Sotrastaurin, a protein kinase C inhibitor, ameliorates ischemia and reperfusion injury in rat orthotopic liver transplantation

Abstract

Sotraustaurin (STN), a small molecule, targeted protein kinase C (PKC) inhibitor that prevents T-lymphocyte activation via a calcineurin-independent pathway, is currently being tested in Phase II renal and liver transplantation clinical trials. We have documented the key role of activated T cells in the inflammation cascade leading to liver ischemia/reperfusion injury (IRI). This study explores putative cytoprotective functions of STN in a clinically relevant rat model of hepatic cold ischemia followed by orthotopic liver transplantation (OLT). Livers from Sprague-Dawley rats were stored for 30 h at 4°C in UW solution, and then transplanted to syngeneic recipients. STN treatment of liver donors/recipients or recipients only prolonged OLT survival to >90% (vs. 40% in controls), decreased hepatocellular damage and improved histological features of IRI. STN treatment decreased activation of T cells, and diminished macrophage/neutrophil accumulation in OLTs. These beneficial effects were accompanied by diminished apoptosis, NF-κB/ERK signaling, depressed proapoptotic cleaved caspase-3, yet upregulated antiapoptotic Bcl-2/Bcl-xl and hepatic cell proliferation. In vitro, STN decreased PKCθ/IκBα activation and IL-2/IFN-γ production in ConA-stimulated spleen T cells, and diminished TNF-α/IL-1β in macrophage-T cell cocultures. This study documents positive effects of STN on liver IRI in OLT rat model that may translate as an additional benefit of STN in clinical liver transplantation.

Figure 1. STN prolongs rat OLT survival, improves hepatocellular function, and ameliorates liver IRI

(A) OLT survival. Donor SD rat livers were stored at 4 °C in UW solution for 30h prior to transplantation to syngeneic rats. Treatment with STN of liver donors (day -1) plus OLT recipients (day 0–3); or OLT recipients only (day 0–3), prolonged animal survival to 90% (9/10) and 100% (6/6), respectively. Forty percent of PBS-treated controls (4/10) remained alive at day 14 post-OLT (p<0.01). (B) Hepatocellular function in OLT recipients. STN treatment decreased sALT levels, compared with controls. *p<0.0005, **p<0.005; n=3–4/group. Mean±SD are shown. (C) Representative liver histology at 6h (a, b) and 24h (c, d) post-OLT. (a & c) control OLTs with severe liver damage and hepatocellular necrosis (Suzuki’s score: 6h = 3.5±0.55; 24h = 3.17±0.75). (b & d) – STN-treated OLTs with well-preserved tissue architecture (Suzuki’s score: 6h = 1.17±0.41; 24h = 0.83±0.41). H&E staining; magnification ×100, n=3–4/group. (D) Neutrophil activity in OLTs. Suppressed MPO activity in STN group, compared with controls. *p<0.01, **p<0.05. n=3–4/group. Mean±SD are shown.

Figure 2. Immunohistochemical staining for CD3 and CD68 in rat OLTs

Left panels: Representative OLTs stained for CD3 (A) and CD68 (B) expression (magnification ×400). Right panels: Cell quantification/HPF. Decreased T cell and macrophage sequestration in OLTs after STN, compared with controls. *p<0.0001, n=3–4/group.

Figure 3. Quantitative RT-PCR-assisted detection of CD25; cytokines (IFN-γ, IL-2, TNF-α, IL-1β); and chemokines (CXCL-10, MCP-1) in OLTs. Data were normalized to β-actin gene expression. *p<0.05, **p<0.005, n=3–4/group. Mean±SD are shown

Figure 4. STN ameliorates apoptosis, depresses NF-κB/ERK signaling and promotes anti-apoptotic function

(A) TUNEL-assisted detection of apoptosis in OLTs. Left panel: Representative staining of apoptotic positive cells (magnification ×400). Right panel: Quantification of apoptotic cells /HPF in STN-treated vs. control OLTs. *p<0.0001, n=3–4/group. (B) Immunohistochemical staining of liver cell proliferation using the BrdU method. Brown-stained nuclei represent actively proliferating hepatocytes. Representative of n=2/group. (C) Western blot-assisted analysis of phospho-Erk, NF-κB, cleaved caspase-3, Bcl-2, and Bcl-xl in OLTs. β-actin was used as an internal control. Representative of n=3–4/group.

Figure 5. STN depresses T cell activation in rat in vitro T cell cultures

(A) Western blot-assisted phospho-PKCθ and phospho-IκBα expression in ConA-stimulated T cells. Representative of n=3. (B) Quantitative RT-PCR-assisted detection of IL-2 in Con A-stimulated T cells. *p<0.005, **p<0.001, n=3/group. (C) IFN-γ production in Con A-stimulated T cells. *p<0.005, n=3/group. (D–E) TNF-α and IL-1β production in Con A-stimulated T cell - macrophages co-cultures. STN suppressed TNF-α/IL-1β levels. *p<0.05, **p<0.005, n=3/group. No effect of STN on TNF-α/IL-1β in macrophage cultures devoid of T cells.