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 (H2S) 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 H2S 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 H2S 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 H2S 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 H2S supplementation. The present study adds to the existing literature by evaluating the effects of H2S 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 H2S 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 (H2S) in prolonged warm renal ischaemia‐reperfusion injury (IRI) using real‐time intravital microscopy (IVM). MATERIALS AND 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+H2S, 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 H2S to 62.8 (0.9) µmol/L (P < 0.05). H2S supplementation during IRI increased renal capillary perfusion on IVM, and improved acute tubular necrosis and apoptotic scores on histology (P < 0.05). Supplemental H2S 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 H2S supplementation: blunting of the ALT and AST surge, decreased hepatic sinusoidal vasodilation, and decreased leukocyte infiltration in post‐sinusoidal venules (P < 0.05). H2S 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 H2S in prolonged periods of warm renal IRI, perhaps acting by decreasing leukocyte migration and limiting inflammatory responses. The protective effects of H2S suggest potential clinical applications in both donors after cardiac death models of renal transplantation and oncological practices requiring vascular clamping.

Hydrogen Sulfide Treatment Mitigates Renal Allograft Ischemia-Reperfusion Injury during Cold Storage and Improves Early Transplant Kidney Function and Survival Following Allogeneic Renal Transplantation

“…Urinary parameters of allograft function were the same regardless of treatment, however H 2 S treatment prolonged the life of the allograft such that 100% of H 2 S treated animals survived for urine to be collected on POD 6, compared to only 40% of UW treated animals. H 2 S treatment significantly reduced early allograft ATN and apoptosis compared to UW, as was shown in previous studies 13,14 . We also noted that UW treated allografts exhibited increased expression of Olr1 and Timp1 mRNA, two stress response genes involved in apoptotic pathway regulation, compared to H 2 S at POD 1-2.…”

Hydrogen sulfide treatment improves long-term renal dysfunction resulting from prolonged warm renal ischemia-reperfusion injury

“…24,25 However, we report that tissue necrosis and apoptosis scores, while still increased compared to Sham, are nearly equal between treatment groups by day 7 post-IRI. This indicates that while H 2 S treatment appears to protect against necrosis and apoptosis associated with warm IRI in the short-term, kidney injury seems to have been resolved to a somewhat similar degree by day 7 regardless of treatment.…”

“…Multiple studies have demonstrated that H 2 S is capable of limiting leukocyte migration, and cytokine expression and release. [22][23][24][30][31][32] We found that H 2 S treatment during warm IRI initially decreased the expression of pro-inflammatory markers, increased the expression of pro-survival marker Bcl-2 and decreased the expression of pro-apoptotic marker BID. While the expression of these markers decreased toward Hydrogen sulfide treatment and long-term renal dysfunction baseline by day 7, H 2 S treatment also resulted in decreased long-term renal inflammation as evidenced by the diminished numbers of infiltrating macrophages in H 2 S treated kidneys at day 7.…”

“…25,26 Previously, we have demonstrated that H 2 S protects against periods of prolonged cold IRI, as well as from shorter periods of warm IRI. 24,27 Recently, H 2 S has also been shown to be reno-protective against warm IRI in large-animals. 28 However, long-term studies on the effects of H 2 S have been limited.…”

“…[19][20][21][22][23] Using a rodent model of warm renal IRI involving uninephric renal clamping with clinically relevant, prolonged warm ischemic times, we have found that exogenous H 2 S treatment during warm renal IRI improves renal function and reduces IRIinduced inflammation in the acute recovery period. 24 To improve clinical outcomes following PN, H 2 S treatment must be shown to provide long-term improvement of renal function and resolution of inflammation. The current study examines the effects of exogenous H 2 S treatment during warm IRI on renal function, injury and inflammation at extended postoperative time points.…”