Han Ro

IL-2/Anti-IL-2 Complex Attenuates Renal Ischemia-Reperfusion Injury through Expansion of Regulatory T Cells

Regulatory T cells (Tregs) can suppress immunologic damage in renal ischemia-reperfusion injury (IRI), but the isolation and ex vivo expansion of these cells for clinical application remains challenging. Here, we investigated whether the IL-2/anti-IL-2 complex (IL-2C), a mediator of Treg expansion, can attenuate renal IRI in mice. IL-2C administered before bilateral renal IRI induced Treg expansion in both spleen and kidney, improved renal function, and attenuated histologic renal injury and apoptosis after IRI. Furthermore, IL-2C administration reduced the expression of inflammatory cytokines and attenuated the infiltration of neutrophils and macrophages in renal tissue. Depletion of Tregs with anti-CD25 antibodies abrogated the beneficial effects of IL-2C. However, IL-2C-mediated renal protection was not dependent on either IL-10 or TGF-β. Notably, IL-2C administered after IRI also enhanced Treg expansion in spleen and kidney, increased tubular cell proliferation, improved renal function, and reduced renal fibrosis. In conclusion, these results indicate that IL-2C-induced Treg expansion attenuates acute renal damage and improves renal recovery in vivo, suggesting that IL-2C may be a therapeutic strategy for renal IRI.

Intra-peritoneal interleukin-6 system is a potent determinant of the baseline peritoneal solute transport in incident peritoneal dialysis patients

BACKGROUND Interleukin-6 (IL-6) is a key player in modulating inflammation. IL-6 and soluble IL-6 receptor (sIL-6R) complex induces the synthesis and secretion of various chemokines, adhesion molecules and angiogenic molecules. We hypothesized that the baseline peritoneal solute transport rate (PSTR) early after commencing peritoneal dialysis (PD) may depend largely on the IL-6/sIL-6R system. We also hypothesized that the dialysate concentrations of IL-6/sIL-6R could be closely related to local inflammation or angiogenesis in the peritoneal cavity. METHODS Fifty incident patients with a modified peritoneal equilibration test result within 3 months after commencing PD and without a previous history of peritonitis were enrolled. Clinical parameters such as age, sex, comorbid disease, body mass index, residual renal function and C-reactive protein were assessed. Serum and dialysate markers including CA125, IL-6, sIL-6R, monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) were measured and correlated with PSTR. RESULTS Dialysate concentrations of IL-6 (r = 0.576, P < 0.001), MCP-1 (r = 0.408, P = 0.003) and Ang-2 (r = 0.408, P = 0.003) correlated with mass transfer area coefficient for creatinine (MTAC(cr)), respectively. Dialysate appearance rate (AR) of albumin correlated with dialysate concentrations of CA125 (r = 0.751, P < 0.001), IL-6 (r = 0.303, P = 0.039), sIL-6R (r = 0.497, P < 0.001), MCP-1 (r = 0.488, P < 0.001), VEGF (r = 0.443, P = 0.004) and Ang-2 (r = 0.488, P < 0.001). Neither MTAC(cr) nor AR of albumin was associated with systemic markers. Multivariate analysis showed that MTAC(cr) is independently associated with dialysate IL-6 and serum albumin. It also showed that AR of albumin is independently predicted by dialysate sIL-6R. Dialysate IL-6 correlated with dialysate concentrations of CA125 MCP-1, VEGF and Ang-2. CONCLUSION Our study from incident PD patients suggested that (i) dialysate the IL-6 system is a potent determinant of baseline PSTR and (ii) elevation of IL-6 in the dialysate is associated with up-regulation of intra-peritoneal inflammatory and angiogenic molecules.

Generation and Characterization of Human Heme Oxygenase-1 Transgenic Pigs

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

The Effect of Renin-Angiotensin-Aldosterone System Blockade on Contrast-Induced Acute Kidney Injury: A Propensity-Matched Study

BACKGROUND The role of the angiotensin-converting enzyme (ACE) inhibitor and angiotensin receptor blocker (ARB) in the pathophysiology of contrast-induced acute kidney injury (AKI) is controversial, and the available literature is contradictory. STUDY DESIGN A retrospective propensity score-matched study to analyze the effect of ACE-inhibitor/ARB therapy on the development of contrast-induced AKI. SETTING & PARTICIPANTS Using propensity score matching, 1,322 ACE-inhibitor/ARB recipients and nonrecipients were paired for analysis from 5,299 patients and fulfilled the inclusion criteria among 11,447 patients receiving coronary angiography (CAG) or percutaneous coronary intervention. PREDICTORS ACE-inhibitor/ARB use based on prescription and risk factors for contrast-induced AKI. OUTCOMES The incidence of contrast-induced AKI defined by AKI Network (AKIN) criteria: an absolute increase in serum creatinine levels ≥0.3 mg/dL or a relative increase ≥50% from baseline values within 48 hours after exposure to the contrast medium. MEASUREMENTS Baseline serum creatinine, hemoglobin, and albumin levels; volume of contrast agents; preprocedural medication; and post-CAG serum creatinine levels. RESULTS An ACE inhibitor/ARB was prescribed for 64.0% of patients receiving CAG. ACE-inhibitor/ARB users showed an increased incidence of contrast-induced AKI after propensity score matching (11.4% vs 6.3%; P < 0.001). In multivariable analysis, use of ACE inhibitors/ARBs remained an independent and significant predictor of contrast-induced AKI in an unmatched cohort (OR, 1.39; 95% CI, 1.10-1.76; P = 0.06). In the matched cohort, use of ACE inhibitors/ARBs also was associated with a higher adjusted OR of contrast-induced AKI (OR, 1.43; 95% CI, 1.06-1.94; P = 0.02). LIMITATIONS A retrospective study at a single center. CONCLUSIONS Use of ACE inhibitors/ARBs during CAG has a possible influence to increase the incidence of contrast-induced AKI. Further randomized clinical trials are warranted to confirm the effect of ACE-inhibitor/ARB therapy on the development of contrast-induced AKI.

Immunosuppressive mechanisms of embryonic stem cells and mesenchymal stem cells in alloimmune response.

Although both embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs) are known to have immunosuppressive effects, the mechanisms of immunosuppression are still controversial. Both types of stem cells suppressed not only the proliferation but also survival of CD4(+) T cells in vitro. They suppressed secretion of various cytokines (IL-2, IL-12, IFN-γ, TNF-α, IL-4, IL-5, IL-1β, and IL-10), whereas there was no change in the levels of TGF-β or IDO. Classic and modified transwell experiments demonstrated that immunosuppressive activities were mainly mediated by cell-to-cell contact. Granzyme B in the ESCs played a significant role in their immunosuppression, whereas PDL-1, Fas ligand, CD30 or perforin was not involved in the contact-dependent immunosuppression. However, none of the above molecules played a significant role in the immunosuppression by the MSCs. Interestingly, both stem cells increased the proportion of Foxp3(+) regulatory T cells. Our results showed that both ESCs and MSCs suppressed the survival as well as the proliferation of T cells by mainly contact-dependent mechanisms and increased the proportion of regulatory T cells. Granzyme B was involved in immunosuppression by the ESCs in a perforin-independent manner.

Impact of polymorphisms of TLR4/CD14 and TLR3 on acute rejection in kidney transplantation

Background. Organ transplantation itself inevitably activates the innate immune system by toll-like receptors (TLRs), potentially leading to allograft rejection and graft failure. We evaluated the possible association between the TLR4/CD14 and TLR3 polymorphisms of donor-recipient pairs, and acute rejection after living donor kidney transplantation. Methods. TLR4 −1607T/C (rs10759932), −2026A/G (rs1927914); CD14 −159C/T (rs2569190); and TLR3 rs3775290, rs3775291, and rs3775296 were genotyped using DNA samples from 216 donor-recipient pairs of adult living donor kidney transplantation between January 1996 and July 2006. Dual luciferase reporter assay was performed to determine the functional significance of promoter single-nucleotide polymorphisms (SNPs) of TLR4. Results. Acute rejection occurred in 42 recipients (19.4%) of 216 adult transplant patients within 1 year. The genotype distributions of both recipient and donor TLR4 rs10759932 differed significantly between the control (no rejection) and acute rejection groups. For recipient rs10759932, the adjusted odds ratio for the TC+CC over TT genotype was 0.25 (95% confidence interval, 0.11–0.57; P =0.001). When the rs10759932 CC genotype was present in the recipient or donor, no episode of acute rejection occurred (Fisher’s exact test, P =0.023). The presence of the rs10759932 C allele was associated with higher rejection-free survival rates (log-rank test, P =0.0053). However, there was no difference in transcriptional activity between wild-type and variant promoters of TLR4. In contrast to TLR4, SNPs of TLR3 or CD14 had no influence on acute rejection. Conclusion. These findings suggest the importance of TLR4 in the pathogenesis of acute rejection in kidney transplantation.

Polymorphisms of the MDR1 and MIF genes in children with nephrotic syndrome

Oral steroid treatment is the first line of therapy for childhood nephrotic syndrome (NS). Nonetheless, some patients are resistant to this treatment. Many efforts have been made to explain the differences in the response to steroid treatment in patients with NS based on the genetic background. We have investigated single nucleotide polymorphisms of the MDR1 [C1236T (rs1128503), G2677T/A (rs2032582), and C3435T (rs1045642)] and MIF (G-173C, rs755622) genes in 170 children with NS. Of these children, 69 (40.6%) were initial steroid non-responders, and 23 (13.5% of total) developed chronic kidney disease. Renal biopsy findings, which were available for 101 patients, showed that 35 patients had minimal change lesion and 66 had focal segmental glomerulosclerosis. The frequencies of the MDR1 1236 CC (18.8 vs 7.2%) or TC (53.5 vs 43.5%) genotype and C allele (45.5 vs 29.0%) were significantly higher in the initial steroid responders than in the non-responders. Analysis of MDR1 three-marker haplotypes revealed that the frequency of the TGC haplotype was significantly lower in the initial steroid responders than in the non-responders (15.8 vs 29.0%). There was no association between the MIF G-173C polymorphism and clinical parameters, renal histological findings, and steroid responsiveness. These data suggest that the initial steroid response in children with NS may be influenced by genetic variations in the MDR1 gene.

Impact of tacrolimus intraindividual variability and CYP3A5 genetic polymorphism on acute rejection in kidney transplantation.

Background: Wide variation in tacrolimus concentrations and low tacrolimus exposure have been reported to be associated with poor renal graft outcomes in non-Asians. The CYP3A5 polymorphism is a representative genetic factor that might affect this association, together with environmental factors. We investigated whether tacrolimus variability or the mean tacrolimus trough concentration can influence kidney allograft outcomes in Asians and whether the CYP3A5 polymorphism (rs776746) can affect this relationship. Methods: Data from renal transplant patients between 2000 and 2010 were analyzed retrospectively. The tacrolimus intraindividual variability (IIV) and the mean tacrolimus trough concentration were calculated from the tacrolimus concentrations between 6 and 12 months after transplantation. Results: A total of 249 renal transplant patients were enrolled. The patients with higher tacrolimus IIV had shorter rejection-free survival (P = 0.002). However, there was no difference in rejection-free survival between CYP3A5 expressers and nonexpressers. The tacrolimus IIV was not associated with the CYP3A5 polymorphism. High IIV of tacrolimus was an independent risk factor of biopsy-proven acute rejection after adjusting for mean tacrolimus concentration, HLA mismatch, induction therapy, donor type, and CYP3A5 polymorphism (hazard ratio 2.655, 95% confidence interval 1.394–5.056). Interestingly, the impact of tacrolimus IIV on acute rejection was significant in CYP3A5 expressers, whereas it was not in CYP3A5 nonexpressers. Conclusions: The IIV of tacrolimus trough concentrations had a significant impact on rejection-free survival. The effect was influenced by CYP3A5 polymorphism, although the tacrolimus variability itself was not determined by the CYP3A5 polymorphism.

Low-Dose Aspirin for Prevention of Cardiovascular Disease in Patients with Chronic Kidney Disease

Background Chronic kidney disease (CKD) is a major risk factor for the development of cardiovascular disease (CVD). Previous trials have investigated the effects of low-dose aspirin on CVD prevention in patients with diabetes; however, patients with CKD were not examined. The role of aspirin in diabetics is controversial, and the available literature is contradictory. Therefore, we studied whether low-dose aspirin would be beneficial for patients with CKD, a group that is at high risk for CVD. Method From a total of 25340 patients with CKD, 1884 recipients of low-dose aspirin (100 mg/day) were paired 1∶1 with non-recipients for analysis using propensity score matching. The primary endpoint was the development of atherosclerotic CVD, including coronary arterial disease, stroke, and peripheral arterial disease. Secondary endpoints included death from any cause, bleeding events, doubling of serum creatinine, and renal death. Results The incidence of a primary endpoint of any atherosclerotic CVD was significantly higher in the aspirin users than in the non-users (P<0.001). Secondary endpoints, including all-cause mortality and composite bleeding events, were not significantly different between the aspirin users and the non-users. However, the doubling of serum creatinine levels (P = 0.001) and renal death (P = 0.042) were significantly associated with the use of aspirin. Conclusion These results suggest that the use of low-dose aspirin in patients with CKD may have harmful consequences related to the development of CVD and renal progression.

Generation of soluble human tumor necrosis factor-α receptor 1-Fc transgenic pig.

Background. Acute humoral xenograft rejection (AHXR) is an important barrier to xenograft survival. Human tumor necrosis factor-&agr; (hTNF-&agr;) is one of the essential mediators of AHXR and induces activation of porcine endothelial cells (PECs), resulting in upregulation of major histocompatibility complex molecules, adhesion molecules, and proinflammatory chemokines. We investigated whether introduction of a soluble human tumor necrosis factor receptor I-Fc (shTNFRI-Fc) fusion gene can suppress activation of PECs and, more importantly, produced shTNFRI-Fc transgenic pigs. Methods. The shTNFRI-Fc gene expression vector was constructed and inserted into PECs. The inhibitory effects of shTNFRI-Fc were tested by luciferase assay, reverse-transcriptase polymerase chain reaction, and flow cytometry. A shTNFRI-Fc transgenic pig was generated by somatic cell nuclear transfer. The expression of shTNFRI-Fc in the transgenic pig was evaluated by PCR, western blot, enzyme-linked immunosorbent assay, and immunohistochemistry. The inhibitory effects of shTNFRI-Fc in the serum obtained from the transgenic pig were also tested. Results. In comparison with control green fluorescent protein, shTNFRI-Fc protein showed much stronger inhibitory effects on NF-&kgr;B activation in the HEK293-NF-&kgr;B-luciferase reporting cell line, expression of chemokines and adhesion molecules in PECs, and TNF-&agr;-mediated cytotoxicity. We successfully generated shTNFRI-Fc transgenic pig. Sera obtained from the transgenic pig inhibited induction of chemokines, and E-selectin in PECs stimulated with Human TNF-&agr;. Conclusions. We have generated transgenic pigs producing shTNFRI-Fc protein that can inhibit TNF-&agr;-mediated activation of PECs. Because TNF-&agr; is an important mediator of xenograft rejection, the use of xenografts that can produce shTNFRI-Fc proteins de novo could be an effective approach in overcoming a considerable component of the xenograft rejection process, especially AHXR.