Ruiming Rong

Erythropoietin Ameliorates Renal Ischemia and Reperfusion Injury via Inhibiting Tubulointerstitial Inflammation

BACKGROUND Tubulointerstitial inflammation is the characteristics of renal ischemia reperfusion injury (IRI) that is inevitable in kidney transplantation. Erythropoietin (EPO) has recently been shown to have protective effects on renal IRI by anti-apoptosis and anti-oxidation. Here, the effect and mechanism of EPO on renal IRI were further investigated, with a focus on tubulointerstitial inflammation. MATERIALS AND METHODS Male Sprague-Dawley rats were administrated with saline or EPO prior to IRI induced by bilateral renal pedicle clamping. Twenty-four hours following reperfusion, the effects of EPO on renal IRI were assessed by renal function and structure, tubulointerstitial myeloperoxidase (MPO) positive neutrophils, and proinflammatory mediator gene expression. The translocation and activity of NF-κB in renal tissues were also evaluated. RESULTS Compared with control groups, the EPO treated group exhibited lower serum urea and creatinine levels, limited tubular necrosis with a lower score of renal histological lesion. MPO positive cells in the tubulointerstitial area were greatly increased by IRI, but significantly reduced by the treatment of EPO. The gene expression of proinflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α) and chemokine (MCP-1) was also significantly decreased by EPO. In addition, less activation and nuclear-translocation of NF-κB was observed in the kidney treated by EPO as well. CONCLUSION EPO improved renal function and structure in IRI rats via reducing neutrophils in the tubulointerstitium, the production of proinflammatory cytokines and chemokine, as well as the activation and nuclear-translocation of NF-κB. EPO may have potential clinical applications as an anti-inflammation agent clinically for a wide range of injury.

Renal telocytes contribute to the repair of ischemically injured renal tubules

Telocytes (TCs), a distinct type of interstitial cells, have been identified in many organs via electron microscopy. However, their precise function in organ regeneration remains unknown. This study investigated the paracrine effect of renal TCs on renal tubular epithelial cells (TECs) in vitro, the regenerative function of renal TCs in renal tubules after ischaemia–reperfusion injury (IRI) in vivo and the possible mechanisms involved. In a renal IRI model, transplantation of renal TCs was found to decrease serum creatinine and blood urea nitrogen (BUN) levels, while renal fibroblasts exerted no such effect. The results of histological injury assessments and the expression levels of cleaved caspase‐3 were consistent with a change in kidney function. Our data suggest that the protective effect of TCs against IRI occurs via inflammation‐independent mechanisms in vivo. Furthermore, we found that renal TCs could not directly promote the proliferation and anti‐apoptosis properties of TECs in vitro. TCs did not display any advantage in paracrine growth factor secretion in vitro compared with renal fibroblasts. These data indicate that renal TCs protect against renal IRI via an inflammation‐independent pathway and that growth factors play a significant role in this mechanism. Renal TCs may protect TECs in certain microenvironments while interacting with other cells.

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.

Baicalin Ameliorates H2O2 Induced Cytotoxicity in HK-2 Cells through the Inhibition of ER Stress and the Activation of Nrf2 Signaling

Renal ischemia-reperfusion injury plays a key role in renal transplantation and greatly affects the outcome of allograft. Our previous study proved that Baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, protects kidney from ischemia-reperfusion injury. This study aimed to study the underlying mechanism in vitro. Human renal proximal tubular epithelial cell line HK-2 cells were stimulated by H2O2 with and without Baicalin pretreatment. The cell viability, apoptosis and oxidative stress level were measured. The expression of endoplasmic reticulum (ER) stress hallmarks, such as binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), were analyzed by western blot and real-time PCR. NF-E2-related factor 2 (Nrf2) expression was also measured. In the H2O2 group, cell viability decreased and cell apoptosis increased. Reactive Oxygen Species (ROS) and Glutathione/Oxidized Glutathione (GSH/GSSG) analysis revealed increased oxidative stress. ER stress and Nrf2 signaling also increased. Baicalin pretreatment ameliorated H2O2-induced cytotoxicity, reduced oxidative stress and ER stress and further activated the anti-oxidative Nrf2 signaling pathway. The inducer of ER stress and the inhibitor of Nrf2 abrogated the protective effects, while the inhibitor of ER stress and the inducer of Nrf2 did not improve the outcome. This study revealed that Baicalin pretreatment serves a protective role against H2O2-induced cytotoxicity in HK-2 cells, where the inhibition of ER stress and the activation of downstream Nrf2 signaling are involved.

Analysis of transcriptional factors and regulation networks in patients with acute renal allograft rejection.

Acute rejection (AR) remains a major clinical challenge, leading to the development of chronic renal allograft failure. The aim of the present study was to explore potential transcriptional factors and regulation networks in the disease to predict the occurrence and process of AR and understand potential strategies to prevent from the disease. Three-hundred fifty-two patients with renal failure had kidney transplantation during March 2006 and March 2010, of which 85 suffered from AR. Plasma from 13 patients with kidney transplantation was collected, of which 5 were from patients with AR and 8 from those without AR. Among the 179 proteins identified by using iTRAQ labeling and quantitative proteomic technology, 66 proteins were at least 2-fold different between patients with or without AR. The results demonstrated that the dominant processes and responses were associated with inflammation and complement activation in AR. A number of transcription factors were identified in AR patients, including nuclear factor-κB, signal transducer and activator of transcription 1, signal transducer and activator of transcription 3. The analysis of transcription regulation networks suggested that the cross-talks among these key transcription factors might contribute to the acute response and coagulation pathway. Thus, our study provides a new description and insight into the molecular events in AR and potential strategies for identifying diagnostic biomarkers.

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.

Urinary Metabolomics in Monitoring Acute Tubular Injury of Renal Allografts: A Preliminary Report

Acute tubular injury (ATI) is very common in biopsy specimens from renal allografts that suffer from delayed graft function (DGF) or dysfunction. Currently there are few reports on investigating small molecule metabolites in urine samples from transplant recipients as a noninvasive method to predict the ATI of renal allografts instead of an allograft biopsy. In our study matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used to analyze small molecule metabolites in urine samples from renal transplant recipients with biopsy-proven slight ATI or moderate ATI or acute tubular necrosis (ATN). To evaluate the ATI-specific value of those small molecules, we applied the Principal Component Analysis (PCA) program. Mass spectra data were imported into the PCA, where loading graphs were constructed to express the constituents of the urine samples. Slight ATI, moderate ATI, or ATN of renal allografts were separated obviously in the loading graph. The position of urine samples in the graph may reflect the tubular injury status of allografts. A farther apart point from the original site may mean the allograft suffered from more severe ATI (even ATN), and vice versa. Detection of small molecule metabolites in urine samples of recipients through MALDI-FTMS may offer a promising noninvasive, high throughput, rapid tool to predict ATI/ATN of renal allografts.