Nakul Datta

Macrophage heme oxygenase-1-SIRT1-p53 axis regulates sterile inflammation in liver ischemia-reperfusion injury

BACKGROUND & AIMS Hepatic ischemia-reperfusion injury (IRI), characterized by exogenous antigen-independent local inflammation and hepatocellular death, represents a risk factor for acute and chronic rejection in liver transplantation. We aimed to investigate the molecular communication involved in the mechanism of liver IRI. METHODS We analyzed human liver transplants, primary murine macrophage cell cultures and IR-stressed livers in myeloid-specific heme oxygenase-1 (HO-1) gene mutant mice, for anti-inflammatory and cytoprotective functions of macrophage-specific HO-1/SIRT1 (sirtuin 1)/p53 (tumor suppressor protein) signaling. RESULTS Decreased HO-1 expression in human post-reperfusion liver transplant biopsies correlated with a deterioration in hepatocellular function (serum ALT; p<0.05) and inferior patient survival (p<0.05). In the low HO-1 liver transplant biopsy group, SIRT1/Arf (alternative reading frame)/p53/MDM2 (murine double minute 2) expression levels decreased (p<0.05) while cleaved caspase 3 and frequency of TUNEL+cells simultaneously increased (p<0.05). Immunofluorescence showed macrophages were the principal source of HO-1 in human and mouse IR-stressed livers. In vitro macrophage cultures revealed that HO-1 induction positively regulated SIRT1 signaling, whereas SIRT1-induced Arf inhibited ubiquitinating activity of MDM2 against p53, which in turn attenuated macrophage activation. In a murine model of hepatic warm IRI, myeloid-specific HO-1 deletion lacked SIRT1/p53, exacerbated liver inflammation and IR-hepatocellular death, whereas adjunctive SIRT1 activation restored p53 signaling and rescued livers from IR-damage. CONCLUSION This bench-to-bedside study identifies a new class of macrophages activated via the HO-1-SIRT1-p53 signaling axis in the mechanism of hepatic sterile inflammation. This mechanism could be a target for novel therapeutic strategies in liver transplant recipients. LAY SUMMARY Post-transplant low macrophage HO-1 expression in human liver transplants correlates with reduced hepatocellular function and survival. HO-1 regulates macrophage activation via the SIRT1-p53 signaling network and regulates hepatocellular death in liver ischemia-reperfusion injury. Thus targeting this pathway in liver transplant recipients could be of therapeutic benefit.

Early cytokine signatures of ischemia/reperfusion injury in human orthotopic liver transplantation

BACKGROUND. Orthotopic liver transplant (OLT) is the primary therapy for end-stage liver disease and acute liver failure. However, ischemia/reperfusion injury (IRI) can severely compromise allograft survival. To understand the evolution of immune responses underlying OLT-IRI, we evaluated longitudinal cytokine expression profiles from adult OLT recipients before transplant through 1 month after transplant. METHODS. We measured the expression of 38 cytokines, chemokines, and growth factors in preoperative and postoperative recipient circulating systemic blood (before transplant and 1 day, 1 week, and 1 month after transplant) and intraoperative portal blood (before and after reperfusion) of 53 OLT patients and analyzed this expression in relation to biopsy-proven IRI (n = 26 IRI+; 27 IRI-), clinical liver function tests early (days 1-7) after transplant, and expression of genes encoding cytokine receptors in biopsies of donor allograft taken before and after reperfusion. RESULTS. Bilirubin and arginine transaminase levels early after transplant correlated with IRI. Fourteen cytokines were significantly increased in the systemic and/or portal blood of IRI+ recipients that shifted from innate to adaptive-immune responses over time. Additionally, expression of cognate receptors for 10 of these cytokines was detected in donor organ biopsies by RNAseq. CONCLUSION. These results provide a mechanistic roadmap of the early immunological events both before and after IRI and suggest several candidates for patient stratification, monitoring, and treatment. FUNDING. Ruth L. Kirschstein National Research Service Award T32CA009120, Keck Foundation award 986722, and a Quantitative & Computational Biosciences Collaboratory Postdoctoral Fellowship.

Heme oxygenase-1 regulates sirtuin-1–autophagy pathway in liver transplantation: From mouse to human

Liver ischemia–reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although graft autophagy is essential for resistance against hepatic IRI, its significance in clinical OLT remains unknown. Despite recent data identifying heme oxygenase‐1 (HO‐1) as a putative autophagy inducer, its role in OLT and interactions with sirtuin‐1 (SIRT1), a key autophagy regulator, have not been studied. We aimed to examine HO‐1–mediated autophagy induction in human OLT and in a murine OLT model with extended (20 hours) cold storage, as well as to analyze the requirement for SIRT1 in autophagy regulation by HO‐1. Fifty‐one hepatic biopsy specimens from OLT patients were collected under an institutional review board protocol 2 hours after portal reperfusion, followed by Western blot analyses. High HO‐1 levels correlated with well‐preserved hepatocellular function and enhanced SIRT1/LC3B expression. In mice, HO‐1 overexpression by genetically modified HO‐1 macrophage therapy was accompanied by decreased OLT damage and increased SIRT1/LC3B expression, whereas adjunctive inhibition of SIRT1 signaling diminished HO‐1–mediated hepatoprotection and autophagy induction. Our translational study confirms the clinical relevance of HO‐1 cytoprotection and identifies SIRT1‐mediated autophagy pathway as a new essential regulator of HO‐1 function in IR‐stressed OLT.

Sirtuin 1 Attenuates Inflammation and Hepatocellular Damage in Liver Transplant Ischemia-Reperfusion: From Mouse-to-Human

Hepatic ischemia/reperfusion injury (IRI), an inevitable antigen‐independent inflammation response in cadaveric liver transplantation, correlates with poor early graft function, rejection episodes, and contributes to donor organ shortage. Sirtuin 1 (SIRT1) is a histone deacetylase that may regulate inflammatory cell activity and manage liver function in IRI, though its functional role and clinical relevance remains to be elucidated. We investigated the efficacy of SIRT1 activation in a murine liver IRI model and verified the concept of putative SIRT1‐mediated hepatoprotection in clinical liver transplantation. In the experimental arm, mice were subjected to 90 minutes of liver partial warm ischemia followed by 6 hours of reperfusion with or without adjunctive SIRT1 activation in vivo (resveratrol [Res]). In parallel, bone marrow–derived macrophage (BMDM) or spleen lymphocyte cultures were treated with Res. In the clinical arm, liver biopsies from 21 adult primary liver transplant patients (2 hours after reperfusion) were divided into “low” (n = 11) versus “high” (n = 10) SIRT1 expression groups, assessed by Western blots. Treatment with Res attenuated murine liver IRI while up‐regulating SIRT1, suppressing leukocyte infiltration, and decreasing proinflammatory cytokine programs. SIRT1 silencing (small interfering RNA) in BMDM cultures enhanced inflammatory cytokine programs, whereas addition of Res decreased proinflammatory response in a SIRT1‐dependent manner. In addition, Res decreased interferon γ production in liver‐infiltrating and spleen lymphocyte cultures. Human liver transplants with high SIRT1 levels showed improved hepatocellular function and superior survival (P = 0.04), accompanied by lower proinflammatory cytokine profile. In conclusion, our translational study is the first to identify SIRT1 as a regulator of hepatocellular function in human liver transplant recipients under ischemia/reperfusion stress. By targeting innate and adaptive immune activation, manipulation of SIRT1 signaling should be considered as a novel means to combat inflammation in liver transplantation. Liver Transplantation 23 1282–1293 2017 AASLD.

Aminoacylase 3 Is a New Potential Marker and Therapeutic Target in Hepatocellular Carcinoma

Ras proteins (HRas, KRas and NRas) are common oncogenes that require membrane association for activation. Previous approaches to block/inhibit Ras membrane association were unsuccessful for cancer treatment in human clinical studies. In the present study we utilized a new approach to decrease Ras membrane association in hepatocellular carcinoma (HCC) cell lines via inhibition of an enzyme aminoacylase 3 (AA3; EC 3.5.1.114). AA3 expression was significantly elevated in the livers of HCC patients and HCC cell lines. Treatment of HepG2 cells with AA3 inhibitors, and HepG2 and HuH7 with AA3 siRNA significantly decreased Ras membrane association and was toxic to these HCC cell lines. AA3 inhibitors also increased the levels of N-acetylfarnesylcysteine (NAFC) and N-acetylgeranylgeranylcysteine (NAGGC) in HepG2 and Huh7 cell lines. We hypothesized that AA3 deacetylates NAFC and NAGGC, and generated farnesylcysteine (FC) and geranylgeranylcysteine (GGC) that are used in HCC cells for the regeneration of farnesylpyrophosphate and geranylgeranylpyrophosphate providing the prenyl (farnesyl or geranylgeranyl) group for Ras prenylation required for Ras membrane association. This was confirmed experimentally where purified human AA3 was capable of efficiently deacetylating NAFC and NAGGC. Our findings suggest that AA3 inhibition may be an effective approach in the therapy of HCC and that elevated AA3 expression in HCC is potentially an important diagnostic marker.

Recombinant relaxin protects liver transplants from ischemia damage by hepatocyte glucocorticoid receptor: From bench‐to‐bedside

Hepatic ischemia‐reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and acute/chronic rejection as well as a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although glucocorticoid receptor (GR) signaling may enhance cytoprotective programs, clinical use of glucocorticoid is limited because of adverse effects, whereas clinical relevance of GR‐facilitated cytoprotection in OLT remains unknown. We aimed to evaluate the significance of hepatic GR in clinical OLT and verify the impact of recombinant human relaxin (rhRLX), which may function as a GR agonist in a tissue/disease‐specific manner. Fifty‐one OLT patients were recruited under an institutional research board (IRB) protocol. Liver biopsies were collected after cold storage (presurgery) and 2 hours postreperfusion (before abdominal closure), followed by western blotting–assisted hepatic analyses. Forty‐three percent of OLTs failed to increase GR perioperatively under surgical stress. Post‐/pre‐GR ratios at postoperative day 1 correlated negatively with serum aspartate aminotransferase (AST)/cleaved caspase‐3 and positively with B‐cell lymphoma‐extra large (Bcl‐xL)/B‐cell lymphoma 2 (Bcl‐2) levels. In a murine OLT model with extended (18‐hour) cold storage, treatment with rhRLX ameliorated ischemia‐reperfusion (IR) damage and improved survival while up‐regulating hepatocyte GR and Bcl‐xL/Bcl‐2 expression in OLT. rhRLX‐induced GR suppressed hepatocyte high‐mobility group box 1 (HMGB1) translocation/release, accompanied by decreased Toll‐like receptor 4 (TLR4)/receptor for advanced glycation end products (RAGE), suppressed interleukin 1 beta (IL1β), chemokine (C‐C motif) ligand 2 (CCL2), C‐X‐C motif chemokine (CXCL)10, tumor necrosis factor alpha (TNFα), CXCL1, and CXCL2 levels, and attenuated neutrophil/macrophage accumulation in OLT. Inhibition of GR in hepatocyte culture and in OLT diminished rhRLX‐mediated cytoprotection. Conclusion: This translational study underscores the role of rhRLX‐GR signaling as a regulator of hepatocellular protection against IR stress in OLT. In the context of a recent phase III clinical trial demonstrating positive outcomes of rhRLX in patients with acute heart failure, studies on rhRLX for the management of IRI in OLT recipients are warranted. (Hepatology 2018;68:258‐273).

Changes in Vitamin D levels After Kidney Transplantation

Introduction Low levels of serum vitamin D are common and associated with progression of chronic kidney disease. However, the frequency of hypovitaminosis D following kidney transplantation (KT) is not well studied. We examined how vitamin D levels change post-transplant and compared the changes between patients with low-and normal-vitamin D pre-transplant. Materials and Methods We examined KT recipients from January 1, 2006, to May 31, 2016, who had 25-hydroxy vitamin D tests both before and within the first year of KT. If there were multiple vitamin D levels, we took the closest value to the transplant date and defined as pre-Tx and post-Tx vitamin D levels. Patients with vitamin D levels greater than 50 were excluded. Patients were divided into two groups according to vitamin D levels, 1) low (<30) and 2) normal (30-50). Mean vitamin D levels were compared between pre-and post-Tx using t-test analysis. Results A total of 398 patients were included in this study. Mean pre-and post-Tx vitamin D levels were 21.12 and 20.39 ng/mL, respectively. We found that 315 patients (79.15%) had low pre-Tx vitamin D levels and 83 patients (20.85%) had normal pre-Tx vitamin D levels. Post-Tx vitamin D levels were 23.13 and 19.67 ng/mL in normal and low pre-Tx vitamin D patients, respectively (p<0.01)(figure1). Patients with low pre-Tx vitamin D levels were more likely to have low post-Tx vitamin D levels (p=0.03)(table1). Furthermore, we found that 60.5% of patients with decreasing post-Tx vitamin D levels had low pre-Tx vitamin D levels while patients with normal pre-Tx vitamin D levels had a greater reduction in post-Tx vitamin D levels than patients with low pre-Tx vitamin D (figure2). Conclusions Hypovitaminosis D was very common in kidney transplant recipients. Preexisting hypovitaminosis D patients were more likely to have persistent low post-transplant vitamin D levels. Moreover, Post-transplant vitamin D levels were still lower than normal values regardless of pre-transplant vitamin D levels. Future studies should include vitamin D supplement and graft survival to support how vitamin D may impact on long-term outcome. Figure. No caption available. Figure. No caption available.

Influence of Hypomagnesemia on New-Onset Diabetes After Kidney Transplantation

Introduction Hypomagnesemia is commonly seen early after kidney transplantation (KT). Several studies have shown hypomagnesemia is an independent risk of new-onset diabetes after transplantation (NODAT) and may have the faster development of NODAT than normomagnesemia. We would examine hypomagnesemia as well as the reduction in serum magnesium (SMg) levels post-transplant on the risk of NODAT within the first year after KT. Materials and Methods We examined 3,014 kidney transplant recipients from January 1, 2006, to May 31, 2016. Patients having serum magnesium levels at baseline, defined as 1-day pre-transplant, and post-transplant period at 14 days, 1 month, and 3 months were included. Patients with a history of pre-transplant diabetes were excluded from this study. NODAT was identified if patients met ADA criteria within 1-year post-transplant. We examined the mean of SMg levels and compared between NODAT and non-NODAT patients by using Wilcoxon rank-sum (Mann-Whitney) test. Patients were also divided into low Mg (SMg <1.4 mEq/L) and normal Mg (SMg≥1.4 mEq/L) group. We also analyzed the risk of NODAT and time to develop NODAT between groups using cox-proportional model. Results 801 (26.58%) patients with a history of pre-transplant diabetes were excluded. A total of 1,487 patients had complete data, among these 236 (15.87%) of them developed NODAT within one-year post-transplant. SMg levels had decreased after KT and went to the lowest levels at 1 month post-transplant (Table 1, Figure 1). Mean SMg level at 14 days and 1 month post-transplant of NODAT patients were greater than non-NODAT patients (Table 1). We found that 1,258 patients (84.60%) had decreased in SMg levels at 1 month and the mean decreasing value was 0.49 mEq/L. Using 0.5 mEq/L as a cut-off decreasing value, patients with lesser reduction in SMg showed association with NODAT risk (p=0.01). As seen in Table 2, at 1 month post-transplant, normomagnesemic patients had greater risk to develop NODAT (30.71% vs. 14.33%; p<0.01) but had a similar time frame to develop NODAT (p=0.11)???. Table. No title available. Figure. No caption available. Table. No title available. Conclusions Majority of patients had decreased SMg levels after KT. However, we did not find independent associations between hypomagnesemia and risk of NODAT. A greater reduction in SMg levels did not show an additional risk of NODAT. On the other hand, post-transplant normomagnesemic patients were more likely to have NODAT. Future studies included various factors affecting NODAT should support evidence how magnesium may impact on NODAT.

The Effect of Diabetes on Serum Magnesium Levels After Kidney Transplantation

Introduction Hypomagnesemia is commonly seen early after kidney transplantation (KT). Pre-transplant diabetes is one of the associated factors that aggravate worsening hypomagnesemia after KT. However, the effect of diabetes on serum magnesium (SMg) levels after KT is not well studied. Therefore, we examined the change in SMg levels post-transplant of KT recipients regarding their diabetes status prior to KT. Materials and Methods We examined 614 kidney transplant recipients from January 1, 2006, to May 31, 2016. Patients having SMg levels at baseline and through 1 year post-transplant were included. We examined the SMg levels at 7 days, 1 month, 3 months, 6 months, 9 months, and 1 year post KT. We also compared the mean change of SMg levels at each time point between diabetes and non-diabetes patients by two-sample Wilcoxon rank-sum (Mann-Whitney) test. Results 141 (23%) patients had a history of pre-transplant diabetes. The mean SMg of diabetes patients were higher than non-diabetes patients (Table 1) in every single time point post KT (Table 1, Figure 1). The lowest SMg levels were at 1 month post KT and significantly dropped from baseline in both DM and non-DM patients. Furthermore, we found that there was a greater SMg levels in diabetes than non-diabetes patients (Table1, Figure 1). Conclusions Most of KT recipients had decreased in the SMg levels post-transplant, regardless of their diabetes status. Pre-transplant diabetes patients had significantly greater SMg levels at every single time point during the first year post KT. Future studies including new onset diabetes after transplantation should support the change in SMg level. Figure. No caption available. Table. No title available.

Screening Coccidioides serology in kidney transplant recipients: A 10-year cross-sectional analysis

Kidney transplant recipients (KTRs) are at risk for reactivation and complicated infection due to Coccidioides. Pre‐transplant serological screening should provide benefit for patients from endemic areas. We evaluated Coccidioides seroprevalence by area of residence in KTRs at a major transplant program in Los Angeles.