Preconditioning with Physiological Levels of Ethanol Protect Kidney against Ischemia/Reperfusion Injury by Modulating Oxidative Stress

Oxidative stress due to excessive production of reactive oxygen species (ROS) and subsequent lipid peroxidation plays a critical role in renal ischemia/reperfusion (IR) injury. The purpose of current study is to demonstrate the effect of antecedent ethanol exposure on IR-induced renal injury by modulation of oxidative stress.Bilateral renal warm IR was induced in male C57BL/6 mice after ethanol or saline administration. Blood ethanol concentration, kidney function, histological damage, inflammatory infiltration, cytokine production, oxidative stress, antioxidant capacity and Aldehyde dehydrogenase (ALDH) enzymatic activity were assessed to evaluate the impact of antecedent ethanol exposure on IR-induced renal injury.After bilateral kidney ischemia, mice preconditioned with physiological levels of ethanol displayed significantly preserved renal function along with less histological tubular damage as manifested by the reduced inflammatory infiltration and cytokine production. Mechanistic studies revealed that precondition of mice with physiological levels of ethanol 3 h before IR induction enhanced antioxidant capacity characterized by significantly higher superoxidase dismutase (SOD) activities. Our studies further demonstrated that ethanol pretreatment specifically increased ALDH2 activity, which then suppressed lipid peroxidation by promoting the detoxification of Malondialdehyde (MDA) and 4-hydroxynonenal (HNE).Our results provide first line of evidence indicating that antecedent ethanol exposure can provide protection for kidneys against IR-induced injury by enhancing antioxidant capacity and preventing lipid peroxidation. Therefore, ethanol precondition and ectopic ALDH2 activation could be potential therapeutic approaches to prevent renal IR injury relevant to various clinical conditions

CD4(+)CD25(-)Nrp1(+) T Cells Synergize with Rapamycin to Prevent Murine Cardiac Allorejection in Immunocompetent Recipients

Besides CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), other immunosuppressive T cells also participated in the regulation of immune tolerance. Reportedly, neuropilin-1 (Nrp1) might be one of the molecules by which regulatory cells exert their suppressive effects. Indeed, CD4(+)CD25(-)Nrp1(+) T cells exhibit potent suppressive function in autoimmune inflammatory responses. Here we investigated the specific role of CD4(+)CD25(-)Nrp1(+) T cells in the setting of the transplant immune response. Through MLR assays, we found that CD4(+)CD25(+)Nrp1(+) T cells suppressed the proliferation of naive CD4(+)CD25(-) T cells activated by allogeneic antigen-stimulation. Adoptive transfer of CD4(+)CD25(-)Nrp1(+) T cells synergized with rapamycin to induce long-term graft survival in fully MHC-mismatched murine heart transplantation, which was associated with decreased IFN-gamma, IL-17 and increased IL-10, TGF-beta, Foxp3 and Nrp1 expression in the grafts. Importantly, our data indicated that CD4(+)CD25(-)Nrp1(+) T cell transfer augments the accumulation of Tregs in the recipient, and creates conditions that favored induction of hyporesponsiveness of the T effector cells. In conclusion, this translational study indicates the possible therapeutic potential of CD4(+)CD25(-)Nrp1(+) T cells in preventing allorejection. CD4(+)Nrp1(+) T cells might therefore be used in bulk as a population of immunosuppressive cells with more beneficial properties concerning ex vivo isolation as compared to Foxp3(+) Treg

CD4⁺CD25⁻Nrp1⁺ T cells induce hyporesponsiveness of the T effector cells.

<p>CD4⁺CD25⁻ T cells were isolated from spleens of recipient mice treated by Rapamycin combined with CD4⁺CD25⁻Nrp1⁺ T cells and from syngeneic transplant recipients on day 70 after transplantation, then primed with irradiated BALB/c (donor) splenocytes in the presence or absence of exogenous IL-2 (100 U/ml). (<b>A</b>) Cell proliferation was determined by ³H thymidine incorporation. (<b>B</b>) Cytokine content of the mixed lymphocyte reaction supernatants with no exogenous IL-2 treatment was evaluated by enzyme-linked immunosorbent assay. Results are presented as mean ± SD values of triplicate wells, and are representative of 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001. SC  =  syngeneic controls, ³H-TdR  =  metabolic incorporation of tritiated thymidine, cpm  =  cells per million, Nrp-1  =  neuropilin-1, rapa  =  Rapamycin, CTR  =  control, NS  =  not significant.</p

CD4⁺CD25⁻Nrp1⁺ T cells augment CD4⁺Foxp3⁺ Treg accumulation in transplant recipients.

<p>(<b>A</b>) Anti-CD4 and anti-Foxp3 intracellular staining were performed on spleen cells harvested from untreated mice on 7d or from Rapamycin and/or CD4⁺CD25⁻Nrp1⁺ T cells on 21d, 42d and 70d. (B) The percentages of CD4⁺Foxp3⁺ T cells were pooled from 4–6 mice from each group. (C) CD4⁺CD25⁺ T cells were purified from each group and used for suppression assays. 2×10⁴ CD4⁺CD25⁻ T cells (C57BL/6) were stimulated by the same amount of irradiated BALB/c splenocytes together with various doses of CD4⁺CD25⁺T cells purified from the indicated group. Cell proliferation was determined by ³H thymidine incorporation. Results are presented as mean ± SD values of triplicate wells, and are representative of 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001. Rapa  =  Rapamycin, Nrp1 = neuropilin-1, ³H-TdR = metabolic incorporation of tritiated thymidine, cpm = cells per million, Treg = T regulatory cells</p

CD4⁺CD25⁻Nrp1⁺ T cells possess potent suppressive function <i>in vitro.</i>

<p>(<b>A</b>) Freshly isolated CD4⁺CD25⁻Nrp1⁺ T cells (10⁵, C57BL/6) were co-cultured with syngeneic responder CD4⁺CD25⁻ T cells (C57BL/6) in different ratios (0, 1∶8, 1∶4, 1∶2, 1∶1) in order to address stimulation induced by irradiated BALB/c (donor) splenocytes (10⁵). Cell proliferation was determined by ³H thymidine incorporation. (<b>B</b>) Cytokine content of the MLRsup where the suppressor cell versus CD4⁺CD25⁻ T cell was 0 (CTR) and 1∶1 (Nrp1) was evaluated by ELISA. Results are presented as mean ± SD values of triplicate wells, and are representative of 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001. Nrp1 = neuropilin-1, MLRsup = mixed-lymphocyte reaction supernatants, CTR  =  control group, ³H-TdR  =  metabolic incorporation of tritiated thymidine, cpm  =  cells per million, NS  =  not significant.</p

Adoptive transfer of CD4⁺CD25⁻Nrp1⁺ T cells changes the expression of inflammatory cytokines and immunomolecules.Both the cardiac allografts and blood samples were harvested 7 days after transplantation.

<p>(<b>A–F</b>) The intragraft gene expression of IFN-γ, IL-17, IL-10, TGF-β, Foxp3 and Nrp1 was analyzed by quantitative real-time polymerase chain reaction. (<b>G–J</b>) Serum levels of IFN-γ, IL-17, IL-10, TGF-β were determined by enzyme-linked immunosorbent assay. Results are presented as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001. SC = syngeneic control, Nrp1 = neuropilin-1, rapa = Rapamycin.</p

Adoptive transfer of CD4⁺CD25⁻Nrp1⁺ T cells synergize with Rapamycin to prevent allograft rejection.

<p>Heterotopic heart grafts were transplanted from BALB/c mice into C57BL/6 recipients. The recipients received a sub-therapeutic regimen of 1 mg/kg/day i.p. Rapamycin for 10 consecutive days (days 0-9), and/or two dose of freshly isolated Nrp1⁺ T cell on day 0 and day 7 (2×10⁶). Rejection was defined as cessation of a palpable impulse. (<b>A</b>) Survival rates were compared using log-rank test. (<b>B</b>) Hematoxylin and eosin staining of representative heart allografts harvested at 7d post transplantation. (<b>C</b>) Quantitative histological evaluation of allografts harvested on 7d post transplantation. SC, syngeneic control, Nrp1⁺ T = neuropilin-1-positive T cells, HPF = high power field, rapa = Rapamycin, NS = not significant. Results are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001.</p

Ethanol pretreatment increased ALDH2 enzymatic activity after renal IR.

<p><b>A.</b> Mice were pretreated with 1 g/kg ethanol and kidney samples were collected for ALDH enzymatic activity assay at different time after ethanol exposure. The CTR group did not receive any ethanol. <b>B.</b> Mice were pretreated with saline or 1 g/kg ethanol, and were exposed to a 30-min bilateral renal IR 3 h later. Kidney samples were collected at different time after reperfusion. <b>C.</b> Immunoprecipitation was performed to separate the total protein from kidney tissues to the ALDH2-enriched part (ALDH2-IP), and the ALDH2-depleted part (ALDH2-DE). <b>D.</b> ALDH enzymatic assay was performed in the ALDH2 enriched or depleted proteins. Results are mean values ± SEM. (<i>n</i> = 4–5). (* <i>p</i><0.05, ** <i>p</i><0.01).</p

Ethanol pretreatment attenuated inflammation after renal IR.

<p>Mice were pretreated with saline or 1 g/kg ethanol, and were exposed to a 30-min bilateral renal IR 3 h later. Kidney samples were collected at 24 h post reperfusion and assessed for: <b>A.</b> CD11b, CD3 mRNA expression; <b>B.</b> MPO activity; and <b>C.</b> TNF-α, IL-6, IL-8 and IL-10 mRNA expression. Results are mean values ± SEM. (<i>n</i> = 4–6). (* <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001).</p