Zitong Zhao

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.

A novel proteolysis-resistant cyclic helix B peptide ameliorates kidney ischemia reperfusion injury.

Helix B surface peptide (HBSP), derived from erythropoietin, displays powerful tissue protection during kidney ischemia reperfusion (IR) injury without erythropoietic side effects. We employed cyclization strategy for the first time, and synthesized thioether-cyclized helix B peptide (CHBP) to improve metabolic stability and renoprotective effect. LC-MS/MS analysis was adopted to examine the stability of CHBP in vitro and in vivo. The renoprotective effect of CHBP in terms of renal function, apoptosis, inflammation, extracellular matrix deposition, and histological injury was also detected in vivo and in vitro. Antibody array and western blot were performed to analyze the signal pathway of involvement by CHBP in the IR model and renal tubular epithelial cells. In this study, thioether-cyclized peptide was significantly stable in vivo and in vitro. One dose of 8nmol/kg CHBP administered intraperitoneally at the onset of reperfusion improved renal protection compared with three doses of 8nmol/kg linear HBSP in a 48h murine IR model. In a one-week model, the one dose CHBP-treated group exhibited remarkably improved renal function over the IR group, and attenuated kidney injury, including reduced inflammation and apoptosis. Interestingly, we found that the phosphorylation of autophagy protein mTORC1 was dramatically reduced upon CHBP treatment. We also demonstrated that CHBP induced autophagy via inhibition of mTORC1 and activation of mTORC2, leading to renoprotective effects on IR. Our results indicate that the novel metabolically stable CHBP is a promising therapeutic medicine for kidney IR injury treatment.

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.

Comparison of Regulatory T Cells and FoxP3-Positive T-Cell Subsets in the Peripheral Blood of Renal Transplant Recipients With Sirolimus Versus Cyclosporine: A Preliminary Study

BACKGROUND Sirolimus-based regimens have recently been introduced as nephron-sparing strategies to avoid the calcineurin inhibitors-induced nephrotoxicity. METHODS To investigate the different effects of these 2 immunosuppressants on CD4(+)CD25(hi)FoxP3(+) regulatory T cells (Tregs) and the newly defined subsets of FoxP3(+) T cells, including CD45RA(-)FoxP3(lo) cytokine-secreting T cells (csT cells), CD45RA(-)FoxP3(hi) activated Treg (aTregs), and CD45RA(+)FoxP3(+) resting Treg (rTregs), 52 cases of renal transplant recipients who have received a maintenance immunosuppressive regimen comprised either cyclosporine (n = 34) or sirolimus (n = 18) were collected and proportion of Tregs and each subset of FoxP3(+) T cells in peripheral blood was detected by flowcytometry. RESULTS Sirolimus significantly prompted the proportion of Tregs (5.92 ± 0.40 vs 2.19 ± 0.18; P < .001) and both of the CD45RA-negative subsets, including csT cells (4.92 ± 0.50 vs 1.83 ± 0.18; P < .001) and aTregs (1.04 ± 0.15 vs 0.23 ± 0.05; P < .05) when compared with cyclosporine. However, the rTregs were not remarkably affected (0.36 ± 0.09 vs 0.79 ± 0.28; P = .063). CONCLUSION Sirolimus plays its immune regulatory role in renal transplantation partly by increasing the proportion of Tregs. However, the increasing of both pro- and anti-inflammatory subsets of FoxP3(+) T cells also indicates the potential compromising of the effects of sirolimus on immune regulation.

The regulatory T cell effector soluble fibrinogen-like protein 2 induces tubular epithelial cell apoptosis in renal transplantation

Acute rejection (AR) hinders renal allograft survival. Tubular epithelial cell (TEC) apoptosis contributes to premature graft loss in AR, while the mechanism remains unclear. Soluble fibrinogen-like protein 2 (sFGL2), a novel effector of regulatory T cells (Treg), induces apoptosis to mediate tissue injury. We previously found that serum sFGL2 significantly increased in renal allograft rejection patients. In this study, the role of sFGL2 in AR was further investigated both in vivo and in vitro. The serum level of sFGL2 and the percentage of CD4+CD25+Foxp3+ Treg in the peripheral blood were measured in renal allograft recipients with AR or stable renal function (n = 30 per group). The human TEC was stimulated with sFGL2, tumor necrosis factor (TNF)-α, or phosphate buffered saline and investigated for apoptosis in vitro. Apoptosis-associated genes expression in TEC was further assessed. Approval for this study was obtained from the Ethics Committee of Fudan University. Our results showed that the serum level of sFGL2, correlated with Treg in the peripheral blood, was significantly increased in the AR patients. In vitro, sFGL2 remarkably induced TEC apoptosis, with a significant up-regulation of proapoptotic genes, including CASP-3, CASP-8, CASP-9, CASP-10, TRADD, TNFSF10, FADD, FAS, FASLG, BAK1, BAD, BAX, and NF-KB1. However, no significant changes were observed in the expression of antiapoptotic genes, including CARD-18, NAIP, BCL2, IKBKB, and TBK1. Therefore, sFGL2, an effector of Treg, induces TEC apoptosis. Our study suggests that sFGL2 is a potential mediator in the pathogenesis of allograft rejection and provides novel insights into the role of Treg in AR.

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.

Skewed T-helper (Th)1/2- and Th17/T regulatory-cell balances in patients with renal cell carcinoma

The characterization of CD4+ T-cell subsets reflects the immune status and is important in the maintenance of tumorigenesis and homeostasis. To identify changes in the balance of T helper (Th)1, Th2, Th17 and regulatory T cells (Treg) in individuals with renal cell carcinoma (RCC), the present study investigated a total of 131 patients with RCC and 36 healthy volunteers. The number of CD4+ T-bet+ cells, CD4+ GATA binding protein 3+ cells, CD4+ RAR-related orphan receptor γt+ cells, CD4+ CD25hi CD127lo CD45RA− cells and CD4+ CD25hi CD127lo CD45RA+ cells, defined as Th1, Th2, Th17, activated and naïve Treg cells, respectively, were detected in the peripheral blood using flow cytometric analysis. In addition, tumor-infiltrating forkhead box P3 (Foxp3)+ cells were examined using immunohistochemistry. Compared with healthy volunteers, a significant decrease in the peripheral percentages of Th1, activated and naïve Treg cells was observed in patients with RCC, while those of the Th2 and Th17 cells were increased. In particular, as the tumor stage and grade progressed, the levels of Th1, activated and naïve Treg cells in the peripheral blood decreased; however, the levels of Th2 and Th17 cells increased. Furthermore, the number of tumor-infiltrating Foxp3+ cells increased with increasing tumor stage. These results demonstrated that the balance of Th1 and Th2 cells was skewed towards the Th2 profile and the balance of Th17 and Treg cells was skewed towards the Th17 profile in the peripheral blood of patients with renal cell carcinoma (RCC) and Treg cells were recruited to the tumor sites. Therefore, dysfunctional host anti-tumor immunity was observed in patients with RCC, with a skewed Th1/Th2 and Th17/Treg balance.

Fighting against kidney diseases with small interfering RNA: opportunities and challenges

The significant improvements in siRNA therapy have been achieved, which have great potential applications in humans. The kidney is a comparatively easy target organ of siRNA therapy due to its unique structural and functional characteristics. Here, we reviewed recent achievements in siRNA design, delivery and application with focuses on kidney diseases, in particular kidney transplant-related injuries. In addition, the strategy for increasing serum stability and immune tolerance of siRNA was also discussed. At last, the future challenges of siRNA therapy including organ/tissue/cell-specific delivery and time-controlled silence, as well as selecting therapeutic targets, were addressed as well.