Quan Hong

Mesenchymal stem cells ameliorate rhabdomyolysis-induced acute kidney injury via the activation of M2 macrophages

IntroductionThe mortality of rhabdomyolysis-induced acute kidney injury (AKI) is still high, as there is no effective therapy. It has been shown that bone marrow-derived mesenchymal stem cells (MSCs) can induce M2 macrophages, which mediate MSC protection in other experimental inflammation-related organ injury. This study was designed to investigate the protective effects of macrophage activation in MSC therapy of rhabdomyolysis-induced AKI.MethodsMSCs were injected into glycerol-induced rhabdomyolysis mice. Renal injury was evaluated using the serum creatinine, urea nitrogen, renal pathology and acute tubular necrosis score. The distribution of MSCs was detected using two-photon fluorescence confocal imaging. Immunofluorescence of anti-F4/80 and anti-CD206 was performed to determine macrophages and M2 macrophages in the tissues of the kidney, and M2 macrophage infiltration was also evaluated using western blotting analyses. After depletion of macrophages using clodronate liposomes at the phase of kidney repair, renal injury was re-evaluated. RAW 264.7 macrophages were incubated with lipopolysaccharide and co-cultured with MSCs and subsequently visualised using immunofluorescence staining and flow cytometry analysis. Finally, disparate phenotype macrophages, including normal macrophages (M0), lipopolysaccharide-stimulated macrophages (M1), and MSC-co-cultured macrophages (M2), were infused into mice with AKI, which were pre-treated with liposomal clodronate.ResultsIn vivo infusion of MSCs protected AKI mice from renal function impairment and severe tubular injury, which was accompanied by a time-dependent increase in CD206-positive M2 macrophage infiltration. In addition, depleting macrophages with clodronate delayed restoration of AKI. In vitro, macrophages co-cultured with MSCs acquired an anti-inflammatory M2 phenotype, which was characterised by an increased expression of CD206 and the secretory cytokine interleukin (IL)-10. The concentrations of IL-10, IL-6 and tumor necrosis factor α were evaluated using enzyme-linked immunosorbent assay. Furthermore, macrophage-depleted mice with intramuscular injection of glycerol were subjected to a single injection of different types of RAW 264.7 macrophages. Mice infused with M0 and M1 macrophages suffered a more severe histological and functional injury, while mice transfused with MSC-educated M2 macrophages showed reduced kidney injury.ConclusionsOur findings suggested that MSCs can ameliorate rhabdomyolysis-induced AKI via the activation of macrophages to a trophic M2 phenotype, which supports the transition from tubule injury to tubule repair.

Rapamycin Upregulates Autophagy by Inhibiting the mTOR-ULK1 Pathway, Resulting in Reduced Podocyte Injury

The podocyte functions as a glomerular filtration barrier. Autophagy of postmitotic cells is an important protective mechanism that is essential for maintaining the homeostasis of podocytes. Exploring an in vivo rat model of passive Heymann nephritis and an in vitro model of puromycin amino nucleotide (PAN)-cultured podocytes, we examined the specific mechanisms underlying changing autophagy levels and podocyte injury. In the passive Heymann nephritis model rats, the mammalian target-of-rapamycin (mTOR) levels were upregulated in injured podocytes while autophagy was inhibited. In PAN-treated podocytes, mTOR lowered the level of autophagy through the mTOR-ULK1 pathway resulting in damaged podocytes. Rapamycin treatment of these cells reduced podocyte injury by raising the levels of autophagy. These in vivo and in vitro experiments demonstrate that podocyte injury is associated with changes in autophagy levels, and that rapamycin can reduce podocyte injury by increasing autophagy levels via inhibition of the mTOR-ULK1 pathway. These results provide an important theoretical basis for future treatment of diseases involving podocyte injury.

Mesenchymal stem cells attenuate ischemic acute kidney injury by inducing regulatory T cells through splenocyte interactions

The mechanism of mesenchymal stem cell therapy in acute kidney injury remains uncertain. Previous studies indicated that mesenchymal stem cells could attenuate inflammation-related organ injury by induction of regulatory T cells. Whether regulatory T-cell induction is a potential mechanism of mesenchymal stem cell therapy in ischemic acute kidney injury and how these induced regulatory T cells orchestrate local inflammation are unknown. Here we found that mesenchymal stem cells decrease serum creatinine and urea nitrogen levels, improve tubular injury, and downregulate IFN-γ production of T cells in the ischemic kidney. In addition to the lung, mesenchymal stem cells persisted mostly in the spleen. Mesenchymal stem cells increased the percentage of regulatory T cells in the spleen and the ischemic kidney. Antibody-dependent depletion of regulatory T cells blunted the therapeutic effect of mesenchymal stem cells, while coculture of splenocytes with mesenchymal stem cells caused an increase in the percentage of regulatory T cells. Splenectomy abrogated attenuation of ischemic injury, and downregulated IFN-γ production and the induction of regulatory T cells by mesenchymal stem cells. Thus, mesenchymal stem cells ameliorate ischemic acute kidney injury by inducing regulatory T cells through interactions with splenocytes. Accumulated regulatory T cells in ischemic kidney might be involved in the downregulation of IFN-γ production.

Hyperuricemia induces endothelial dysfunction via mitochondrial Na+/Ca2+ exchanger-mediated mitochondrial calcium overload.

BACKGROUND Uric acid (UA) has proven to be a causal agent in endothelial dysfunction in which ROS production plays an important role. Calcium overload in mitochondria can promote the mitochondrial production of ROS. We hypothesize that calcium transduction in mitochondria contributes to UA-induced endothelial dysfunction. METHODS AND RESULTS We first demonstrated that high concentrations of UA cause endothelial dysfunction, marked by a reduction in eNOS protein expression and NO release in vitro. We further found that a high concentration of UA increased levels of [Ca2+]mito, total intracellular ROS, H2O2, and mitochondrial O2·-, and Δψmito but not the [Ca2+]cyt level. When the mitochondrial calcium channels NCXmito and MCU were blocked by CGP-37157 and Ru360, respectively, the UA-induced increases in the levels of [Ca2+]mito and total intracellular ROS were significantly reduced. Mitochondrial levels of O2·- and Δψmito were reduced by inhibition of NCXmito but not of MCU. Moreover, inhibition of NCXmito, but not of MCU, blocked the UA-induced reductions in eNOS protein expression and NO release. CONCLUSIONS The increased generation of mitochondrial O2·- induced by a high concentration of UA is triggered by mitochondrial calcium overload and ultimately leads to endothelial dysfunction. In this process, the activation of NCXmito is the major cause of the influx of calcium into mitochondria. Our results provide a new pathophysiological mechanism for UA-induced endothelial dysfunction and may offer a new therapeutic target for clinicians.

Downregulation of Connexin 43 Expression by High Glucose Induces Senescence in Glomerular Mesangial Cells

Connexin 43 (Cx43) plays an important role in cell differentiation and growth control, but whether it can be regulated by high glucose and whether it can mediate in glomerular mesangial cells (GMC) the phenotype alterations that are induced by high glucose still remain to be explored. In this study, RNA interference and gene transfer techniques were used to knock down and overexpress Cx43 gene in rat GMC to determine the contribution of Cx43 to GMC senescence that was induced by high glucose. The results show that high glucose (30 mM) not only downregulated Cx43 mRNA and protein expression (P<0.05) but also increased the percentage of senescence-associated beta-galactosidase (SA-beta-gal) stained cells and expression of p21cip1 and p27kip1 (P<0.05), indicating that high glucose promoted rat GMC senescence. Knocking down Cx43 gene expression significantly increased the percentage of SA-beta-gal stained cells and p27kip1 and p21cip1 expression in GMC (P<0.05), whereas overexpression of Cx43 significantly decreased the percentage of SA-beta-gal stained cells (P<0.05). These results demonstrate for the first time that downregulation of Cx43 expression by high glucose promotes the senescence of GMC, which may be involved in the pathogenesis of diabetic nephropathy.

Mesenchymal stem cells ameliorate sepsis-associated acute kidney injury in mice.

ABSTRACT Objective: Significant progress has been made in critical care medicine during the past several decades. However, the mortality rate is still high in patients with sepsis, especially with acute kidney injury (AKI). Mesenchymal stem cells (MSCs) possess an ability to ameliorate renal injury from ischemia-reperfusion, but it is still unknown whether they have the ability to reduce sepsis-associated AKI. Methods: Male C57BL/6 mice underwent cecal ligation and puncture operation to induce sepsis and then received either normal saline or MSCs (1 × 106 cells intravenously) 3 h after surgery. Results: Within 24 h after cecal ligation and puncture operation, the septic mice developed kidney injury and exhibited a higher mortality. Treatment with MSCs decreased serum creatinine and blood urea nitrogen levels and improved recovery of tubular function. mRNA levels of interleukin 6 (IL-6), IL-17, tumor necrosis factor &agr;, interferon &ggr;, CXCL1, CXCL2, CXCL5, CCL2, and CCL3 in kidney tissue were dramatically decreased after MSC treatment. Neutrophil infiltration in kidney and blood bacterial loads were attenuated after MSC injection. Moreover, mice treated with MSCs had a higher survival rate than the saline treatment group. Injected MSCs were mainly localized in the lungs, spleen, and abdominal cavity lymph node, but not in the kidneys. Conclusions: Treatment with MSCs can alleviate sepsis-associated AKI and improve survival in mice with polymicrobial sepsis. These effects may be mediated by the inhibition of IL-17 secretion and balance of the proinflammatory and anti-inflammatory states. Mesenchymal stem cells may be a potential new therapeutic agent for the prevention or reduction of sepsis-associated AKI.

Age-related changes in the function of autophagy in rat kidneys

Autophagy is a highly regulated intracellular process for the degradation of cytoplasmic components, especially protein aggregates and damaged organelles. It is essential for maintaining healthy cells. Impaired or deficient autophagy is believed to cause or contribute to aging and age-related disease. In this study, we investigated the effects of age on autophagy in the kidneys of 3-, 12-, and 24-month-old Fischer 344 rats. The results revealed that autophagy-related gene (Atg)7 was significantly downregulated in kidneys of increasing age. The protein expression level of the autophagy marker light chain 3/Atg8 exhibited a marked decline in aged kidneys. The levels of p62/SQSTM1 and polyubiquitin aggregates, representing the function of autophagy and proteasomal degradation, increased in older kidneys. The level of 8-hydroxydeoxyguanosine, a marker of mitochondrial DNA oxidative damage, was also increased in older kidneys. Analysis by transmission electron microscope demonstrated swelling and disintegration of cristae in the mitochondria of aged kidneys. These results suggest that autophagic function decreases with age in the kidneys of Fischer 344 rats, and autophagy may mediate the process of kidney aging, leading to the accumulation of damaged mitochondria.

Tissue inhibitor of metalloproteinase-1 exacerbated renal interstitial fibrosis through enhancing inflammation

BACKGROUND Tissue inhibitor of metalloproteinase-1 (TIMP-1) is associated with renal fibrosis. Furthermore, it is a multi-functional protein, and whether it has other roles in renal fibrosis is unknown. As several inflammatory mediators are substrates of matrix metalloproteinases (MMPs), TIMP-1 might affect renal fibrosis via inflamatory pathways. METHODS Plasmids containing the sense and antisense human TIMP-1 sequence were stably transfected into the human kidney proximal tubular epithelial cell line (HKC), MMP-2 and MMP-9 siRNA were transiently transfected into HKC and the transfected cells were stimulated with phorbol 12-myristate 13-acetate (PMA). In vivo, we established unilateral ureteral obstruction (UUO) models by using homozygote human TIMP-1 transgenic mice. The expression of intercellular adhesion molecule-1 (ICAM-1) in transfected cells and F4/80-positive cells in the renal interstitium were examined by indirect immunofluorescence. Protein levels in the cells and UUO models were examined by western blot, and the activities of the gelatinases and TIMP-1 were examined by gelatin zymography and reverse zymography, respectively. RESULTS After stimulation with PMA, the activities of the gelatinases were decreased, ICAM-1 was upregulated, and soluble ICAM-1 in the supernatant was decreased, in HKC transfected with sense TIMP-1, and ICAM-1 was increased in HKC transfected with MMP-9 siRNA. At 14 days after UUO, it was found that compared with wild-type mice, in transgenic mice, with upregulation of TIMP-1, activities of gelatinases were downregulated, ICAM-1, transforming growth factor-beta1 (TGF-beta1), collagens I and III were upregulated, and the extent of renal fibrosis and infiltration of macrophages was more severe. CONCLUSION Overexpression of TIMP-1 could promote renal interstitial fibrosis through the inflammatory pathway, which might be partly induced by upregulating ICAM-1.

Expression and mechanism of mammalian target of rapamycin in age-related renal cell senescence and organ aging

The mammalian target of rapamycin (mTOR) is relevant to cell senescence and organismal aging. This study firstly showed that the level of mTOR expression increased with aging in rat kidneys, rat mesangial cells and WI-38 cells (P < 0.05). The levels of phosphorylated-mTOR (p-mTOR), cyclin D1 and p21(WAF1/CIP1/SDI1) expression were significantly higher in WI-38 cells treated with l-leucine (an activator of mTOR) (P < 0.05). The positive staining ratio of senescence-associated beta-galactosidase, number of cells in G1 phase, and cellular volume were all increased in WI-38 cells treated with l-leucine when the cellular population doubling (PD) number was 34, while the above phenotypes did not appear in control group until its PD number reached 40 (P < 0.05). The levels of p-mTOR, cyclin D1, and p21(WAF1/CIP1/SDI1) as well as the aging-related phenotypes were all reduced in cells treated with rapamycin (an inhibitor of mTOR) than in control cells (P < 0.05). These results demonstrated that the level of mTOR was increased in kidney with aging, and that mTOR may promote cellular senescence by regulating the cell cycle through p21(WAF1/CIP1/SDI1), which might provide a new target for preventing renal aging.

TIMP-1 Transgenic Mice Recover From Diabetes Induced by Multiple Low-Dose Streptozotocin

Type 1 diabetes results from autoimmune destruction of the insulin-producing β-cells of pancreatic islets, of which the capacity for self-replication in the adult is too limited to restore following extensive tissue injury. Tissue inhibitor of metalloproteinase (TIMP)-1 inhibits matrix metalloproteinase activity and regulates proliferation and apoptosis of a variety of cells types, depending on the context. Here, we show that overexpression of human TIMP-1 in pancreatic β-cells of transgenic mice counteracts the cytotoxicity and insulitis induced by multiple low-dose streptozotocin (MLDS). Nontransgenic mice developed severe hyperglycemia, hypoinsulinemia, and insulitis 2 weeks after streptozotocin administration and died within 17 weeks. However, MLDS-treated transgenic mice gradually normalized the metabolic parameters and survived. β-Cell mass increased in parallel as a result of enhancement of β-cell replication. Thus, our results have demonstrated for the first time that overexpression of TIMP-1 in β-cells enhances the replication of pancreatic islets β-cells and counteracts type 1 diabetes, indicating that the TIMP-1 gene may be a potential target to prevent, or even reverse, type 1 diabetes.