Yichen Jia

Helix B surface peptide administered after insult of ischemia reperfusion improved renal function, structure and apoptosis through beta common receptor/erythropoietin receptor and PI3K/Akt pathway in a murine model:

Erythropoietin (EPO) has been well recognized as a tissue protective agent by inhibiting apoptosis and inflammation. The tissue protective effect of EPO, however, only occurs at a high dosage, which may elicit severe side-effects at the meantime. Helix B surface peptide (HBSP), a novel peptide derived from the non-erythropoietic helix B of EPO, plays a specific role in tissue protection. We investigated effects of HBSP and the expression of its heterodimeric receptor, beta common receptor (βcR)/EPO receptor (), in a murine renal ischemia reperfusion (IR) injury model. HBSP significantly ameliorated renal dysfunction and tissue damage, decreased apoptotic cells in the kidney and reduced activation of caspase-9 and 3. The βcR/EPOR in the kidney was up-regulated by IR, but down-regulated by HBSP. Further investigation revealed that the expression and phosphorylation of Akt was dramatically enhanced by HBSP, but strongly reversed by wortmannin, the PI3K inhibitor. Wortmannin intervention improved βcR/EPOR expression, promoted caspase-9 and 3 activation, and increased active caspase-3 positive cells, while renal function and structure, and apoptotic cell counts scarcely changed. This result indicates a significant contribution of PI3K/Akt signaling pathway in the renoprotection of HBSP. The therapeutic effects of HBSP in this study suggest that HBSP could be a better candidate for renal protection.

Naked caspase 3 small interfering RNA is effective in cold preservation but not in autotransplantation of porcine kidneys.

BACKGROUND Caspase 3 associated with apoptosis and inflammation plays a key role in ischemia-reperfusion injury. The efficacy of naked caspase 3 small interfering RNA (siRNA) has been proved in an isolated porcine kidney perfusion model but not in autotransplantation. MATERIALS AND METHODS The left kidney was retrieved from mini pigs and infused with the University of Wisconsin solution with or without 0.3mg of caspase 3 siRNA into the renal artery with the renal artery and vein clamped for 24-h cold storage (CS). After right nephrectomy, the left kidney was autotransplanted into the right for 48 h without systemic treatment of siRNA. RESULTS Fluorescent dye-labeled caspase 3 siRNA was visualized in the post-CS kidneys but was weakened after transplantation. The expression of caspase 3 messenger RNA and precursor was downregulated by siRNA in the post-CS kidneys. In the siRNA-preserved posttransplant kidneys, however, the caspase 3 messenger RNA and active subunit were upregulated with further decreased precursor but increased active caspase 3+ cells, apoptotic cells, and myeloperoxidase+ cells. Moreover, the renal tissue damage was aggravated by siRNA, whereas the renal function was not significantly changed. CONCLUSIONS Naked caspase 3 siRNA administered into the kidney was effective in cold preservation but not enough to protect posttransplant kidneys, which might be because of systemic complementary responses overcoming local effects.

Mycophenolate mofetil inhibits macrophage infiltration and kidney fibrosis in long-term ischemia–reperfusion injury ☆

Immunosuppressants have been widely used in renal transplantation, in which ischemia-reperfusion injury is inevitable. Mycophenolate mofetil (MMF) is a relative novel immunosuppressant and also attenuates ischemia-reperfusion injury in the acute phase, but its long-term effects are still obscure. Unilateral renal ischemia-reperfusion injury model was established in Sprague-Dawley rats and 30 mg/kg/day MMF or natural saline was administered a day before the surgery. Renal function was monitored, and histological changes and fibrosis in the kidney were evaluated in both short and long terms. TGF-β1 secretion and MCP-1 expression were determined by immunohistochemistry and real-time PCR respectively. The infiltration of macrophages in renal tissues was also assessed by fluorescence activated cell sorting (FACS). MMF treatment significantly improved renal function in ischemia-reperfusion injury rats in the short and long-term and also effectively prevented interstitial fibrosis. TGF-β1 secretion and MCP-1 expression in the renal tissue of MMF-treated rats were much lower than those in natural saline-treated rats, with much less macrophage infiltration as well. MMF treatment effectively prevented the deterioration of renal function and interstitial fibrosis in ischemia-reperfusion injury rats, which may be associated with decreased TGF-β1, MCP-1 and macrophages. These results provide evidence for the choice of MMF in the renal transplant patients not only for acute renal injury but also for long-term survival of renal allograft.

Serum-stabilized Naked Caspase-3 siRNA Protects Autotransplant Kidneys in a Porcine Model

The naked small interfering RNA (siRNA) of caspase-3, a key player in ischemia reperfusion injury, was effective in cold preserved and hemoreperfused kidneys, but not autotransplanted kidneys in our porcine models. Here, chemically modified serum stabilized caspase-3 siRNAs were further evaluated. The left kidney was retrieved and infused by University of Wisconsin solution with/without 0.3 mg caspase-3 or negative siRNA into the renal artery for 24-hour cold storage (CS). After an intravenous injection of 0.9 mg siRNA and right-uninephrectomy, the left kidney was autotransplanted for 2 weeks. The effectiveness of caspase-3 siRNA was confirmed by caspase-3 knockdown in the post-CS and/or post-transplant kidneys with reduced apoptosis and inflammation, while the functional caspase-3 siRNA in vivo was proved by detected caspase-3 mRNA degradation intermediates. HMGB1 protein was also decreased in the post-transplanted kidneys; correlated positively with renal IL-1β mRNA, but negatively with serum IL-10 or IL-4. The minimal off-target effects of caspase-3 siRNA were seen with favorable systemic responses. More importantly, renal function, associated with active caspase-3, HMGB1, apoptosis, inflammation, and tubulointerstitial damage, was improved by caspase-3 siRNA. Taken together, the 2-week autotransplanted kidneys were protected when caspase-3 siRNA administrated locally and systemically, which provides important evidence for future clinical trials.

Innate immunity activation involved in unprotected porcine auto-transplant kidneys preserved by naked caspase-3 siRNA

BackgroundThe naked caspase-3 small interfering RNA (siRNA) infused into the renal artery during cold preservation was effective, but did not protect auto-transplant porcine kidneys with increased inflammation and apoptosis in our previous study. The mechanisms involved, in particular, whether siRNA or complementary systemic feedback eliciting innate immune responses are worthy to be further investigated.MethodsThe protein and mRNA expression of innate immunity-related molecules were detected by western blotting and quantitative PCR in the tissues previously collected from 48 h auto-transplant kidneys. The donor kidneys were retrieved from mini pigs and cold preserved by University of Wisconsin solution with/without 0.3 mg caspase-3 siRNA for 24 h.ResultsThe protein level of Toll like receptor (TLR) 3, TLR7, and their main adapters, TRIF and MyD88, was up-regulated in the siRNA preserved auto-transplant kidneys. The mRNA level of NF-κB and c-Jun was increased, as well as pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α and interferon (IFN)-α, β and γ. In addition, the non-TLR RNA sensor PKR protein, but not RIG1, was also increased in the siRNA preserved auto-transplant kidneys.ConclusionsThe activation of innate immunity with amplified inflammatory responses in the caspase-3 siRNA preserved auto-transplant kidneys are associated with increased TLR3, TLR7 and PKR, which might be due to complementary systemic feedback, although persistent actions initiated by short-acting caspase-3 siRNA cannot be completely ruled out. These results provided valuable evidence to guide future siRNA design and pre-clinic studies.

Prevention of renal ischemia-reperfusion injury by short hairpin RNA of endothelin A receptor in a rat model.

Endothelin A receptor (ETaR) is a key molecule involved in a variety of biological events such as vessel contraction and inflammatory response in ischemia-reperfusion (I/R) injury. RNA interference using short hairpin RNA (shRNA) is a powerful tool to silence gene expression. Here, the effect of ETaR shRNA on I/R injury in rats was studied. A more effective shRNA sequence out of two constructed into plasmid vectors was selected using the A-10 cell line, and was then applied to a rat model. Twenty-eight male Sprague-Dawley rats were randomized into four groups: Sham, shRNA, vector and phosphate-buffered saline (PBS). Renal I/R injury was induced by clamping the left renal pedicle for one hour followed by reperfusion for 24 h. ETaR shRNA (100 μg) plasmid was administered by renal vein injection 48 h before clamping. The expression of both ETaR mRNA and protein was lowered by ETaR shRNA treatment compared with that in the vector and PBS groups; serum creatinine and blood urea nitrogen were significantly decreased; the semi-quantitative score of renal structural damage was improved; the mRNA level of endothelin 1 (ET-1), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), macrophage inflammatory protein 2 (MIP-2) and monocyte chemoattractant protein 1 (MCP-1) was reduced, but nitric oxide (NO) production in kidney tissues was increased (P < 0.05). In conclusion, ETaR shRNA partially silenced ETaR expression in I/R injury kidneys, reduced the mRNA level of ET-1, inflammatory mediators including TNF-α, IL-6, MIP-2 and MCP-1, increased NO production, and ultimately improved renal function and structure.

Serum Level of Soluble Fibrinogen-Like Protein 2 in Renal Allograft Recipients With Acute Rejection: A Preliminary Study

BACKGROUND Soluble fibrinogen-like protein 2 (sfgl2), which is mainly secreted by T cells, is a novel effector of regulatory T cells with immunosuppressive functions. The aim of this study was to investigate serum levels of sfgl2 among renal allograft recipients. METHODS From November 2010 to August 2011 we retrospectively divided 47 renal allograft recipients into an acute rejection (n = 19) versus a stable group (n = 28) according to allograft biopsy results, using the Banff 2007 classification. The acute rejection group was subdivided into grade I (n = 8) versus grade II T-cell-mediated (n = 6) or antibody-mediated rejection episodes (n = 5). Peripheral blood samples were collected at the time of biopsy. Fourteen healthy volunteers were included as normal group controls. Serum levels of sfgl2 were analyzed by enzyme-linked immunosorbent assay. RESULTS Serum levels of sfgl2 were increased among renal allograft recipients suffering from biopsy-proven acute rejection episodes (61.91 ± 45.68 ng/mL), versus those with stable allografts (38.59 ± 19.92 ng/mL, P < .05) or healthy volunteers (29.10 ± 18.08 ng/mL, P < .05). The sfgl2 level was significantly higher among patients with antibody-mediated (118.48 ± 55.54 ng/mL) than T-cell-mediated acute rejection episodes (41.71 ± 16.44 ng/mL, P < .01). Serum sfgl2 levels were remarkably elevated in patients with grade II (51.87 ± 19.13 ng/mL) versus grade I T-cell-mediated rejection (34.10 ± 9.26 ng/mL, P < .05). CONCLUSIONS Serum sfgl2 levels were increased among renal allograft recipients with acute rejection episodes to an extent dependent upon the pathological type and severity of the response.

Resveratrol Alleviates Inflammatory Responses and Oxidative Stress in Rat Kidney Ischemia-Reperfusion Injury and H 2 O 2 -Induced NRK-52E Cells via the Nrf2/TLR4/NF-κB Pathway

Background: Ischemia-reperfusion injury (IRI) is one of the major causes of postoperative renal allograft dysfunction, which is mainly the result of proinflammatory reactions including inflammatory responses, oxidative stress, and metabolic disorders. Resveratrol (RSV) plays an important role in protecting various organs in IRI because it reduces oxidative stress, lessens the inflammatory response, and exerts anti-apoptotic effects. The aim of this study was to demonstrate the renoprotective effect of RSV in inhibiting inflammatory responses, reducing oxidative stress, and decreasing cell apoptosis in vivo and in vitro. Methods: RSV was administered before renal ischemia and H2O2 induction. Serum and kidneys were harvested 24 h after reperfusion and NRK-52E cells were collected 4 h after H2O2 stimulation. Serum creatinine and blood urea nitrogen were used to assess renal function. Hematoxylin and eosin staining was performed to assess histological injury. Quantitative real-time PCR and enzyme-linked immunosorbent assay were used to assess proinflammatory cytokine expression. Oxidative stress–related proteins, such as Nrf2 and TLR4, were evaluated by western blot. Terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling assay was used to detect apoptotic cells in tissues, and western blot was used to evaluate the expression of caspase-3, -8, and -9 in this study. Results: RSV inhibited inflammatory responses and improved renal function after renal IRI. Additionally, RSV decreased oxidative stress and reduced cell apoptosis by upregulating Nrf2 expression, downregulating the TLR4/NF-κB signaling pathway, and by decreasing caspase-3 activity and caspase cascades. Conclusion: Our study demonstrated the mechanisms underlying RSV renoprotection. We found that RSV exerts its greatest effects by blocking inflammatory responses, lowering oxidative stress, and reducing apoptosis via the Nrf2/TLR4/NF-κB pathway.

Endothelin Receptor Down-Regulation Mediated Ligand Regulation Mechanisms Protect Against Cellular Hypoxia Injury in Rat Vascular Endothelial Cells

Objective: Investigation of the effect of endothelin receptor A (ETaR)-targeting small interfering RNA (siRNA) on rat vascular endothelial cellular hypoxia injury, as well as its underlying mechanism. Methods: An in vitro rat vascular smooth muscle cells - endothelial cells co-culture model was established and transfected with ETaR siRNA before hypoxia treatment. Cell culture supernatant, cellular protein and RNA were collected and examined at 0.5hrs, 1hrs, 2hrs, 4hrs, 8hrs, 16hrs, 24hrs and 48hrs of hypoxia with 1% oxygen. The time point at which the best silencing effect was achieved was chosen, eNOS inhibitor L-NAME was added, and post hypoxia cell culture supernatant, cellular protein and RNA was collected for further examination. Results: After hypoxic treatment, endothelial-1 (ET-1) and ETaR expression levels gradually increased as oxygen deprivation extended. ET-1 and ETaR expression levels were significantly lower in the ETaR siRNA group compared with the Hypoxia group (P<0.001). Such difference peaked at 4hrs of hypoxia. ELISA examination of cell culture supernatant revealed that the amount of ET-1 and TGF-βin the ETaR siRNA group were significantly lower compared to the Hypoxia group at all times, while the amount of NO and eNOS was higher. After 4 hrs of hypoxia, Smad2, Smad3, HIF-1, TNF-α, IFN-γ, IL-6, MCP-1, NF-κb, ET-1 and ANG II mRNA expression in endothelial cells and ETaR mRNA expression in A-10 cells of the ETaR siRNA group were lower than those of the Hypoxia siRNA group, while such results were much higher in the L-NAME group. Western Blot results showed lower expression of ETaR in the ETaR siRNA group compared with the hypoxia and negative siRNA groups, as well as significantly higher ETaR expression in the L-NAME group compared with the ETaR siRNA group. PI3K and p-AKT expression levels were mildly elevated after mild oxygen deprivation, and ETaR siRNA was able to enhance such elevation induced by hypoxia. In the L-NAME group, PI3K and p-AKT expression was much higher than the ETaR siRNA group. PKG and sGC expression levels significantly descended after mild oxygen deprivation. While such levels were higher in the ETaR siRNA group, compared with the hypoxia and negative siRNA groups, the L-NAME group had lower levels of PKG and sGC compared with the ETaR siRNA group. Conclusion: ETaR siRNA is capable of down-regulating the expression of inflammatory and transcription factors among endothelial cells treated with hypoxia. Down-regulation of ET-1 is triggered by altered nucleus transcription factor activity through the sGC/PKG signal pathway, and results in enhanced eNOS activity through the PI3K/Akt signal pathway. We suspect this to be the mechanism of the protective effect of ETaR siRNA.

Estimation of Mycophenolic Acid Area Under the Curve With Limited-Sampling Strategy in Chinese Renal Transplant Recipients Receiving Enteric-Coated Mycophenolate Sodium

Background: The enteric-coated mycophenolate sodium (EC-MPS), whose active constituent is mycophenolic acid (MPA), has been widely clinically used for organ transplant recipients. However, its absorption is delayed due to its special designed dosage form, which results in difficulty to monitor the exposure of the MPA in patients receiving the EC-MPS. This study was aimed at developing a relatively practical and precise model with limited sampling strategy to estimate the 12-hour area under the concentration–time curve (AUC0–12 h) of MPA for Chinese renal transplant recipients receiving EC-MPS. Methods: A total of 36 Chinese renal transplant recipients receiving the EC-MPS and tacrolimus were recruited in this study. The time point was 2 weeks after the transplantation for all the patients. The MPA concentrations were measured with enzyme-multiplied immunoassay technique for 11 blood specimens collected predose and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 12 hours after the morning dose of EC-MPS. The measured AUC was calculated with these 11 points of MPA concentrations with the linear trapezoidal rule. Limited sampling strategy was used to develop models for estimated AUC in the model group (n = 18). The bias and precision of different models were evaluated in the validation group (n = 18). Results: C4 showed the strongest correlation with the measured AUC. The best 3 time point equation was 6.629 + 8.029 × C0 + 0.592 × C3 + 1.786 × C4 (R2 = 0.910; P < 0.001), whereas the best 4 time point equation was 3.132 + 5.337 × C0 + 0.735 × C3 + 1.783 × C4 + 3.065 × C8 (R2 = 0.959; P < 0.001). When evaluated in the validation group, the 4 time point model had a much better performance than the 3 time point model: for the 4 time point model: R2 = 0.873, bias = 0.505 [95% confidence interval (CI), −10.159 to 11.170], precision = 13.370 (95% CI, 5.186–21.555), and 77.8% of estimated AUCs was within 85%–115% of the measured AUCs; for the 3 time point model: R2 = 0.573, bias = 6.196 (95% CI, −10.627 to 23.018), precision = 21.286 (95% CI, 8.079–34.492), and 50.0% of estimated AUCs was within 85%–115% of the measured AUCs. Conclusions: It demanded at least 4 time points to develop a relatively reliable model to estimate the exposure of MPA in renal transplant recipients receiving the EC-MPS. The long time span needed restricted its application, especially for the outpatients, but it could be a useful tool to guide the personalized prescription for the inpatients.