Single-Cell Sequencing Informs That Mesenchymal Stem Cell Alleviates Renal Injury Through Regulating Kidney Regional Immunity in Lupus Nephritis.

Lupus nephritis (LN) is the common complication of systemic lupus erythematosus. The pathogenesis of LN kidney injury is unclear. In addition to systemic (extrarenal) immune cells, local (intrarenal) immune cells residing in "kidney regional immunity" are momentous in LN. Mesenchymal stem cell (MSC) therapy is effective for LN. However, mechanisms of MSC therapy remains unclear. In this study, we first systematically investigated the effects of MSC on immune cells in kidney regional immunity in LN using single-cell sequencing. We found that MSC reduced proinflammatory central memory CD4+ T cells, cytotoxic tissue-resident memory CD8+ T cells and exhausted CD8+ T cells, increased anti-inflammatory Naive/Effector CD8+ T cells and type 1 regulatory T cells; reduced infiltrating proinflammatory Ly6c hi/inter/lo era2+ macrophages, increased anti-inflammatory resident macrophage and Ly6c lo ear2- macrophage; and reduced long-lived plasma cells and proinflammatory neutrophils and dendritic cells. This study laid a foundation for clinical applications of MSC.

Transient upregulation of EGR1 signaling enhances kidney repair by activating SOX9+ renal tubular cells.

Background: Acute kidney injury (AKI) is associated with damage to the nephrons and tubular epithelial cells (TECs), which can lead to chronic kidney disease and end-stage renal disease. Identifying new biomarkers before kidney dysfunction will offer crucial insight into preventive and therapeutic options for the treatment of AKI. Early growth response 1 (EGR1) has been found to be a pioneer transcription factor that can sequentially turn on/off key downstream genes to regulate whole-body regeneration processes in the leopard worm. Whether EGR1 modulates renal regeneration processes in AKI remains to be elucidated. Methods: AKI models of ischemia-reperfusion injury (IRI) and folic acid (FA) were developed to investigate the roles of EGR1 in kidney injury and regeneration. To further determine the function of EGR1, Egr1-/- mice were applied. Furthermore, RNA sequencing of renal TECs, Chromatin Immunoprecipitation (ChIP) assay, and Dual-luciferase reporter assay were carried out to investigate whether EGR1 affects the expression of SOX9. Results: EGR1 is highly expressed in the kidney after AKI both in humans and mice through analysis of the Gene Expression Omnibus (GEO) database. Furthermore, we verified that EGR1 rapidly up-regulates in the very early stage of IRI and nephrotoxic models of AKI, and validation studies confirmed the essential roles of EGR1 in renal tubular cell regeneration. Further experiments affirmed that genetic inhibition of Egr1 aggravates the severity of AKI in mouse models. Furthermore, our results revealed that EGR1 could increase SOX9 expression in renal TECs by directly binding to the promoter of the Sox9 gene, thus promoting SOX9+ cell proliferation by activating the Wnt/ß-catenin pathway. Conclusions: Together, our results demonstrated that rapid and transient induction of EGR1 plays a renoprotective role in AKI, which highlights the prospects of using EGR1 as a potential therapeutic target for the treatment of AKI.

Establishment of the induced pluripotent stem cell line PLAFMCi006-A from peripheral blood mononuclear cells of polycystic kidney disease patients with PKD2 gene mutation.

Polycystic kidney disease (PKD) caused by PKD2 mutation is an important type of autosomal dominant PKD. In this study, peripheral blood mononuclear cells from a patient with PKD2 polycystic kidney disease were reprogrammed to obtain induced pluripotent stem cells (iPSCs). After stable amplification, the pluripotency of the iPSCs was determined by identifying their cell-surface markers, their expression of pluripotency-related genes, and their ability to form teratomas with three germ layers in vivo. The establishment of the iPSC line could provide a basis for a kidney-like organ model of human PKD caused by PKD2 mutation for use in studying the pathogenesis of PKD along with relevant screening and testing intervention drugs.

Low-dose 2-deoxyglucose and metformin synergically inhibit proliferation of human polycystic kidney cells by modulating glucose metabolism.

Polycystic kidney disease (PKD) is a common hereditary kidney disease with abnormal proliferation and apoptosis of kidney cystic epithelial cells, eventually leading to chronic renal failure. Currently, there are no effective treatment methods. Similar to tumor cells, cystic epithelial cells have abnormal glycolysis and over-activation of proliferation signaling pathways. In the present study, for the first time, we investigated the effects of low-dose combinational use of 2-deoxyglucose (2-DG) and metformin (MET) on the proliferation and apoptosis in the human cystic kidney epithelial cells. Cystic epithelia cells were divided into control group, 2-DG group, MET group and 2-DG+MET group. Cell Proliferation, apoptosis and glucose metabolism were measured in each group. The results showed that low-dose combinational treatment of 2-DG and MET significantly inhibited the proliferation of renal cystic epithelial cells by suppressing the activities of PKA, mTOR and ERK signaling pathways and upregulating PI3K/Akt pathway. Combination of both drugs increased the apoptosis rates of cystic epithelial cells. Two drugs inhibited glucose metabolic phenotypes, glycolysis and oxidative phosphorylation, and significantly lowered the intracellular ATP level in cystic epithelial cells. 2-DG could also neutralize excessive production of lactate (lactic acidosis) caused by MET and both drugs had complementary effect for cystic epithelial cells. These results reveal that combinational use of low-dose 2-DG and MET can markedly inhibit proliferation via modulating glucose metabolic phenotypes in human polycystic kidney epithelial cells, low-dose combinational use of both drugs can also lower the toxic effects of each drug, and is a novel strategy for future treatment of human polycystic kidney disease.

N-Acetylcysteine Ameliorates Gentamicin-Induced Nephrotoxicity by Enhancing Autophagy and Reducing Oxidative Damage in Miniature Pigs.

The clinical use of gentamicin over prolonged periods is limited because of dose and time-dependent nephrotoxicity, in which intracellular oxidative stress and heightened inflammation have been implicated. Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. The aim of this study was to determine changes in autophagy in response to the antioxidant N-acetylcysteine (NAC), via its effects on oxidative stress, inflammation, apoptosis, and renal function, following treatment with gentamicin in mini pigs. Adult mini pigs were divided into isotonic saline solution, gentamicin, and gentamicin plus NAC combination treatment groups. Gentamicin-induced histopathological changes, including inflammatory cell infiltration and tubular necrosis, were attenuated by NAC. NAC ameliorated the gentamicin-induced decreases in the levels of autophagy-related proteins, such as LC3 (microtubule-associated protein 1 light chain 3), PINK1 (phosphatase and tensin homologue deleted on chromosome10-induced kinase 1), phospho-parkin, AMBRA1 (activatingmolecule in Beclin 1-regulated autophagy), p62/SQSTM1 (sequestosome protein 1), and polyubiquitinated protein aggregates. NAC also caused a significant reduction in oxidative damage markers, including 4-hydroxy-2-nonenal, protein carbonyls, γ-H2AX (gamma histone variant H2AX), and 8-hydroxy-2'-deoxyguanosine, in gentamicin-treated animals. These data show that the protective effects of NAC might be related, at least in part, to a reduced inflammatory response, as observed in animals treated with both gentamicin and NAC. These results suggest that autophagy could be a new therapeutic target for preventing gentamicin-induced kidney injury, and that NAC might ameliorate gentamicin-induced nephrotoxicity by autophagy.

The combination of metformin and 2-deoxyglucose significantly inhibits cyst formation in miniature pigs with polycystic kidney disease.

BACKGROUND AND PURPOSE: The pathogenic mechanism of autosomal dominant polycystic kidney disease (ADPKD) is unclear. Similar to tumour cells, polycystic kidney cells are primarily dependent on aerobic glycolysis for ATP production. Compared with rodents, miniature pigs are more similar to humans. This study is the first time to investigate the effects of the combination of metformin and 2-deoxyglucose (2DG) in a pig model of chronic progressive ADPKD. EXPERIMENTAL APPROACH: A miniature pig ADPKD model was established by inducible deletion of the PKD1 gene. Blood, urine and kidney biopsy specimens were collected for analysis at specific times. The renal vesicle index was analysed by three-dimensional reconstruction of CT scans. Markers of the mammalian target of rapamycin (mTOR) and ERK signalling pathways and associated metabolism were detected by Western blots and colorimetry. KEY RESULTS: The three-dimensional reconstruction of CT scans indicated a markedly lower renal vesicle index in the combination therapy group. Each drug intervention group showed a significantly lower serum creatinine and urinary protein/creatinine ratio. This treatment regimen also inhibited the activities of markers of the proliferation-related mTOR and ERK pathways, and the expression of key enzymes involved in glycolysis, as well as reducing the production of ATP and lactic acid. CONCLUSIONS AND IMPLICATIONS: This study showed that the combination of metformin and 2DG blocked the formation of renal cysts and improved the renal function in ADPKD miniature pigs. Our results indicate that the combination of metformin and 2DG may be a promising therapeutic strategy in human ADPKD.

Modulation of Macrophage Polarization by Human Glomerular Mesangial Cells in Response to the Stimuli in Renal Microenvironment.

Mesangial cell (MC) activation and macrophage infiltration are 2 major events closely related with each other in mesangial proliferative glomerulonephritis. In the anti-Thy 1 nephritis model, macrophages mediate the damage and also the expansion of mesangium through secreting various inflammatory factors; however, in glomerular microenvironment how MCs affect macrophage activity in the presence of various stimuli have not yet been understood. In the present study, we found that resting human MCs (HMCs) constitutively expressed chemokine [C-C motif] ligand 2 (CCL-2) and interleukin (IL)-6 and induced M2 polarization of macrophages in the coculture system. HMC proliferation and migration and expression of IL-6, CCL-2, and macrophage colony-stimulating factor in HMCs were enhanced after platelet-derived growth factor (PDGF)-BB stimulation, among which CCL-2 was responsible for inducing the M2 polarization of macrophages. Furthermore, PDGF-BB-stimulated HMCs alleviated the classical activation of macrophages and drove more intensified M2 polarization of macrophages than resting HMCs did. However, lipopolysaccharide and interferon-γ (IFN-γ) stimulated HMCs maintained the M1 phenotype of cocultured macrophages. In conclusion, MCs actively participated in glomerular inflammation through influencing macrophage polarization. The interplay between MCs and infiltrated macrophages is finely modulated by secretory factors such as PDGF-BB and IFN-γ in response to the renal inflammatory microenvironment.

Urinary metabolites of organophosphate flame retardants in China: Health risk from tris(2-chloroethyl) phosphate (TCEP) exposure.

Organophosphate esters (OPs) are substitutes for polybrominated diphenyl ether (PBDE) flame retardants. China is the largest producer of OPs globally, with the production rate increasing at 15% annually. Since some OPs are neurodevelopmental and/or carcinogenic toxicants, human exposure is a concern. In this study, concentrations of eight OP metabolites (mOPs) were measured in human urine samples collected from 13 cities located in Northern, Eastern, Southern, and Southwestern China. All target mOPs were frequently detected with detection rates of 50% to 100%, indicating widespread human exposure to OPs. Bis(2-chloroethyl) phosphate (BCEP; median: 0.68 ng/mL), bis(1-chloro-2-propyl) phosphate (BCIPP; 0.30 ng/mL), diphenyl phosphate (DPHP; 0.30 ng/mL), and dibutyl phosphate (DBP; 0.29 ng/mL) were the dominant mOPs across all participants. Regional differences in concentrations (ΣmOPs varied from 0.86 to 3.7 ng/mL) and composition profiles (contribution of chlorinated mOPs to ΣmOPs varied from 35% to 95%) of mOPs were observed within China. In comparison to the concentrations reported worldwide, urinary DPHP and bis(1,3-dichloro-2-propyl) phosphate (BDCIPP) levels in China were lower, whereas BCEP and DBP levels were comparable or higher. The total daily intake (TDI) of tris(2-chloroethyl) phosphate (TCEP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and triphenyl phosphate (TPHP) were estimated from daily urine excretion rate and the fraction of OP metabolized in human liver microsomes (TDIHLM) or S9 fraction (TDIS9). The intake estimates showed that Chinese residents were exposed to TCEP from 96.9 to 46,700 (or 52.2 to 25,200) ng/kg bw/day. Depending on the reference dose, we found that approximately 5% of the individuals exceeded the limit (i.e., 2200 ng/kg bw/day) for TCEP intake. To our knowledge, this is the first nationwide baseline survey to determine urinary levels of mOPs in Chinese residents.

The changes in glucose metabolism and cell proliferation in the kidneys of polycystic kidney disease mini-pig models.

The pathogenic mechanism of polycystic kidney disease (PKD) is unclear. Abnormal glucose metabolism is maybe involved in hyper-proliferation of renal cyst epithelial cells. Mini-pigs are more similar to humans than rodents and therefore, are an ideal large animal model. In this study, for the first time, we systematically investigated the changes in glucose metabolism and cell proliferation signaling pathways in the kidney tissues of chronic progressive PKD mini-pig models created by knock-outing PKD1gene. The results showed that in the kidneys of PKD mini-pigs, the glycolysis is increased and the expressions of key oxidative phosphorylation enzymes Complexes I and IV significantly decreased. The activities of mitochondrial respiration chain Complexes I and IV significantly decreased; the phosphorylation level of key metabolism-modulating molecule AMP-activated protein kinase (AMPK) significantly decreased; and the mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) signaling pathway are activated obviously. This study showed that in the kidneys of PKD mini-pigs, the level of glycolysis significantly increased, oxidative phosphorylation significantly decreased, and cell proliferation signaling pathways significantly activated, suggesting that metabolic changes in PKD may result in the occurrence and development of PKD through the activation of proliferation signaling pathways.

Characterization and immunostimulating effects on murine peritoneal macrophages of a novel protein isolated from Panax quinquefolius L.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax quinquefolius L. has been used as a proverbial tonic in oriental countries for hundreds of years. It is used as a traditional medicinal herb to nourish vitality. AIM OF THE STUDY: The purpose of our study was to inquiry the activation effects on murine peritoneal macrophages of a novel protein separated from the roots of Panax quinquefolius L. MATERIALS AND METHODS: In our work, a novel protein of the roots of American ginseng (AGNP) was separated and purified from the roots of Panax quinquefolius L. The characteristic was investigated with SDS-PAGE, high pressure gel filtration chromatography (HPGFC) and matrix-assisted laser desorption ionization/time-of-flight mass (MALDI-TOF-MS) spectrometry method. The method of neutral red was carried out to investigate the phagocytosis of peritoneal macrophages. And Griess method and colorimetry were executed to detect the level of nitric oxide and iNOS activity respectively. Tumor necrosis factor-α and interleukin-6 were analyzed by enzyme linked immunosorbent assay (ELISA). RESULTS: Our results demonstrated that the subunit molecular weight of AGNP determined by SDS-PAGE was 15kD and the content of proteins determined by Bradford assay was 2.31mg/mL. The molecular weight of the AGNP was15, 114Da both of electrophoresis and MS purity. And the result of HPGFC showed that the molecular weight of AGNP was 31,086Da, Immunological studied indicated that AGNP could conspicuously increase phagocytosis of macrophages, facilitate the nitric oxide production, Tumor necrosis factor-α and interleukin-6 production. What is more, AGNP dose-dependently stimulated NO formation through the up-regulation of iNOS activity. CONCLUSIONS: In conclusion, AGNP had good immunoregulatory effects supporting the traditional claims and may provide a valuable therapeutic strategy to promoting immune function and metabolism.

Hydrogen sulfide mediates the protection of dietary restriction against renal senescence in aged F344 rats.

Renal aging is always accompanied by increased oxidative stress. Hydrogen sulfide (H2S) can be up-regulated by 50% dietary restriction (DR) for 7-day and can block mitochondrial oxidative stress. H2S production exerts a critical role in yeast, worm, and fruit fly models of DR-mediated longevity. In this study, we found that renal aging could be attenuated by 30% DR for 6-month (DR-6M) and life-long (DR-LL), but not for 6-week (DR-6W). The expressions of cystathionine-γ-lyase (CGL) and cystathionine-ß- synthase (CBS) were improved by DR-6M and DR-LL. Endogenous H2S production shared the same trend with CBS and CGL, while glutathione (GSH) didn't. When comparing efficiencies of DR for different durations, more evident production of H2S was found in DR-6M and DR-LL than in DR-6W. Finally the level of oxidative stress was improved by DR-6M and DR-LL rather than by DR-6W. It concluded that aged rats had the ability to produce enough H2S on 30% DR interventions protecting against renal aging, and the effect of DR for long-term were more significant than that of DR for short-term.

The Expression Changes of Inflammasomes in the Aging Rat Kidneys.

The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammation is related to kidney aging. Inflammasomes are important components of innate immune system in the body. However, the function of inflammasomes and their underlying mechanisms in renal aging remain unclear. In this study, for the first time, we systematically investigated the role of the inflammasomes and the inflammatory responses activated by inflammasomes during kidney aging. We found that during kidney aging, the expression levels of the molecules associated with the activation of inflammasomes, including toll-like receptor-4 and interleukin-1 receptor (IL-1R), were significantly increased; their downstream signaling pathway molecule interleukin-1 receptor-associated kinase-4 (IRAK4) was markedly activated (Phospho-IRAK4 was obviously increased); the nuclear factor-κB (NF-κB) signaling pathway was activated (the activated NF-κB pathway molecules Phospho-IKKß, Phospho-IκBα, and Phospho-NF-κBp65 were significantly elevated); the levels of the inflammasome components NOD-like receptor P3 (NLRP3), NLRC4, and pro-caspase-1 were prominently upregulated; and the proinflammatory cytokines IL-1ß and IL-18 were notably increased in the kidneys of 24-month-old (elderly group) rats. These results showed that inflammasomes are markedly activated during the renal aging process and might induce inflamm-aging by promoting the maturation and secretion of the proinflammatory cytokines IL-1ß and IL-18.

Anti-Inflamm-Aging Effects of Long-Term Caloric Restriction via Overexpression of SIGIRR to Inhibit NF-κB Signaling Pathway.

BACKGROUND: Chronic inflammation is thought to be a determinant of the aging rate and longevity. Caloric restriction (CR) attenuates age-related increases in the systemic levels of several pro-inflammatory mediators, but the anti-inflammatory mechanisms of CR in the aging process remain unclear. METHODS: Fisher 344 rats in a CR group were fed an amount of food corresponding to 60% of that fed to an ad libitum-fed (AL) group for 8 months. Biochemical analyses and renal pathological grading were used to analyze physiological status. Important signaling molecules in the Toll-like receptor/nuclear factor kappa-light-chain-enhancer of activated B cells (TLR/NF-κB) pathway were also analyzed by western blotting, immunofluorescence and immunohistochemistry. RESULTS: 1) Compared with AL feeding, CR decreased aging-mediated increases in both biochemical marker levels and renal pathological grading. 2) Single immunoglobulin IL-1 (IL-1)-related receptor (SIGIRR) expression decreased with increasing age, but CR led to overexpression. 3) The expression of TLR4 was significantly higher in the CR group than in the AL group. 4) SIGIRR overexpression decreased the expression of the adaptor molecules myeloid differentiation factor 88 (MyD88), IL-1 receptor-associated kinase 4 (IRAK4) and tumor necrosis factor receptor-associated factor 6 (TRAF6). 5) The levels of the inflammatory markers phospho-IκBα and phospho-NF-κB p65 decreased in the CR group. CONCLUSIONS: The inflammatory response might be alleviated by SIGIRR via blockade of the TLR4/NF-κB signaling pathway. Therefore, CR can decrease inflammation via SIGIRR overexpression, and SIGIRR might be a new target to delay aging.

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.

Validation of a differential diagnostic model of diabetic nephropathy and non-diabetic renal diseases and the establishment of a new diagnostic model.

BACKGROUND: The aims of the present study were to validate the differential diagnostic model of diabetic nephropathy (DN) and non-diabetic renal diseases (NDRD) established in 2003 and to establish a new diagnostic model suitable for the current clinical characteristics of DN. METHODS: We examined 200 patients with Type 2 diabetes who underwent kidney biopsy from 2004 to 2012. The 2003 differential diagnostic model based on the data collected from 1993 to 2003 was evaluated by the diagnostic test and changes in the clinical differentiation parameters of DN and NDRD were analyzed. Logistic regression, receiver operating characteristics (ROC) curve, net reclassification improvement (NRI), and integrated discrimination improvement (IDI) analysis were applied. RESULTS: The 2003 diagnostic model showed an accuracy of 77.5%. A significantly elevated incidence of hematuria, longer history of diabetes, and reduced level of glycated hemoglobin (HbA1c) were observed in the DN group from 2004 to 2012 compared with DN group from 1993 to 2003. Histories of diabetes mellitus (Dm), systolic blood pressure (Bp), HbA1c (Gh), hematuria (Hu), diabetic retinopathy (Dr), and hemoglobin (Hb) are independently related to DN. Thus, a new diagnostic model was constructed as follows: PDN = exp (0.846 + 0.022 Dm + 0.033Bp + 2.050 Gh-2.664 Hu-0.078 Hb + 2.942Dr)/[1 + exp (0.846 + 0.022 Dm + 0.033 Bp + 2.050 Gh-2.664 Hu-0.078 Hb + 2.942 Dr)].Validation tests determined that the accuracy of the new model were 90.9%. CONCLUSIONS: Changes in people with DN, clinical characteristics have reduced the diagnostic efficacy of the 2003 diagnostic model. The newly established model can provide a better, more current differentiation between DN and NDRD.

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.

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.

Membrane topology structure of human high-affinity, sodium-dependent dicarboxylate transporter.

High-affinity, sodium-dependent dicarboxylate transporter (NaDC3) is responsible for transport of Krebs cycle intermediates and may involve in regulation of aging and life span. Hydropathy analysis predicts that NaDC3 contains 11 or 12 hydrophobic transmembrane (TM) domains. However, the actual membrane topological structure of NaDC3 remains unknown. In this study, confocal immunofluorescence microscopy and membrane biotinylation of epitope-tagged N and C termini of NaDC3 provide evidence of an extracellular C terminus and an intracellular N terminus, indicating an odd number of transmembrane regions. The position of hydrophilic loops within NaDC3 was identified with antibodies against the loops domains combined with cysteine accessibility methods. A confocal image of membrane localization and transport activity assay of the cysteine insertion mutants show behavior similar to that of wild-type NaDC3 in transfected HEK293 cells, suggesting that these mutants retain a native protein configuration. We find that NaDC3 contains 11 transmembrane helices. The loops 1, 3, 5, 7, and 9 face the extracellular side, and loops 2, 4, 6, and 10 face the cytoplasmic side. A re-entrant loop-like structure between TM8 and TM9 may protrude into the membrane. Our results support the topography of 11 transmembrane domains with an extracellular C terminus and an intracellular N terminus of NaDC3, and for the first time provide experimental evidence for a novel topological model for NaDC3.

[p16INK4a expression mediated by recombinant adenovirus can induce senescence of A549 cells].

OBJECTIVE: To construct E1-deletion and replication-defective human type 5 recombinant adenovirus vector and to study the effect of p16INK4a on proliferation and aging of A549 cells. METHODS: p16INK4a cDNA was cloned into pAdCMV to construct recombinant pAdCMV p16INK4a, which was co-transfected into 293 cell together with pJM17. The recombinant p16INK4a adenovirus (Ad-p16INK4a) was generated by homologous recombination and identified with duplex PCR. Lung cancer cell A549, which has a homozygous deletion of p16INK4a gene, was infected with the prepared Ad-p16INK4a virus. X-gal staining and TRAP-ELISA were used for detecting senescence-associated beta-galactosidase and telomerase activities in A549 cells. RESULTS: Immunohistochemical staining and Western blot indicated that p16INK4a gene was transferred into A549 cell with more than 95% efficiency by recombinant adenovirus and p16INK4a protein was expressed at a high level- p16INK4a could markedly inhibit growth of A549 cells, induced expression of senescence-associated beta-galactosidase and suppressed telomerase activity in A549 cells. CONCLUSION: Recombinant adenovirus vector could efficiently mediate transfer and expression of foreign genes in human cell and could be used for gene immunization and gene therapy; p16INK4a could inhibit A549 cell growth and induce its replicative senescence.