Outcomes of primary membranous nephropathy based on serum anti-phospholipase A2 receptor antibodies and glomerular phospholipase A2 receptor antigen status: a retrospective cohort study.

INTRODUCTION: Primary membranous nephropathy (PMN) is associated with the anti-phospholipase A2 receptor (anti-PLA2R) antibody in 70% of cases. Some anti-PLA2R-negative patients have the PLA2R antigen in renal tissue. This study examined the prognosis of patients with PMN according to their serum anti-PLA2R antibody (SAb) and glomerular PLA2R antigen (GAg) status. METHODS: Patients diagnosed with PMN were included retrospectively. Patients were grouped according to their PLA2R status into the SAb-/GAg-, SAb-/GAg+, and SAb+/GAg + groups. Baseline data, renal biopsy results, treatment, and clinical data were compared among the groups. Cox univariable and multivariable analyses examined the factors related to complete remission (CR). RESULTS: A total of 114 patients were enrolled; 10 (9%) in the SAb-/GAg-, 23 (20%) in the SAb-/GAg+, and 81 (71%) in the SAb+/GAg+ groups. Cumulative CR rate showed a significant difference between the SAb-/GAg - and SAb+/GAg+ groups (log-rank p = 0.003). The multivariable Cox proportional hazard analysis showed that age (HR = 0.968; 95%CI = 0.946-0.990; p = 0.005), SAb+/GAg+ versus SAb-/GAg- (HR = 0.387; 95%CI = 0.190-0.788; p = 0.009), SAb-/GAg+ versus SAb-/GAg- (HR = 0.398; 95%CI = 0.169, 0.939; p = 0.035), total renal chronicity score ≥2 (HR = 0.461, 95%CI: 0.277-0.766, p = 0.003), and IgA deposition (HR = 2.596; 95%CI = 1.227-5.492; p = 0.013) were all independently related (p < 0.05) to CR. CONCLUSIONS: The SAb and GAg status was an indicator of PMN prognosis. The patients with SAb-/GAg - had an increased likelihood of achieving CR than those with SAb-/GAg+ and SAb+/GAg+.

Advanced glycation end-products suppress autophagic flux in podocytes by activating mammalian target of rapamycin and inhibiting nuclear translocation of transcription factor EB.

Insufficient autophagy in podocytes is related to podocyte injury in diabetic nephropathy (DN). Advanced glycation end-products (AGEs) are major factors of podocyte injury in DN. However, the role and mechanism of AGEs in autophagic dysfunction remain unknown. We investigated autophagic flux in AGE-stimulated cultured podocytes using multiple assays: western blotting, reverse transcription-quantitative PCR, immunofluorescence staining, and electron microscopy. We also utilized chloroquine and a fluorescent probe to monitor the formation and turnover of autophagosomes. Mice of the db/db strain were used to model diabetes mellitus (DM) with high levels of AGEs. To mimic DM with normal levels of AGEs as a control, we treated db/db mice with pyridoxamine to block AGE formation. AGEs impaired autophagic flux in the cultured podocytes. Compared with db/db mice with normal AGEs but high glucose levels, db/db mice with high AGEs and high glucose levels exhibited lower autophagic activity. Aberrant autophagic flux was related to hyperactive mammalian target of rapamycin (mTOR), a major suppressor of autophagy. Pharmacologic inhibition of mTOR activity restored impaired autophagy. AGEs inhibited the nuclear translocation and activity of the pro-autophagic transcription factor EB (TFEB) and thus suppressed transcription of its several autophagic target genes. Conversely, TFEB overexpression prevented AGE-induced autophagy insufficiency. Attenuating mTOR activity recovered TFEB nuclear translocation under AGE stimulation. Co-immunoprecipitation assays further demonstrated the interaction between mTOR and TFEB in AGE-stimulated podocytes and in glomeruli from db/db mice. In conclusion, AGEs play a crucial part in suppressing podocyte autophagy under DM conditions. AGEs inhibited the formation and turnover of autophagosomes in podocytes by activating mTOR and inhibiting the nuclear translocation of TFEB. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

DNA methyltransferase 1 may be a therapy target for attenuating diabetic nephropathy and podocyte injury.

The contribution of DNA methylation to diabetic nephropathy, especially the effect on podocyte integrity, is not clarified. Here we found that albuminuria in a db/db mouse model was markedly attenuated after treatment with a DNA methylation inhibitor. This was accompanied by alleviation of glomerular hypertrophy, mesangial matrix expansion, and podocyte injury. The expression of DNA methyltransferase 1 (Dnmt1), nuclear factor Sp1, and nuclear factor kappa B (NFκB)-p65 markedly increased in podocytes in vivo and in vitro under the diabetic state. The increased expression of Dnmt1 was attenuated after treatment with 5-azacytidine or 5-aza-2'-deoxycytidine or Dnmt1 knockdown, accompanied by restored decreased podocyte slit diaphragm proteins resulting from hypermethylation and improved podocyte motility. Further studies found that increased Sp1 and NFκB-p65 interacted in the nucleus of podocytes incubated with high glucose, and Sp1 bound to the Dnmt1 promoter region. The involvement of the Sp1/NFκB-p65 complex in Dnmt1 regulation was confirmed by the observation that Sp1 knockdown using mithramycin A or siRNA decreased Dnmt1 protein levels. The luciferase reporter assay further indicated that Dnmt1 was a direct target of Sp1. Thus, inhibition of DNA methylation may be a new therapeutic avenue for treating diabetic nephropathy. Hence, the Sp1/NFκB p65-Dnmt1 pathway may be exploited as a therapeutic target for protecting against podocyte injury in diabetic nephropathy.

Succinylation proteomic analysis identified differentially expressed succinylation sites affecting porcine muscle fiber type function.

Muscle fiber type is a major factor in pork meat quality, however, the role of post-translational protein modifications, especially succinylation, in the regulation of muscle fiber type is not fully understood. Here we performed protein succinylation profiles of fast-type biceps femoris (BF) and slow-type soleus (SOL) muscles. A total of 4,221 succinylation sites were identified from these samples, of which 294 sites were differentially expressed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that these succinylated proteins were mainly involved in glycolysis, tricarboxylic acid cycle, and fatty acid metabolism. Succinylation modification of the CRAT and RAB10 proteins was verified by co-immunoprecipitation. Protein-protein interaction (PPI) network analysis unveiled the interactions of these succinylated proteins that regulate pig myofiber type conversion. This investigation offers fresh perspectives into the molecular roles of protein succinylation in the regulation of pig myofiber type transformation and meat quality.

Comparative Proteomic Analysis of Glycolytic and Oxidative Muscle in Pigs.

The quality of meat is highly correlated with muscle fiber type. However, the mechanisms via which proteins regulate muscle fiber types in pigs are not entirely understood. In the current study, we have performed proteomic profiling of fast/glycolytic biceps femoris (BF) and slow/oxidative soleus (SOL) muscles and identified several candidate differential proteins among these. We performed proteomic analyses based on tandem mass tags (TMTs) and identified a total of 26,228 peptides corresponding to 2667 proteins among the BF and SOL muscle samples. Among these, we found 204 differentially expressed proteins (DEPs) between BF and SOL muscle, with 56 up-regulated and 148 down-regulated DEPs in SOL muscle samples. KEGG and GO enrichment analyses of the DEPs revealed that the DEPs are involved in some GO terms (e.g., actin cytoskeleton, myosin complex, and cytoskeletal parts) and signaling pathways (PI3K-Akt and NF-kappa B signaling pathways) that influence muscle fiber type. A regulatory network of protein-protein interaction (PPI) between these DEPs that regulates muscle fiber types was constructed, which demonstrates how three down-regulated DEPs, including PFKM, GAPDH, and PKM, interact with other proteins to potentially control the glycolytic process. This study offers a new understanding of the molecular mechanisms in glycolytic and oxidative muscles as well as a novel approach for enhancing meat quality by transforming the type of muscle fibers in pigs.

Phosphoproteomic analysis identifies differentially expressed phosphorylation sites that affect muscle fiber type in pigs.

Skeletal muscle of livestock is composed of both fast- and slow-twitch muscle fibers, which are key factors in their meat quality. However, the role of protein phosphorylation in muscle fiber type is not completely understood. Here, a fast-twitch (biceps femoris, BF) and slow-twitch (soleus, SOL) muscle tissue sample was collected from three male offspring of Duroc and Meishan pigs. We demonstrate that the meat quality of SOL muscle is significantly better than that of BF muscle. We further used phosphoproteomic profiling of BF and SOL muscles to identify differences between these muscle types. A total of 2,327 phosphorylation sites from 770 phosphoproteins were identified. Among these sites, 287 differentially expressed phosphorylation sites (DEPSs) were identified between BF and SOL. GO and KEGG enrichment analysis of proteins containing DEPSs showed that these phosphorylated proteins were enriched in the glycolytic process GO term and the AMPK signaling pathway. A protein-protein interaction (PPI) analysis reveals that these phosphorylated proteins interact with each other to regulate the transformation of muscle fiber type. These analyses reveal that protein phosphorylation modifications are involved in porcine skeletal muscle fiber type transformation. This study provides new insights into the molecular mechanisms by which protein phosphorylation regulates muscle fiber type transformation and meat quality in pigs.

Comparative analysis of differentially abundant proteins between high and low intramuscular fat content groups in donkeys.

Intramuscular fat (IMF) is an important regulator that determines meat quality, and its content is closely related to flavor, tenderness, and juiciness. Many studies have used quantitative proteomic analysis to identify proteins associated with meat quality traits in livestock, however, the potential candidate proteins that influence IMF in donkey muscle are not fully understood. In this study, we performed quantitative proteomic analysis, with tandem-mass-tagged (TMT) labeling, with samples from the longissimus dorsi (LD) muscle of the donkey. A total of 585,555 spectra were identified from the six muscle samples used in this study. In total, 20,583 peptides were detected, including 15,279 unique peptides, and 2,540 proteins were identified. We analyzed differentially abundant proteins (DAPs) between LD muscles of donkeys with high (H) and low (L) IMF content. We identified 30 DAPs between the H and L IMF content groups, of which 17 were upregulated and 13 downregulated in the H IMF group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis of these DAPs revealed many GO terms (e.g., bone morphogenetic protein (BMP) receptor binding) and pathways (e.g., Wnt signaling pathway and Hippo signaling pathway) involved in lipid metabolism and adipogenesis. The construction of protein-protein interaction networks identified 16 DAPs involved in these networks. Our data provide a basis for future investigations into candidate proteins involved in IMF deposition and potential new approaches to improve meat quality in the donkey.

Circular RNA Profiling Identifies Novel circPPARA that Promotes Intramuscular Fat Deposition in Pigs.

Intramuscular fat (IMF) content plays an important role in pork quality. Circular RNAs (circRNAs) implicate various biological processes; however, the regulatory mechanisms and functions of circRNAs in porcine IMF remains elusive. Hence, the study assessed the circRNA expression profiling in the longissimus dorsi muscle of pigs with high (H) and low (L) IMF content to unravel their regulatory functions in improving meat quality. The RNA sequencing analysis identified 29,732 circRNAs from six sampled pigs, most of which were exon-derived. In the muscle, 336 were differentially expressed (DE) between the H and L IMF groups; 196 circRNAs were upregulated, and 140 were downregulated. Subsequent qRT-PCR validation of 10 DE circRNAs revealed expression patterns consistent with the RNA-seq data. Gene ontology and KEGG enrichment analysis revealed that most significantly enriched DE circRNAs' host genes were linked to lipid metabolism and adipogenesis processes. The circRNA-miRNA regulatory network analysis found several circRNAs targeting miRNAs associated with adipogenesis. Finally, a novel circRNA, circPPARA, was identified with the expression positively correlated with the IMF content. Detailed analysis revealed that circPPARA was formed via head-to-tail splicing and was more stable than the linear PPARA, predominantly located in the cytoplasm. Functional studies using overexpression and siRNA constructs demonstrated that circPPARA promotes differentiation and hinders the proliferation of porcine intramuscular preadipocytes. Moreover, the dual-luciferase assay revealed that circPPARA adsorbed miR-429 and miR-200b, thereby promoting intramuscular adipogenesis in pigs. Our results identified a candidate circRNA, circPPARA, that affects porcine IMF content. The study provides knowledge of the regulatory functions of circRNAs in intramuscular adipogenesis and abundant resource for future research on circRNAs in pigs.

Lipopolysaccharide-induced podocyte injury is mediated by suppression of autophagy.

High-level autophagy has an important role in maintaining the stable state of podocytes. The present study explored the influence of lipopolysaccharide (LPS) on autophagic activity in podocytes and demonstrated its mechanistic involvement in LPS-induced injury. Conditionally immortalized podocytes were cultured in vitro and were treated with chloroquine (CQ), LPS, LPS+rapamycin or LPS+3­methyladenine (3­MA). The autophagic vesicles and endoplasmic reticulum were observed using transmission electron microscopy. The tandem mRFP­GFP­LC3 adenovirus was used to detect autophagosomes and autolysosomes. The expression levels of light chain 3­II (LC3 II), beclin­1, P62, CCAAT­enhancer­binding protein homologous protein (CHOP) and podocin were determined by western blot analysis. Autophagic vesicles were detected in podocytes under basic conditions. CQ was found to increase the protein levels of LC3 II in a time­dependent manner (2, 4 or 6 h), confirming the high activity of autophagy in podocytes. Compared with the control group, LPS induced the expansion of the endoplasmic reticulum and high expression levels of CHOP, while decreasing the protein expression of podocin. Notably, podocytes treated with LPS showed decreases in LC3 II and beclin­1 levels and autophagosome/autolysosome numbers, which was accompanied by high P62 levels. Furthermore, the autophagy enhancer rapamycin reversed the downregulation of LC3 II and podocin, and the upregulation of CHOP induced by LPS, while the autophagy inhibitor 3­MA aggravated the effects of LPS. In conclusion, the present study demonstrated that LPS inhibited podocyte autophagy, which contributed to LPS-induced injury of podocytes.

Spironolactone inhibits podocyte motility via decreasing integrin ß1 and increasing integrin ß3 in podocytes under high-glucose conditions.

Integrin ß1 and ß3 expression by podocytes is required to maintain glomerular structural integrity. Previous studies have shown that aldosterone (ALD) is involved in glomerular podocyte injury, and mineralocorticoid receptor (MR) blocker spironolactone effectively reduces proteinuria in patients with diabetic nephropathy. The present study was designed to observe the effects of spironolactone on ß1 and ß3 integrin expression and podocyte motility under in vitro diabetic conditions. Immortalized mouse podocytes were cultured in media containing normal glucose (NG) levels, high glucose (HG) or HG plus spironolacton. The expression of ß1 and ß3 integrin in podocytes was detected by reverse transcription quantitative polymerase chain reaction, immunofluorescence and western blot analyses. The effects of spironolacton on podocyte motility was further evaluated using a wound healing assay. HG stimulation markedly decreased mRNA and protein expression of integrin ß1, and significantly increased mRNA and protein expression of integrin ß3 in cultured podocytes. However, simultaneous treatment with spironolacton (10­7 mol/l) significantly attenuated HG-mediated increases in integrin ß3 and decreases in integrin ß1 expression. Furthermore, the migration of podocytes induced by HG was abrogated by concomitant treatment with spironolacton. In conclusion, the present study suggested that HG decreased the expression of integrin ß1 in cultured podocytes, accompanied with an increase of integrin ß3. Spironolactone inhibited cell motility and stabilized podoctyes treated with HG, probably through partly normalizing the expression of integrin ß1 and decreasing the expression of integrin ß3.

NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice.

BACKGROUND AND PURPOSE: Podocyte injury plays a key role in the development of diabetic nephropathy (DN). We have recently shown that 11R-VIVIT, an inhibitor of cell-permeable nuclear factor of activated T-cells (NFAT), attenuates podocyte apoptosis induced by high glucose in vitro. However, it is not known whether 11R-VIVIT has a protective effect on DN, especially podocyte injury, under in vivo diabetic conditions. Hence, we examined the renoprotective effects of 11R-VIVIT in diabetic db/db mice and the possible mechanisms underlying its protective effects on podocyte injury in vivo and in vitro. EXPERIMENTAL APPROACH: Type 2 diabetic db/db mice received i.p. injections of 11R-VIVIT (1 mg·kg(-1)) three times a week and were killed after 8 weeks. Immortalized mouse podocytes were cultured under different experimental conditions. KEY RESULTS: 11R-VIVIT treatment markedly attenuated the albuminuria in diabetic db/db mice and also alleviated mesangial matrix expansion and podocyte injury. However, body weight, food and water intake, and glucose levels were unaffected. It also attenuated the increased NFAT2 activation and enhanced urokinase-type plasminogen activator receptor (uPA receptor) expression in glomerulor podocytes. In cultured podocytes, the increased nuclear accumulation of NFAT2 and uPA receptor expression induced by high glucose treatment was prevented by 11R-VIVIT or NFAT2-knockdown; this was accompanied by improvements in the filtration barrier function of the podocyte monolayer. CONCLUSIONS AND IMPLICATIONS: The NFAT inhibitor 11R-VIVIT might be a useful therapeutic strategy for protecting podocytes and treating DN. The calcinerin/NFAT2/uPA receptor signalling pathway should be exploited as a therapeutic target for protecting podocytes from injury in DN.

Gene expression and antitumor effect following im electroporation delivery of human interferon alpha 2 gene.

AIM: To investigate the gene expression and antitumor effect following im electroporation delivery of human interferon alpha 2 (hIFN-alpha 2) gene. METHODS: The pcD2/hIFN-alpha 2 was injected into the middle of the quadriceps muscle of female BALB/c mice or the leukemia-bearing female BALB/c nude mice, and then electroporation was given to the injection site. Optimal electrical parameters and the efficiency of gene transfer was studied with hIFN-alpha 2 ELISA kit. The HL-60 tumor model in BALB/c nude mice was used to investigate therapeutic effects of im electroporation delivery of pcD2/hIFN-alpha 2. RESULTS: The optimal conditions for the electric pulses were as follows: voltage at 200 V/cm; pulse duration at 40 ms per pulse; number of pulse at 6 pulses and frequency at 1 Hz. Under optimal conditions, the serum hIFN-alpha 2 levels in electroporation group (160 microg/L+/-31 microg/L) were 45-fold higher than those of nonelectroporation group (3.6 microg/L+/-1.6 microg/L, P<0.01). The growth of leukemia was inhibited more obviously and the survival time of the leukemia-bearing nude mice was prolonged after im electroporation delivery of pcD2/hIFN-alpha 2 100 microg or 200 microg. CONCLUSION: Electroporation was an efficient method for the delivery of plasmid DNA and im electroporation delivery of pcD2/hIFN-alpha 2 was effective in treating leukemia.