Comparison of two fluid solutions for resuscitation in a rabbit model of crush syndrome.

BACKGROUND: Crush syndrome is a common injury, the main characteristics of which include acute kidney injury. However, there is still lack of reliable animal model of crush syndrome, and it also remains controversial as to which type of fluid should be chosen as a more appropriate treatment option for prevention and treatment of acute kidney injury. METHODS: The rabbits were crushed at the lower limbs for 6 h with 36 times the body weight, which means the pressure of each leg was also 36 times the body weight. Fluid resuscitation was performed from 1 h prior to the end of the crush treatment until 24 h after the reperfusion. Tissue, blood and urine samples were collected at predetermined time points before and after reperfusion. Twelve rabbits in each group were taken for survival observation for 72 h. RESULTS: The model group showed elevated serum creatine kinase, aspartate aminotransferase, alanine aminotransferase, and K(+) level, reduced serum Ca(2+) level and Na(+) level, and increased serum creatinine and blood urea nitrogen levels, neutrophil gelatinase-associated lipocalin, and kidney injury molecule-1 (p < 0.05). The 0.9 % normal saline (SAL) group and SAL plus 6 % hydroxyethyl starch 130/0.4 SAL/HES group showed reduced serum creatinine and blood urea nitrogen levels (p < 0.05). The SAL/HES group also showed reduced serum IL-6 and IL-10 levels (p < 0.05). The 72 h survival rate of the SAL/HES group was higher than that of the model group (p < 0.05). CONCLUSION: The rabbit model of crush syndrome showed clinical features consistent with those of crush syndrome. There was no significant difference in the ability of preventing AKI after a crush injury between the two fluid solutions, while SAL/HES can improve the survival rate.

Analysis of pathological data of renal biopsy at one single center in China from 1987 to 2012.

BACKGROUND: In China, the prevalence of chronic kidney disease has increased significantly. Many studies shows that the spectrum of kidney disease had changed in recent years. We retrospectively analyzed the pathological types of renal biopsy and its spectrum change at the General Hospital of the Chinese People's Liberation Army from December 1987 to December 2012, in order to offer new supporting evidences for further specifying the distribution of renal pathological types in China. METHODS: According to the "Revised Protocol for the Histological Typing of Glomerulopathy" (WHO, 1995), pathological diagnosis of renal biopsy was classified, detection rate of each pathological type was summarized (i.e., percentage of total renal biopsy cases), study period was divided at an interval of 5 years, and age-stratified distribution change of main pathological types was analyzed. RESULTS: The proportion of pathological types in 11 618 cases of renal biopsy was as follows: primary glomerulonephritis (PGN, 70.7%), secondary glomerulonephritis (SGN, 20.7%), tubular-interstitial nephropathy (4.0%), hereditary/rare nephropathy (0.3%), end-stage renal disease (0.9%), and unclassified renal disease (3.3%). Among PGN, there was IgA nephropathy (IgAN, 37.0%), membranous nephropathy (MN, 11.8%), mesangial proliferative glomerulonephritis (MsPGN, 8.9%), minimal change disease (MCD, 6.6%), and focal segmental glomerulosclerosis (3.9%). Among SGN there was lupus nephritis (LN, 5.5%), Henoch-Schönlein purpura glomerulonephritis (5.3%), hepatitis B virus-associated nephritis (HBVAN, 3.03%), diabetic nephropathy (2.2%), and hypertension/malignant hypertension-associated renal damage (1.9%). Pathological data were analyzed from 1987-1992 to 2008-2012 (after age adjustment). Detection rate of IgAN tended to rise (P < 0.001). Detection rates of MN and MCD rose significantly (P < 0.001), but detection rate of MsPGN dropped significantly (P < 0.001). Among SGN, detection rate of HBVAN tended to drop (P < 0.001). CONCLUSION: In China, PGN was the most common glomerulopathy (mostly IgAN), LN was the most common SGN, and detection rate of MN and MCD rose significantly.

miR-34a regulates mesangial cell proliferation via the PDGFR-ß/Ras-MAPK signaling pathway.

The main pathological characteristic of glomerulonephritis is diffuse mesangial cell proliferation. MiR-34a is associated with the proliferation of various organs and cancer cells. However, the role of miR-34a in renal proliferation diseases is not clear. Therefore, this study aimed to elucidate the mechanism of miR-34a in the regulation of renal mesangial cell proliferation. The miR-34a expression level at different time points in an anti-Thy1 mesangial proliferative nephritis rat model was determined by qRT-PCR. The cell proliferation rate and cell cycle changes were measured in the in vitro cultured rat mesangial cells (RMCs). Our results suggested that miR-34a expression was negatively correlated with the degree of cell proliferation in the anti-Thy1 nephritis model. MiR-34a could extend the G0/G1 phase and block cell proliferation in RMCs. Dual-luciferase assay results showed that there were binding sites of miR-34a at 3'-UTR of platelet-derived growth factor receptor-ß (PDGFR-ß). MiR-34a can inhibit PDGFR-ß protein expression at a post-transcriptional level, suppress Ras/MAPK signaling pathways, and down-regulate expression of cell cycle proteins at the G0/G1 phase, such as cyclin D1, CDK4/CDK6. In addition, miR-34a may also inhibit RMC proliferation by directly targeting cyclin E and CDK2. MiR-34a inhibits exogenous stimuli-induced proliferation of mesangial cells. Expression levels of phospho-PDGFR-ß and phospho-MEK1 (an important downstream molecule in PDGFR-ß-induced signaling pathway) were significantly increased in the anti-Thy-1 nephritis rat model. These results suggest that miR-34a may regulate RMC proliferation by directly inhibiting expressions of PDGFR-ß, MEK1, and cell cycle proteins, cyclin E and CDK2.

Effects of compound Shenhua tablet on renal tubular Na+-K+-ATPase in rats with acute ischemic reperfusion injury.

OBJECTIVE: To observe the effect of Compound Shenhua Tablet (, SHT) on the sodium-potassium- exchanging adenosinetriphosphatase (Na(+)-K(+)-ATPase) in the renal tubular epithelial cells of rats with acute ischemic reperfusion and to investigate the mechanisms underlying the effects of SHT on renal ischemic reperfusion injury (RIRI). METHODS: Fifty male Wistar rats were randomly divided into the sham surgery group, model group, astragaloside group [150 mg/(kg·d)], SHT low-dose group [1.5 g/(kg·d)] and SHT high-dose group [3.0 g/(kg·d)], with 10 rats in each group. After 1 week of continuous intragastric drug administration, surgery was performed to establish the model. At either 24 or 72 h after the surgery, 5 rats in each group were sacrificed, blood biochemistry, renal pathology, immunoblot and immunohistochemical examinations were performed, and double immunofluorescence staining was observed under a laser confocal microscope. RESULTS: Compared with the sham surgery group, the serum creatinine (SCr) and blood urea nitrogen (BUN) levels were significantly increased, Na(+)-K(+)-ATPase protein level was decreased, and kidney injury molecule-1 (KIM-1) protein level was increased in the model group after the surgery (P<0.01 or P<0.05). Compared with the model group, the SCr, BUN, pathological scores, Na(+)-K(+)-ATPase, and the KIM-1 protein level of the three treatment groups were significantly improved at 72 h after the surgery (P<0.05 or P<0.01). And the SCr, BUN of the SHT low- and high-dose groups, and the pathological scores of the SHT high-dose group were significantly lower than those of the astragaloside group (P<0.05). The localizations of Na(+)-K(+)-ATPase and megalin of the model group were disrupted, with the distribution areas overlapping with each other and alternately arranged. The severity of the disruption was slightly milder in three treatment groups compared with that of the model group. The results of immunofluorescence staining showed that the SHT high-dose group had a superior effect as compared with the astragaloside group and the SHT low-dose group. CONCLUSIONS: The SHT effectively alleviated RIRI caused by ischemic reperfusion, promoted the recovery of the polarity of renal tubular epithelial cells, and protected the renal tubules. The therapeutic effects of SHT were superior to those of astragaloside as a single agent.

Short-term calorie restriction protects against renal senescence of aged rats by increasing autophagic activity and reducing oxidative damage.

To explore the effect of short-term calorie restriction (CR) on renal aging, 8-week CR with 60% of the food intake of the ad libitum group was administered in 25-month-old male Sprague-Dawley rats. Aged rats subjected to short-term CR had lower body weight, level of triglycerides and ratio of urine protein to urine creatinine, respectively. Short-term CR blunted the increased glomerular volume, the degree of fibrosis, p16 and the positive rate of senescence-associated ß-galactosidase staining of the kidneys in old ad libitum group. Light chain 3/Atg8 as an autophagy marker exhibited a marked decline in aged kidneys, which was increased by short-term CR. The levels of p62/SQSTM1 and polyubiquitin aggregates, which were increased in older kidneys, were blunted by short-term CR. Short-term CR retarded the level of 8-hydroxydeoxyguanosine, a marker of mitochondrial DNA oxidative damage. Moreover, we found an increased level of SIRT1 and AMPK, and a decreased level of mTOR in aged kidneys after short-term CR. These results suggested that short-term CR could be considered as a potential intervention for retardation of renal senescence by increasing autophagy and subsequently reducing oxidative damage. Three master regulators of energy metabolism, SIRT1, AMPK and mTOR are associated with these effects.

Effects of Aging on Kidney Graft Function, Oxidative Stress and Gene Expression after Kidney Transplantation.

Conflicting results have been reported regarding the effects of donor age, recipient age and donor-recipient age difference on short- and long-term outcomes after kidney transplantation. The aim of this study was to evaluate the effects of recipient age on graft function, oxidative stress, and gene expression after renal transplantation. Fifty male Fischer 344 rats [25 young (Y, 4 months), 25 senior (S, 16 months)] were randomized to 6 groups: 2 sham groups (Y and S, n = 5 in each group) and 4 renal transplant groups[young-to-young (Y-Y), young-to-senior (Y-S), senior-to-young (S-Y), senior-to-senior (S-S), (n = 10 in each group)]. The left kidneys were transplanted from donor to recipient. After 12 weeks, systematic blood pressure, graft weight, graft function, histology and oxidative stress were measured. Microarray analysis and quantitative real-time PCR confirmation were performed to study gene expression in the grafts. There were no differences in renal graft function between young and senior kidney cross-transplantation. Transplanted kidneys showed no significant differences in glomerulosclerosis index compared to non-transplanted kidneys but had significantly different tubulointerstitium scores compared to age-matched controls. Senior rats had lower SOD activity and higher MDA content than young rats. SOD activity was significantly lower and MDA content significantly higher in the Y-S group than in the Y-Y group. There were 548 transcript differences between senior and young kidneys with 36 upregulated and 512 downregulated transcripts. There were 492 transcript differences between Y-S and Y-Y groups with 127 upregulated and 365 downregulated transcripts. There were 1244 transcript differences between the S-Y and S-S groups with 680 upregulated and 574 downregulated transcripts. Oxidative stress and gene expression profile was significantly different in the Y-S compared to the S-Y group. The identified differences were mainly in the MAPK and insulin signal pathways, making these potential targets for therapeutic intervention.

Mitochondrial autophagy involving renal injury and aging is modulated by caloric intake in aged rat kidneys.

BACKGROUND: A high-calorie (HC) diet induces renal injury and promotes aging, and calorie restriction (CR) may ameliorate these responses. However, the effects of long-term HC and CR on renal damage and aging have been not fully determined. Autophagy plays a crucial role in removing protein aggregates and damaged organelles to maintain intracellular homeostasis and function. The role of autophagy in HC-induced renal damage is unknown. METHODS: We evaluated the expression of LC3/Atg8 as a marker of the autophagosome; p62/SQSTM1; polyubiquitin aggregates as markers of autophagy flux; Ambra1, PINK1, Parkin and Bnip3 as markers of mitophagy; 8-hydroxydeoxyguanosine (8-OHdG) as a marker of DNA oxidative damage; and p16 as a marker of organ aging by western blot and immunohistochemical staining in the kidneys of 24-month-old Fischer 344 rats. We also observed mitochondrial structure and autolysosomes by transmission electron microscopy. RESULTS: Expression of the autophagosome formation marker LC3/Atg8 and markers of mitochondrial autophagy (mitophagy) were markedly decreased in the kidneys of the HC group, and markedly increased in CR kidneys. p62/SQSTM1 and polyubiquitin aggregates increased in HC kidneys, and decreased in CR kidneys. Transmission electron microscopy demonstrated that HC kidneys showed severe abnormal mitochondrial morphology with fewer autolysosomes, while CR kidneys exhibited normal mitochondrial morphology with numerous autolysosomes. The level of 8-hydroxydeoxyguanosine was increased in HC kidneys and decreased in CR kidneys. Markers of aging, such as p16 and senescence-associated-galactosidase, were increased significantly in the HC group and decreased significantly in the CR group. CONCLUSION: The study firstly suggests that HC diet inhibits renal autophagy and aggravates renal oxidative damage and aging, while CR enhances renal autophagy and ameliorates oxidative damage and aging in the kidneys.

[Effects of TIMP-1 on PTEN expression on renal tubular epithelial cells].

OBJECTIVE: To investigate the role of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in regulating both angiogenesis and the expressions of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and vascular endothelial growth factor (VEGF)/Flk-1 expression in human proximal tubular epithelial cells (HKC). METHODS: HKC cells were transfected with two recombinant plasmids containing sense and antisense full-length TIMP-1 cDNA (TIMP-1S-pcDNA3.0 and TIMP-1AS-pcDNA3.0, respectively) constructed previously, or treated with 100 µmol/L MMP-2/MMP-9 inhibitor III (with similar cellular enzyme suppression activity with sense TIMP-1 plasmid). The mRNA expression of TIMP-1, MMP-2, MMP-9, PTEN, VEGF and Flk-1 were examined by RT-PCR. In each group, the expression of PTEN, VEGF and Flk-1 were also detected using an indirect immunofluorescence assay. RESULTS: Compared with non-transfected cells and cells transfected with the empty vector, sense TIMP-1-transfected cells showed obviously upregulated PTEN expression (P<0.05) and significantly lowered gelatinase activity (P<0.05) and VEGF and Flk-1 expressions (P<0.05). Transfection with the antisense TIMP-1 plasmid produced the reverse results (P<0.05). MMP-2/MMP-9 inhibitor III did not obviously affected the expression of PTEN, VEGF or Flk-1 as compared with the non-transfected or empty vector-transfected cells. CONCLUSION: In the aging progress, the renal tissues express high levels of TIMP-1 to upregulate PTEN expression via a MMP-independent pathway, and subsequently down-regulates the expression of VEGF and Flk-1 to cause aging-related impairment of renal angiogenesis. These findings provide new evidence for understanding the role of TIMP-1 in renal aging.

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.

Immunohistochemistry of deparaffinised sections using antigen retrieval with microwave combined pressure cooking versus immunofluorescence in the assessment of human renal biopsies.

BACKGROUND: Immunofluorescence of frozen tissue sections (IF-F) is a traditional technique used in renal biopsy. However, IF-F has certain disadvantages, such as a few or even no glomeruli in the section, and limited long-term preservation of the fluorescently labelled samples. METHODS: We compared two-step immunohistochemistry (IHC) staining of deparaffinised sections for antigen retrieval with microwave combined high-pressure cooking to IF-F used to detect antigens of IgG, IgA, IgM, C3, C1q, κ and λ in patient renal biopsy samples. The number of glomeruli detected, sensitivity and specificity of positive staining, tissue structure, and location staining of the antigens were determined using the two methods in 285 patients diagnosed with different renal diseases. RESULTS: Concordant observations between IF-F and IHC were 99% for all antigen staining (1969 of 1995 observations) and 100% for IgG, IgA and IgM (all 285 observations). The number of glomeruli in IHC sections was significantly greater compared with IF-F sections (p<0.001). IHC provided clearer images of tissue structure, more precise localisation of positive-staining antigens, and IHC staining allowed simultaneous evaluation of tissue by light microscopy. Correlation between tissue structure and immune deposits are not readily attained by IF-F. CONCLUSIONS: IHC is superior to IF-F for immunopathological diagnosis of renal biopsy tissue and is a reliable replacement for the more traditional IF-F method.

Immunofluorescence with dual microwave retrieval of paraffin-embedded sections in the assessment of human renal biopsy specimens.

Immunofluorescence of frozen tissue sections (IF-F) is a classic technique for renal immunopathologic examination. However, it has certain disadvantages, such as diffuse antigen distribution and few or even no glomeruli in the section. We developed a new technique of immunofluorescence staining using dual microwave retrieval in paraffin-embedded renal tissue sections (IF-DMP) and compared IF-DMP with IF-F in 406 renal biopsy samples. IF-DMP detected significantly more glomeruli than did IF-F (P< .001). There was no significant difference for the specificity and sensitivity in the detection of immunoglobulins, complements, κ, and λ between IF-F and IF-DMP. Concordant observations were 98% for all immunofluorescence, complements, κ, and λ staining and 100% for immunoglobulin staining. Both techniques were completely accurate in confirming diagnoses of various glomerular diseases. IF-DMP provided clearer images of tissue structure and more precise localization of antigens, and it is a suitable alternative for traditional IF-F in clinical renal immunopathologic diagnosis.

Erythropoietin protects against cisplatin-induced nephrotoxicity by attenuating endoplasmic reticulum stress-induced apoptosis.

BACKGROUND: The anticancer drug cisplatin (CP)-induced nephrotoxicity associated with apoptosis plays crucial roles in tumor patients. Erythropoietin (EPO) has recently been shown to enhance recovery from CP-induced acute kidney injury (AKI) in rats by exerting anti-apoptotic effects. However, the molecular mechanisms of Erythropoietin protects against CP-induced AKI are not very clear. The present study investigated the protective effects of erythropoietin (EPO) against CP-induced nephrotoxicity and the possible mechanism in rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into four groups: (1) Control group (n=16): which received a single intraperitoneal injection of vehicle (0.9% saline; 5 mL/kg); (2) CP group (n=16): which received a single intraperitoneal injection of 10.0 mg/kg CP (previously dissolved at 2.0 mg/mL in 0.9% saline solution); (3) CP+rHuEPO group (n=16): which received rHuEPO (5000 U/kg) with co-injection of the LY294002 vehicle dimethyl sulfoxide (DMSO, 33.3 µL/kg; Sigma, St. Louis, MO, USA) by tail vein injection 2 days before CP administration, 15 min before CP administration, and 2 days after CP administration; (4) CP+rHuEPO+LY group (n=16): which received LY294002 (0.3 mg/kg) 10 min before rHuEPO administration by three injections into the tail vein at 2-day intervals beginning 2 days before CP administration. Apoptosis was assessed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. The expressions of C/EBP-homologous protein (CHOP), glucose-regulated protein 78 (GRP78), caspase-12, the phosphorylation of Akt, cleaved-caspase-3 and EPO receptor (EPOR) were measured after CP-treated. In addition, light microscopy and immunohistochemistry examinations were performed. RESULTS: The levels of serum urea and creatinine were increased at 96 h after CP-administered group. The eHuEPO-administered group had significantly lower serum creatinine levels. CP caused an increase in TUNEL-positive cells that was accompanied and apoptotic cell death induced by CP was significantly abrogated by rHuEPO at 96h by morphological evidence of apoptosis. The over-expression of CP-induced endoplasmic reticulum (ER) stress markers (CHOP and GRP78) and activation of caspase-12 were suppressed by rHuEPO, which also attenuated the CP-induced suppression of phosphatidylinositol-3kinase/Akt (PI3K/Akt) signaling in rat kidneys. In addition, LY294002 diminished the effect of rHuEPO on renal protection and antiapoptosis. CONCLUSIONS: Injection of rHuEPO enhance recovery from CP-induced AKI in rats by ameliorating renal functional impairment and exerting important anti-apoptotic effects. However, rHuEPO inhibited CP-induced AKI by a possible mechanism involving PI3K/Akt activation and the inhibition of ER stress-mediated apoptosis.

Impaired autophagic function in rat islets with aging.

Type 2 diabetes is characterized by a deficit in ß-cell function and mass, and its incidence increases with age. Autophagy is a highly regulated intracellular process for degrading cytoplasmic components, particularly protein aggregates and damaged organelles. Impaired or deficient autophagy is believed to cause or contribute to aging and age-related disease. Autophagy may be necessary to maintain structure, mass, and function of pancreatic ß-cells. In this study, we investigated the effects of age on ß-cell function and autophagy in pancreatic islets of 4-month-old (young), 14-month-old (adult), and 24-month-old (old) male Wistar rats. We found that islet ß-cell function decreased gradually with age. Protein expression of the autophagy markers LC3/Atg8 and Atg7 exhibited a marked decline in aged islets. The expression of Lamp-2, a good indicator of autophagic degradation rate, was significantly reduced in the islets of old rats, suggesting that autophagic degradation is decreased in the islets of aged rats. However, protein expression of beclin-1/Atg6, which plays an important role in the induction and formation of the pre-autophagosome structure by associating with a multimeric complex of autophagy regulatory proteins (Atg14, Vps34/class 3 PI3 kinase, and Vps15), was most prominent in the islets of adult rats, and was higher in 24-month-old islets than in 4-month-old islets. The levels of p62/SQSTM1 and polyubiquitin aggregates, representing the functions of autophagy and proteasomal degradation, were increased in aging islets. 8-Hydroxydeoxyguanosine, a marker of mitochondrial and nuclear DNA oxidative damage, exhibited strong immunostaining in old islets. Analysis by electron microscopy demonstrated swelling and disintegration of cristae in the mitochondria of aged islets. These results suggest that ß-cell and autophagic function in islets decline simultaneously with increasing age in Wistar rats, and that impaired autophagy in the islets of older rats may cause accumulation of misfolded and aggregated proteins and reduce the removal of abnormal mitochondria in ß-cells, leading to reduced ß-cell function. Dysfunctional autophagy in islets during the aging process may be an important mechanism leading to the development of type 2 diabetes.

Beneficial effects of short-term calorie restriction against cisplatin-induced acute renal injury in aged rats.

BACKGROUND: The therapeutic use of the antineoplastic drug cisplatin (DDP) in the elderly is limited by its nephrotoxic effects. The aim of this study was to examine the effect of short-term calorie restriction (CR) on DDP-induced nephrotoxicity in aged rats. METHODS: A group of 25-month-old male Sprague-Dawley rats were divided into two groups: ad libitum (AL) and CR, which were fed 60% of the food consumed by AL rats for 8 weeks. The two groups were each further randomly divided into two subgroups: OAL control, OAL+DDP, OCR control, and OCR+DDP. A single dose of DDP (6 mg/kg) was injected intraperitoneally. Functional and structural changes of the kidneys were evaluated quantitatively by biochemical, histopathological, and morphometric analyses. RESULTS: At the end of the 8 weeks, rats in the OCR group lost 14.8% more body mass than rats in the OAL group. Pretreatment with CR had several effects: (1) it reduced the levels of blood urea nitrogen and serum creatinine, (2) it reduced the magnitude of the renal tubular epithelial damage, and (3) it significantly reduced the incidence of activated caspase-3 and TUNEL-positive cells in kidneys injured by DDP. However, SIRT1 had the opposite trend after DDP application between the two groups. CONCLUSIONS: Short-term CR exhibits a renoprotective effect in experimental DDP-induced renal injury, the mechanism of which may involve CR antiapoptotic effects and promotion of SIRT1.

Effects of Fufang Shenhua Tablet on the expression of toll-like receptors during acute kidney injury induced by ischemia-reperfusion in rats.

OBJECTIVE: To investigate the impact of a traditional Chinese medicinal compound known as Fufang Shenhua Tablet (SHP) on the expression of Toll-like receptors (TLRs) during renal ischemia-reperfusion injury (IRI)-induced acute kidney injury (AKI) in rats. METHODS: A total of 28 Wistar rats were randomly divided into five groups: (1) pseudo-operation control group, (2) ischemia-reperfusion model group, (3) Astragaloside group, (4) high-dose SHP group, and (5) low-dose SHP group. There were four rats in the pseudo-operation group and six rats in each of the other groups. The accepted ischemia-reperfusion model was established after a 7-day gavage intervention, and pathological changes and renal function were observed, using an enzyme-linked immunosorbent assay (ELISA) to detect interleukin 8 (IL-8) and interferon gamma (IFN-γ) levels, as well as immunohistochemical staining to detect altered levels of TLR2 and TLR4 expression in renal tissue. RESULTS: After 24 h, renal pathological damage and the expression levels of serum creatinine (Scr), IL-8, IFN-γ, TLR2, and TLR4 were significantly higher in the model group as compared with the pseudo-operation group (P<0.05). In addition, at 24 h the above indicators decreased significantly in the Astragaloside group, high-dose SHP group and low-dose SHP group as compared with the ischemia-reperfusion model group (P< 0.05). TLR2 and TLR4 expression levels were significantly reduced in the SHP treatment and Astragaloside group as compared with the pseudo-operation group (P<0.05). Further, the high-dose SHP group showed significantly less renal damage score and decreased levels of TLR expression than those of low-dose SHP group and Astragaloside group (all P<0.05). CONCLUSION: SHP can alleviate the renal structural and functional damage caused by IRI-induced AKI in rats by reducing the damage of renal pathology, which may reduce inflammatory cytokine levels by downregulating the expression of TLRs in renal tissue in a dose-dependent manner.

Novel mechanism for mesenchymal stem cells in attenuating peritoneal adhesion: accumulating in the lung and secreting tumor necrosis factor α-stimulating gene-6.

INTRODUCTION: We previously found that mesenchymal stem cells (MSCs) injected intravenously could attenuate peritoneal adhesion by secreting tumor necrosis alpha-stimulating gene (TSG)-6, while MSCs injected intraperitoneally could not. However, the underlying mechanism remains unclear. This study was designed to investigate the means by which MSCs exert their effects. METHODS: Rat bone marrow-derived MSCs/red fluorescent protein (RFP) were injected either intraperitoneally or intravenously into Sprague-Dawley (SD) rats at different time points after peritoneal scraping. Peritoneal adhesions were evaluated macroscopically at day 14 after scraping. The distribution of MSCs injected intraperitoneally or intravenously was traced by two-photon fluorescence confocal imaging and immunofluorescence microscopy. The co-localization of MSCs and macrophages in the lung and the spleen, and the expression of TSG-6 in MSCs trapped in the lung or the spleen were evaluated by immunofluorescence microscopy. The concentration of TSG-6 in serum was evaluated by ELISA. After intravenous injection of TSG-6- small interfering (si) RNA-MSCs, the expression of TSG-6 in MSCs and the concentration of TSG-6 in serum were reevaluated, and peritoneal adhesions were evaluated macroscopically and histologically. RESULTS: MSCs injected intraperitoneally failed to reduce peritoneal adhesion, and MSCs injected intravenously markedly improved peritoneal adhesion. Two-photon fluorescence confocal imaging showed that MSCs injected intravenously accumulated mainly in the lung, where they remained for seven days, and immunofluorescence microscopy showed few MSCs phagocytosed by macrophages. In contrast, large numbers of MSCs accumulated in the spleen with obvious phagocytosis by macrophages even at 4 hours after intraperitoneal injection. Immunofluorescence microscopy showed that MSCs that accumulated in the lung after intravenous injection could express TSG-6 within 12 hours, but TSG-6-siRNA-MSCs or MSCs accumulated in the spleen after intraperitoneal injection did not. ELISA showed that the concentration of TSG-6 in serum was increased at 4 hours after intravenous injection of MSCs, while there was no increase after injection of TSG-6-siRNA-MSCs or after intraperitoneal injection of MSCs. Moreover, intravenous injection of TSG-6-siRNA-MSCs failed to attenuate peritoneal adhesion. CONCLUSIONS: Our findings suggest that intravenously injected MSCs accumulated in the lung and attenuated peritoneal adhesion by secreting TSG-6, but intraperitoneally injected MSCs were phagocytosed by macrophages in the spleen and failed to attenuate peritoneal adhesion.

Mesenchymal stem cells attenuate peritoneal injury through secretion of TSG-6.

BACKGROUND: Mesothelial cell injury plays an important role in peritoneal fibrosis. Present clinical therapies aimed at alleviating peritoneal fibrosis have been largely inadequate. Mesenchymal stem cells (MSCs) are efficient for repairing injuries and reducing fibrosis. This study was designed to investigate the effects of MSCs on injured mesothelial cells and peritoneal fibrosis. METHODOLOGY/PRINCIPAL FINDINGS: Rat bone marrow-derived MSCs (5 × 10(6)) were injected into Sprague-Dawley (SD) rats via tail vein 24 h after peritoneal scraping. Distinct reductions in adhesion formation; infiltration of neutrophils, macrophage cells; number of fibroblasts; and level of transforming growth factor (TGF)-ß1 were found in MSCs-treated rats. The proliferation and repair of peritoneal mesothelial cells in MSCs-treated rats were stimulated. Mechanically injured mesothelial cells co-cultured with MSCs in transwells showed distinct increases in migration and proliferation. In vivo imaging showed that MSCs injected intravenously mainly accumulated in the lungs which persisted for at least seven days. No apparent MSCs were observed in the injured peritoneum even when MSCs were injected intraperitoneally. The injection of serum-starved MSCs-conditioned medium (CM) intravenously reduced adhesions similar to MSCs. Antibody based protein array of MSCs-CM showed that the releasing of TNFα-stimulating gene (TSG)-6 increased most dramatically. Promotion of mesothelial cell repair and reduction of peritoneal adhesion were produced by the administration of recombinant mouse (rm) TSG-6, and were weakened by TSG-6-RNA interfering. CONCLUSIONS/SIGNIFICANCE: Collectively, these results indicate that MSCs may attenuate peritoneal injury by repairing mesothelial cells, reducing inflammation and fibrosis. Rather than the engraftment, the secretion of TSG-6 by MSCs makes a major contribution to the therapeutic benefits of MSCs.

Kruppel-like factor-15 inhibits the proliferation of mesangial cells.

BACKGROUND/AIMS: The Kruppel-like factor-15 (KLF15), a DNA-binding transcription factor, is highly expressed in endothelial and mesangial cells of the kidney. However, its effects on mesangial cell proliferation have not previously been investigated. In this study, we investigated the effect of KLF15 on mesangial cell proliferation. METHODS: We established a classic rat anti-Thy1 mesangial proliferative nephritis model. Affymetrix rat U230 2.0 chip was used to detect the gene expression profiles at different time point in the model. The different expression of KLF15 was shown during mesangial cell proliferation period and proliferation declined period of anti-Thy1 nephritis model by microarray analysis, Real-time PCR and Western blotting. Then we determined the effects of KLF15 and its downstream target, cell cycle regulation factor E2F1 on the proliferation of mesangial cells and the expression of the positive-acting cell cycle regulatory proteins, cyclinD1 and CDK2, by means of positive and negative interference experiments in cultured rat mesangial cells. We detected also protein expression of E2F1, cyclinD1 and CDK2 in vivo. RESULTS: By real-time PCR, Western blotting, and microarray analysis, KLF15 expression was shown to be lower during mesangial cell proliferation period and higher during proliferation declined period and under normal conditions. The mesangial cell proliferation was reduced and the expression of E2F1, cyclin D1 and CDK2 was downregulated in mesangial cells overexpressing KLF15. When KLF15 expression was inhibited by siRNA, the expression of E2F1, cyclin D1 and CDK2 and mesangial cell proliferation were increased. When E2F1 was inhibited by siRNA, protein level of CDK2 and cyclin D1 were lower than control. When siE2F1 was co-transfected with siKLF15 into mesangial cells, the increase of cell proliferation induced by siKLF15 was eliminated partly by siE2F1. Moreover, E2F1, cyclin D1 and CDK2 were higher expression during mesangial cell proliferation period, and were downregulated during proliferation declined period in vivo. CONCLUSIONS: These results suggest that KLF15 inhibits mesangial cell proliferation, possibly by regulating the expression of cell cycle regulation proteins through E2F1. Thus, KLF15 may be a useful target for therapeutic intervention in mesangial proliferative glomerulonephritis.

Overexpression of Robo2 causes defects in the recruitment of metanephric mesenchymal cells and ureteric bud branching morphogenesis.

Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.

[Experimental study of immunological function regulated by Fufang Hongjingtian capsule in mice].

The aim of this study was to investigate the immunological function regulated by Fufang Hongjingtian capsule (HJT) in mice. The mice were given ig HJT 25, 250 and 750 mg/kg, once daily, for 30 - 38 d, respectively. The mice in control group were given ig corresponding solvent. After the last time of administration, the immunological parameters of the mice were measured. The results showed that compared with negative control group, the delayed type hypersensitivity, spleen lymphocyte proliferation and number of spleen IgM antibody forming cells increased in HJT groups. In conclusion the HJT has the effect to improve the immunological functions of mice.