title: Detrimental effects of prolonged warm renal ischaemia-reperfusion injury are abrogated by supplemental hydrogen sulphide: an analysis using real-time intravital microscopy and polymerase chain reaction.
abstract: What's known on the subject? and What does the study add? Hydrogen sulphide (H(2) S) has recently been classified as a member of the gasotransmitter family. Its physiological and pathophysiological effects are rapidly expanding with numerous studies highlighting the protective effects of H(2) S on ischaemia-reperfusion injury (IRI) in various organ systems, e.g. heart, liver, CNS and lungs. The mechanisms behind its protective effects reside in its vasodilatory, anti-inflammatory and anti-oxidant characteristics. These specific mechanistic profiles appear to be different across different tissues and models of IRI. We recently showed that supplementation of preservation solutions with H(2) S during periods of prolonged cold renal storage and subsequent renal transplantation leads to a massive and significant survival, functional and tissue protective advantage compared with storage in standard preservation solution alone. However, there have only been a few studies that have evaluated the effects of H(2) S against warm renal IRI; although these studies have focused primarily upon shorter periods of warm renal pedicle clamping, they have shown a clear survival benefit to H(2) S supplementation. The present study adds to the existing literature by evaluating the effects of H(2) S in a model of warm IRI with clinically relevant, prolonged warm ischaemia-reperfusion times (1 h ischaemia, 2 h reperfusion). We show an unprecedented view into real-time renal and hepatic perfusion with intravital microscopy throughout the reperfusion period. We show, for the first time, that supplemental H(2) S has multiple protective functions against the warm IRI-induced tissue damage, which may be clinically applicable to both donation after cardiac death models of renal transplantation, as well as to uro-oncological practices requiring surgical clamping of the renal pedicle, e.g. during a partial nephrectomy.
  
  
  OBJECTIVE
  • To determine the protective role of supplemental hydrogen sulphide (H(2) S) in prolonged warm renal ischaemia-reperfusion injury (IRI) using real-time intravital microscopy (IVM).
  
  
  METHODS
  • Uninephrectomised Lewis rats underwent 1 h of warm ischaemia and 2 h of reperfusion during intraperitoneal treatment with phosphate buffer saline (IRI, n = 10) or 150 µmol/L NaHS (IRI+H(2) S, n = 12) and were compared with sham-operated rats (n = 9). • Blood was collected for measurement of serum creatinine (Cr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). • IVM was performed to assess renal and hepatic microcirculation. • Kidneys were sectioned for histology and real-time quantitative polymerase chain reaction for markers of inflammation.
  
  
  RESULTS
  • The mean (sd) Cr concentration raised to 72.8(2.5) µmol/L after IRI from 11.0 (0.7) µmol/L (sham) but was partially inhibited with H(2) S to 62.8 (0.9) µmol/L (P < 0.05). • H(2) S supplementation during IRI increased renal capillary perfusion on IVM, and improved acute tubular necrosis and apoptotic scores on histology (P < 0.05). • Supplemental H(2) S decreased expression of the pro-inflammatory markers toll-like receptor 4, tumour necrosis factor α, interleukin 8, C-C chemokine receptor type 5, interferon γ and interleukin 2 (P < 0.05). • Distant organ (liver) dysfunction after renal IRI was limited with H(2) S supplementation: blunting of the ALT and AST surge, decreased hepatic sinusoidal vasodilation, and decreased leukocyte infiltration in post-sinusoidal venules (P < 0.05). • H(2) S supplementation directly inhibited interleukin 8-induced neutrophil chemotaxis in vitro (P < 0.05).
  
  
  CONCLUSIONS
  • These findings are the first to show the real-time protective role of supplemental H(2) S in prolonged periods of warm renal IRI, perhaps acting by decreasing leukocyte migration and limiting inflammatory responses. • The protective effects of H(2) S suggest potential clinical applications in both donors after cardiac death models of renal transplantation and oncological practices requiring vascular clamping.
authors.affiliation: Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Justin X G
authors.initials: JX
authors.lastname: Zhu
authors.forename: Melanie
authors.initials: M
authors.lastname: Kalbfleisch
authors.forename: Yi Xin
authors.initials: YX
authors.lastname: Yang
authors.forename: Relka
authors.initials: R
authors.lastname: Bihari
authors.forename: Ian
authors.initials: I
authors.lastname: Lobb
authors.forename: Michael
authors.initials: M
authors.lastname: Davison
authors.forename: Amy
authors.initials: A
authors.lastname: Mok
authors.forename: Gedaminas
authors.initials: G
authors.lastname: Cepinskas
authors.forename: Abdel-Rahman
authors.initials: AR
authors.lastname: Lawendy
authors.forename: Alp
authors.initials: A
authors.lastname: Sener
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D019587:Dietary Supplements; D004195:Disease Models, Animal; D006862:Hydrogen Sulfide / Q000008:administration & dosage / Q000627:therapeutic use*; D007668:Kidney / Q000098:blood supply* / Q000187:drug effects / Q000378:metabolism; D016030:Kidney Transplantation; D008297:Male; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D011917:Rats, Inbred Lew; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015427:Reperfusion Injury / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control*; D052096:Warm Ischemia / Q000379:methods*
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger; D006862:Hydrogen Sulfide