W, Schuetz G, Libert C, Reichardt HM, Tuckermann JP: Glucocorticoid receptor dimerization is required for survival in septic shock via suppression of interleukin-1 in macrophages. FASEB J. 2012 Feb;26(2):722-9.
Recknagel P, Claus RA, Neugebauer U, Bauer M, Gonnert FA: In vivo imaging of hepatic excretory function in the rat by fluorescence microscopy. J Biophotonics. 2012 Jan 23, [Epub ahead of print]
Recknagel P, Gonnert FA, Westermann M., Lambeck S, Lupp A, Rudiger A, Dyson A, Carré JE, Kortgen A, Krafft C, Popp J, Sponholz C, Fuhrmann V, Hilger I, Claus RA, Riedemann NC, Wetzker R, Singer M, Trauner M, Bauer M: Liver Dysfunction and Phosphatidylinositol-3-kinase Signaling in Early Sepsis: Experimental Studies in Rodent Models of Peritonitis. PLoS Medicine, 2012 accepted.
Sponholz C, Huse K, Kramer M, Giamarellos-Bourboulis EJ, Claus RA, Engel C, Kuhnt E, Kiehntopf M, Brunkhorst FM, Routsi C, Mylona V, Tsangaris I, Heinemann SH, Reinhart K, Platzer M, Bauer M: Gene polymorphisms in the heme degradation pathway and outcome of severe human sepsis. Shock in press
Weber M, Lambeck S, Ding N, Henken S, Kohl M, Deigner HP, Enot DP, Igwe EI, Frappart L, Kiehntopf M, Claus RA, Kamradt T, Weih D, Vodovotz Y, Briles DE, Ogunniyi AD, Paton JC, Maus UA, Bauer M: Hepatic induction of cholesterol biosynthesis reflects a remote adaptive response to pneumococcal pneumonia. FASEB J 2012 Jun, 26(6):2424-36.