tRF3-IleAAT reduced extracellular matrix synthesis in diabetic kidney disease mice by targeting ZNF281 and inhibiting ferroptosis.

It is well established that the synthesis of extracellular matrix (ECM) in mesangial cells is a major determinant of diabetic kidney disease (DKD). Elucidating the major players in ECM synthesis may be helpful to provide promising candidates for protecting against DKD progression. tRF3-IleAAT is a tRNA-derived fragment (tRF) produced by nucleases at tRNA-specific sites, which is differentially expressed in the sera of patients with diabetes mellitus and DKD. In this study we investigated the potential roles of tRFs in DKD. Db/db mice at 12 weeks were adapted as a DKD model. The mice displayed marked renal dysfunction accompanied by significantly reduced expression of tRF3-IleAAT and increased ferroptosis and ECM synthesis in the kidney tissues. The reduced expression of tRF3-IleAAT was also observed in high glucose-treated mouse glomerular mesangial cells. We administered ferrostatin-1 (1 mg/kg, once every two days, i.p.) to the mice from the age of 12 weeks for 8 weeks, and found that inhibition of the onset of ferroptosis significantly improved renal function, attenuated renal fibrosis and reduced collagen deposition. Overexpression of tRF3-IleAAT by a single injection of AAV carrying tRF3-IleAAT via caudal vein significantly inhibited ferroptosis and ECM synthesis in DKD model mice. Furthermore, we found that the expression of zinc finger protein 281 (ZNF281), a downstream target gene of tRF3-IleAAT, was significantly elevated in DKD models but negatively regulated by tRF3-IleAAT. In high glucose-treated mesangial cells, knockdown of ZNF281 exerted an inhibitory effect on ferroptosis and ECM synthesis. We demonstrated the targeted binding of tRF3-IleAAT to the 3'UTR of ZNF281. In conclusion, tRF3-IleAAT inhibits ferroptosis by targeting ZNF281, resulting in the mitigation of ECM synthesis in DKD models, suggesting that tRF3-IleAAT may be an attractive therapeutic target for DKD.

MicroRNA-26a alleviates tubulointerstitial fibrosis in diabetic kidney disease by targeting PAR4.

Our previous study found that miR-26a alleviates aldosterone-induced tubulointerstitial fibrosis (TIF). However, the effect of miR-26a on TIF in diabetic kidney disease (DKD) remains unclear. This study clarifies the role and possible mechanism of exogenous miR-26a in controlling the progression of TIF in DKD models. Firstly, we showed that miR-26a was markedly decreased in type 2 diabetic db/db mice and mouse tubular epithelial cells (mTECs) treated with high glucose (HG, 30 mM) using RT-qPCR. We then used adeno-associated virus carrying miR-26a and adenovirus miR-26a to enhance the expression of miR-26a in vivo and in vitro. Overexpressing miR-26a alleviated the TIF in db/db mice and the extracellular matrix (ECM) deposition in HG-stimulated mTECs. These protective effects were caused by reducing expression of protease-activated receptor 4 (PAR4), which involved in multiple pro-fibrotic pathways. The rescue of PAR4 expression reversed the anti-fibrosis activity of miR-26a. We conclude that miR-26a alleviates TIF in DKD models by directly targeting PAR4, which may provide a novel molecular strategy for DKD therapy.

tRF-003634 alleviates adriamycin-induced podocyte injury by reducing the stability of TLR4 mRNA.

Podocyte injury plays a key role in the production of proteinuria and is closely related to the progression of chronic kidney disease (CKD). Alleviating podocyte injury is beneficial to prevent the occurrence and development of CKD. tRNA-derived RNA fragments (tRFs) are associated with podocytes injury processes such as protein binding, cell adhesion, synapses, the actin cytoskeleton. Our previous data showed that tRF-003634 tightly correlated with podocyte injury, while its effect remains unclear. This study aimed to investigate the role of tRF-003634 in podocyte injury and the potential mechanisms. The expression level of tRF-003634, nephrin, podocin and tRF-003634 targeted toll-like receptor 4 (TLR4) in podocytes and kidney tissues were examined by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. The biochemical indices were monitored and renal pathological changes were assessed by hematoxylin and eosin PAS staining. Furthermore, potential target genes of tRF-003634 were screened using high-throughput mRNA sequencing, and then confirmed by RNA pulse-chase analysis. The results showed that tRF-003634 was downregulated in adriamycin (Adr)-induced podocyte injury. Overexpression of tRF-003634 increased the expression of nephrin and podocin in vivo and in vitro and alleviated podocyte injury. Meanwhile, overexpression of tRF-003634 alleviated proteinuria and renal pathological damage. In addition, high-throughput sequencing after overexpression of tRF-003634 showed that TLR4 might be a downstream target gene. tRF-003634 can alleviate podocyte injury by reducing the stability of TLR4 mRNA, possibly by competing with TLR4 mRNA to bind to YTH domain-containing protein 1 (YTHDC1). In conclusion, tRF-003634 was underexpressed in Adr-induced podocyte injury, and its overexpression alleviated podocyte injury in vitro and in vivo by reducing the stability of TLR4 mRNA.

Effects of liposoluble components of highland barley spent grains on physiological indexes, intestinal microorganisms, and the liver transcriptome in mice fed a high-fat diet.

The purpose of this study was to investigate the effects of the active ingredients of barley lees on the physiological indexes, intestinal flora, and liver transcriptome of mice fed a high-fat diet. Twenty-four male C57BL/6J mice were randomly divided into 4 groups and fed the experimental diets for 5 weeks. The results showed that the fat-soluble components of distillers' grains significantly reduced body weight, abdominal fat, perirenal fat, blood glucose, low-density lipoprotein cholesterol, triglycerides, and total cholesterol in the high-fat diet-fed mice (p < .05), significantly decreased alanine aminotransferase and malondialdehyde levels, and significantly increased total superoxide dismutase, catalase, reduced glutathione and glutathione peroxidase levels (p < .05). At the phylum level, lipid-soluble components significantly increased the abundance of Bacteroidetes and decreased the Firmicutes/Bacteroidetes ratio. At the genus level, the relative abundances of Bacteroidetes and Clostridium were increased. Transcriptomic analysis showed that lipid-soluble components of spent grains reduced the mRNA expression of ANGPTL8, CD36, PLTP, and SOAT1 and increased the mRNA expression of CYP7A1 and ABCA1 in the cholesterol metabolism pathway, promoted the transport of cholesterol, and inhibited the absorption of cholesterol, which can decrease cholesterol levels by speeding up the conversion of cholesterol into bile acids.

Molecular Organization and Functional Analysis of a Novel Plasmid-Borne cps Gene Cluster from Lactiplantibacillus plantarum YC41.

Capsular polysaccharide (CPS) can tightly attach to bacterial surfaces and plays a critical role in protecting microorganisms from environmental stresses. However, the molecular and functional properties of some plasmid-borne cps gene clusters are poorly understood. In this study, comparative genomics of the draft genomes of 21 Lactiplantibacillus plantarum strains revealed that the specific gene cluster for CPS biosynthesis was observed only in the 8 strains with a ropy phenotype. Furthermore, the complete genomes showed that the specific gene cluster cpsYC41 was located on the novel plasmid pYC41 in L. plantarum YC41. In silico analysis confirmed that the cpsYC41 gene cluster contained the dTDP-rhamnose precursor biosynthesis operon, the repeating-unit biosynthesis operon, and the wzx gene. The insertional inactivation of the rmlA and cpsC genes abolished the ropy phenotype and reduced the CPS yields by 93.79% and 96.62%, respectively, in L. plantarum YC41 mutants. These results revealed that the cpsYC41 gene cluster was responsible for CPS biosynthesis. Moreover, the survival rates of the YC41-rmlA- and YC41-cpsC- mutants under acid, NaCl, and H2O2 stresses were decreased by 56.47 to 93.67% compared to that of the control strain. Furthermore, the specific cps gene cluster was also confirmed to play a vital role in CPS biosynthesis in L. plantarum MC2, PG1, and YD2. These findings enhance our understanding of the genetic organization and gene functions of plasmid-borne cps gene clusters in L. plantarum. IMPORTANCE Capsular polysaccharide is well known to protect bacteria against various environmental stresses. The gene cluster for CPS biosynthesis is typically organized in the chromosome in bacteria. It is worth noting that complete genome sequencing showed that a novel plasmid pYC41-borne cpsYC41 gene cluster was identified in L. plantarum YC41. The cpsYC41 gene cluster included the dTDP-rhamnose precursor biosynthesis operon, the repeating-unit biosynthesis operon, and the wzx gene, which was verified by the significantly decreased CPS yield and the absent ropy phenotype in the corresponding mutants. The cpsYC41 gene cluster plays an important role in bacterial survival under environmental stress, and the mutants had decreased fitness under stress conditions. The vital role of this specific cps gene cluster in CPS biosynthesis was also confirmed in other CPS-producing L. plantarum strains. These results advanced a better understanding of the molecular mechanisms of plasmid-borne cps gene clusters and the protective functionality of CPS.

Expression profiles of tRNA­derived fragments in high glucose­treated tubular epithelial cells.

Transfer RNA-derived fragments (tRFs), a novel class of small non-coding RNA produced by the cleavage of pre- and mature tRNAs, are involved in various diseases. Renal tubulointerstitial fibrosis is a common final pathway in diabetic nephropathy (DN) in which hyperglycemia-induced tubular extracellular matrix (ECM) accumulation serves a vital role. The present study aimed to detect and investigate the role of tRFs in the accumulation of tubular ECM. Differentially expressed tRFs were analysed with high-throughput sequencing in primary mouse tubular epithelial cells treated with high glucose (HG). The Gene Ontology (GO) was used to analyze the potential molecular functions of these differentially expressed tRFs, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the associated signaling pathways involved in these differentially expressed tRFs. tRF-1:30-Gln-CTG-4 was overexpressed using tRF-1:30-Gln-CTG-4 mimic, followed by HG treatment. A total of 554 distinct tRFs were detected and 64 differentially expressed tRFs (fold change >2; P<0.05) were identified in tubular epithelial cells following high glucose (HG) treatment, among which 27 were upregulated and 37 were downregulated. Ten selected tRFs with the greatest difference (fold change >2; P<0.05) were verified to be consistent with small RNA-sequencing data, of which tRF-1:30-Gln-CTG-4 showed the most pronounced difference in expression and was significantly decreased in response to HG. GO analysis indicated that the differentially expressed tRFs were associated with 'cellular process', 'biological regulation' and 'metabolic process'. An analysis of the KEGG database suggested that these differentially expressed tRFs were involved in 'autophagy' and signaling pathways for 'forkhead box O', 'the mammalian target of rapamycin' and 'mitogen-activated protein kinase'. Finally, the overexpression of tRF-1:30-Gln-CTG-4 ameliorated HG-induced ECM accumulation in tubular epithelial cells. Therefore, the present study demonstrated that there may be a significant association between tRFs and HG-induced ECM accumulation in tubular epithelial cells; these differentially expressed tRFs warrant further study to explore the pathogenesis of DN.

Rapamycin attenuated podocyte apoptosis via upregulation of nestin in Ang II-induced podocyte injury.

BACKGROUND: Angiotensin II (Ang II) contributes to the progression of glomerulosclerosis, mainly by inducing podocyte injury. Convincing evidence indicates that the mTOR inhibitor rapamycin could play a fundamental role in protection against podocyte injury. Nestin, a major cytoskeletal protein, is stably expressed in podocytes and correlates with podocyte damage. The purpose of this study was to investigate the effect of rapamycin on podocyte injury induced by Ang II and to clarify the role and mechanism of nestin in the protective effect of rapamycin of podocyte injury. METHODS AND RESULTS: We established an Ang II perfusion animal model, and the effects of rapamycin treatment on podocytes were investigated in vivo. In vitro, podocytes were stimulated with Ang II and rapamycin to observe podocyte injury, and nestin-siRNA was transfected to investigate the underlying mechanisms. We observed that Ang II induced podocyte injury both in vivo and in vitro, whereas rapamycin treatment relieved Ang II-induced podocyte injury. We further found that nestin co-localized with p-mTOR in glomeruli, and the protective effect of rapamycin was reduced by nestin-siRNA in podocytes. Moreover, co-IP indicated the interaction between nestin and p-mTOR, and nestin could affect podocyte injury via the mTOR/P70S6K signaling pathway. CONCLUSION: We demonstrated that rapamycin attenuated podocyte apoptosis via upregulation of nestin expression through the mTOR/P70S6K signaling pathway in an Ang II-induced podocyte injury.

High quality haplotype-resolved genome assemblies of Populus tomentosa Carr., a stabilized interspecific hybrid species widespread in Asia.

Populus has a wide ecogeographical range spanning the Northern Hemisphere, and interspecific hybrids are common. Populus tomentosa Carr. is widely distributed and cultivated in the eastern region of Asia, where it plays multiple important roles in forestry, agriculture, conservation, and urban horticulture. Reference genomes are available for several Populus species, however, our goals were to produce a very high quality de novo chromosome-level genome assembly in P. tomentosa genome that could serve as a reference for evolutionary and ecological studies of hybrid speciation throughout the genus. Here, combining long-read sequencing and Hi-C scaffolding, we present a high-quality, haplotype-resolved genome assembly. The genome size was 740.2 Mb, with a contig N50 size of 5.47 Mb and a scaffold N50 size of 46.68 Mb, consisting of 38 chromosomes, as expected with the known diploid chromosome number (2n = 2x = 38). A total of 59,124 protein-coding genes were identified. Phylogenomic analyses revealed that P. tomentosa is comprised of two distinct subgenomes, which we deomonstrate is likely to have resulted from hybridization between Populus adenopoda as the female parent and Populus alba var. pyramidalis as the male parent, with an origin of approximately 3.93 Ma. Although highly colinear, significant structural variation was found between the two subgenomes. Our study provides a valuable resource for ecological genetics and forest biotechnology.

Investigation of PtSGT1 and PtSGT4 Function in Cellulose Biosynthesis in Populus tomentosa Using CRISPR/Cas9 Technology.

Cellulose synthesis is a complex process in plant cells that is important for wood processing, pulping, and papermaking. Cellulose synthesis begins with the glycosylation of sitosterol by sitosterol glycosyltransferase (SGT) to produce sitosterol-glucoside (SG), which acts as the guiding primer for cellulose production. However, the biological functions of SGTs in Populus tomentosa(P. tomentosa) remain largely unknown. Two full-length PtSGT genes (PtSGT1 and PtSGT4) were previously isolated from P. tomentosa and characterized. In the present study, CRISPR/Cas9 gene-editing technology was used to construct PtSGT1-sgRNA and PtSGT4-sgRNA expression vectors, which were genetically transformed into P. tomentosa using the Agrobacterium-mediated method to obtain transgenic lines. Nucleic acid and amino acid sequencing analysis revealed both base insertions and deletions, in addition to reading frame shifts and early termination of translation in the transgenic lines. Sugar metabolism analysis indicated that sucrose and fructose were significantly downregulated in stems and leaves of mutant PtSGT1-1 and PtSGT4-1. Glucose levels did not change significantly in roots and stems of PtSGT1-1 mutants; however, glucose was significantly upregulated in stems and downregulated in leaves of the PtSGT4-1 mutants. Dissection of the plants revealed disordered and loosely arranged xylem cells in the PtSGT4-1 mutant, which were larger and thinner than those of the wild-type. This work will enhance our understanding of cellulose synthesis in the cell walls of woody plants.

miR-199b-5p mediates adriamycin-induced podocyte apoptosis by inhibiting the expression of RGS10.

Podocyte apoptosis is a key risk factor for the progression of kidney diseases. MicroRNA (miR)-199b-5p has been shown to be involved in cell apoptosis. However, the molecular mechanisms of miR-199b-5p in podocyte apoptosis remain uncertain. Thus, the present study aimed to investigate whether miR-199b-5p participates in the regulation of podocyte apoptosis and to elucidate the involved mechanisms of this process. A podocyte apoptosis model was constructed using adriamycin (ADR) in vitro. miR-199b-5p mimic and inhibitor were transfected in podocytes to change the expression level of miR-199b-5p. RNA expression was examined by reverse transcription-quantitative PCR. Western blotting was used to measure protein expression. Apoptosis was monitored via flow cytometry and detection of apoptosis-associated proteins. The results from the present study demonstrated that miR-199b-5p was upregulated and that regulator of G-protein signaling 10 (RGS10) was downregulated in ADR-stimulated podocytes. Overexpression of miR-199b-5p could inhibit RGS10 expression and stimulate podocyte apoptosis, whereas miR-199b-5p knockdown restored the levels of RGS10 and ameliorated podocyte apoptosis in ADR-induced podocytes. Furthermore, the effects of miR-199b-5p overexpression could be significantly reversed by RGS10 overexpression. In addition, podocyte transfection of miR-199b-5p activated the AKT/mechanistic target of rapamycin (mTOR) signaling, which was blocked following RGS10 overexpression. Taken together, the present study demonstrated that miR-199b-5p upregulation could promote podocyte apoptosis by inhibiting the expression of RGS10 through the activation of AKT/mTOR signaling.

Comparative transcriptomic analysis reveals the coordinated mechanisms of Populus × canadensis 'Neva' leaves in response to cadmium stress.

Cadmium (Cd), a heavy metal element has strong toxicity to living organisms. Excessive Cd accumulation directly affects the absorption of mineral elements, inhibits plant tissue development, and even induces mortality. Populus × canadensis 'Neva', the main afforestation variety planted widely in northern China, was a candidate variety for phytoremediation. However, the genes relieving Cd toxicity and increasing Cd tolerance of this species were still unclear. In this study, we employed transcriptome sequencing on two Cd-treated cuttings to identify the key genes involved in Cd stress responses of P. × canadensis 'Neva' induced by 0 (CK), 10 (C10), and 20 (C20) mg/L Cd(NO3)2 4H2O. We discovered a total of 2,656 (1,488 up-regulated and 1,168 down-regulated) and 2,816 DEGs (1,470 up-regulated and 1,346 down-regulated) differentially expressed genes (DEGs) between the CK vs C10 and CK vs C20, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses in response to the Cd stress indicated that many DEGs identified were involved in the catalytic activity, the oxidoreductase activity, the transferase activity, and the biosynthesis of secondary metabolites. Based on the enrichment results, potential candidate genes were identified related to the calcium ion signal transduction, transcription factors, the antioxidant defense system, and transporters and showed divergent expression patterns under the Cd stress. We also validated the reliability of transcriptome data with the real-time PCR. Our findings deeper the understanding of the molecular responsive mechanisms of P. × canadensis 'Neva' on Cd tolerance and further provide critical resources for phytoremediation applications.

tRNA-Derived Fragments in Podocytes with Adriamycin-Induced Injury Reveal the Potential Mechanism of Idiopathic Nephrotic Syndrome.

Idiopathic nephrotic syndrome (INS) is a disease involving injury to podocytes in the glomerular filtration barrier, and its specific causes have not been elucidated. Transfer RNA-derived fragments (tRFs), products of precise tRNA cleavage, have been indicated to play critical roles in various diseases. Currently, there is no relevant research on the role of tRFs in INS. This study intends to explore the changes in and importance of tRFs during podocyte injury in vitro and to further analyze the potential mechanism of INS. Differentially expressed tRFs in the adriamycin-treated group were identified by high-throughput sequencing and further verified by quantitative RT-PCR. In total, 203 tRFs with significant differential expression were identified, namely, 102 upregulated tRFs and 101 downregulated tRFs (q < 0.05, ∣log2FC | ≥2). In particular, AS-tDR-008924, AS-tDR-011690, tDR-003634, AS-tDR-013354, tDR-011031, AS-tDR-001008, and AS-tDR-007319 were predicted to be involved in podocyte injury by targeting the Gpr, Wnt, Rac1, and other genes. Furthermore, gene ontology analysis showed that these differential tRFs were strongly associated with podocyte injury processes such as protein binding, cell adhesion, synapses, the actin cytoskeleton, and insulin-activate receptor activity. KEGG pathway analysis predicted that they participated in the PI3K-Akt signaling pathway, Wnt signaling pathway, and Ras signaling pathway. It was reported that these pathways contribute to podocyte injury. In conclusion, our study revealed that changes in the expression levels of tRFs might be involved in INS. Seven of the differentially expressed tRFs might play important roles in the process of podocyte injury and are worthy of further study.

tRNA-derived fragments (tRFs) contribute to podocyte differentiation.

Loss of glomerular podocytes is the crucial event in the progression of chronic kidney disease (CKD). tRNA-derived fragments (tRFs), a newfangled branch of small non-coding RNA (sncRNA), recently reported to play a vital part in several diseases. In present study, we aimed to detect and reveal the role of tRFs in podocyte differentiation. The expression levels of tRFs between undifferentiated and differentiated podocytes were sequenced by illumina nextseq 500, and further verified by quantitative RT-PCR. 69 upregulated and 70 downregulated tRFs in total were singled out (Fold change > 2, P < 0.05). Gene ontology (GO) analysis indicated they are involved in the biological processes of transcription, DNA-templated, positive regulation of transcription from RNA polymerase II promoter, angiogenesis, cell adhesion. Besides, KEGG analysis suggested that these differentially tRFs are associated with PI3K-Akt signaling pathway, Rap1 signaling pathway, Ras signaling pathway, MAPK signaling pathway, and Wnt signaling pathway. Therefore, the differentially tRFs might regulate the differentiation of podocyte and the process of CKD. The functions and mechanisms of tRFs in podocytes are needed to be further explored.

Gene expression profiling analysis reveals that the long non­coding RNA uc.412 is involved in mesangial cell proliferation.

Hyperproliferation of mesangial cells (MCs) is the central pathological feature observed in certain human renal diseases. Furthermore, the long non­coding RNA uc.412 is regulated by transforming growth factor ß1 in mesangial cells in vitro. The present study aimed to investigate whether uc.412 serves a role in renal fibrosis and whether it may be considered as a therapeutic target in mesangial proliferative kidney diseases. The results demonstrated that uc.412 overexpression significantly increased MC proliferation. The transcriptional profile of MCs overexpressing uc.412 was assessed by RNA sequencing. A total of 462 up­ and 843 downregulated genes were identified (|fold change| ≥1.5), and reverse transcription­quantitative PCR was used to determine the expression of these differentially expressed genes (DEGs). Subsequently, the potential function of these DEGs was determined by bioinformatics analyses. The results indicated that these DEGs were involved in numerous signaling pathways associated with MC proliferation. The downstream association between up­ and downregulated genes was constructed via the STRING database. The protein­protein interaction network indicated that serpin family E member 1 and matrix metallopeptidase 3 may be hub proteins. In conclusion, the present study provided novel insight into the role of uc.412 in MC proliferation, which may aid in the development of novel treatment for mesangial proliferative kidney diseases.

Raw Material Regulates Flavor Formation via Driving Microbiota in Chinese Liquor Fermentation.

Raw material is important for flavors in fermented foods. Here, the effect of hulless barley on the microbiota in Chinese liquor was studied using two main cultivars (heilaoya and dulihuang). Six genera (Lactobacillus, Saccharomyces, Komagataella, Aspergillus, Pichia, and Weissella) were identified as flavor producers. Komagataella, mainly correlated with esters, dominated in heilaoya, and Pichia, mainly correlated with carbonyls, dominated in dulihuang. The Mantel test indicated reducing sugar drove the succession of microbiota (heilaoya: P = 0.001; dulihuang: P = 0.006). Especially, glucose (P = 0.0226) and fructose (P = 0.0168) presented the most significant correlations with Pichia and Komagataella, respectively. The simulative fermentation confirmed Komagataella phaffii QK2 grew better in heilaoya with more fructose, whereas Pichia fermentans PF grew better in dulihuang with more glucose. This work highlighted the effect of raw material on microbiota, which would be beneficial for regulating the quality of fermented foods.

Alterations in the long non­coding RNA transcriptome in mesangial cells treated with aldosterone in vitro.

Clinical and experimental reports indicate that aldosterone (ALD) contributes to the progression of renal failure independent of its hemodynamic effects. However, the mechanisms remain to be completely elucidated. The aim of the present study was to investigate the alterations of long non­coding RNA (lncRNA) in mesangial cells (MCs) treated with ALD. The present study used MCs treated with 10­6 M ALD as experimental cells. Microarray techniques performed by Agilent Technologies were used to identify the profiles of differentially expressed lncRNAs between the ALD group and the control group. Pathway and gene ontology analysis were applied to determine the roles of the differentially expressed lncRNAs. Reverse transcription quantitative polymerase chain reaction (RT­qPCR) was used to quantify the differentially expressed lncRNAs. A total of 8,459 lncRNA and 13,214 mRNAs with differential expression between MCs treated with and without ALD were identified. The expression of lncRNAs was confirmed by RT­qPCR and the results were consistent with the lncRNA array. The biological functions of lncRNAs are associated with responding to external stimuli, positive regulation of biological and apoptotic processes, cell division, mitosis and nuclear division. The pathways include cell cycle and peroxisome proliferator­activated receptor signaling pathways. The present study revealed distinct sets of lncRNA expressed in MCs treated with ALD, suggesting that this class of transcripts may be involved in the pathogenesis of chronic kidney diseases.

Beyond Gap Junction Channel Function: the Expression of Cx43 Contributes to Aldosterone-Induced Mesangial Cell Proliferation via the ERK1/2 and PKC Pathways.

AIMS: This study aimed to explore the precise mechanism and signaling pathways of mesangial cell (MC) proliferation from a new point of view considering Connexin 43 (Cx43). METHODS: MC proliferation was measured by the incorporation of 3H-thymidine (3H-TdR). Cx43 was over-expressed in MC cells using lipofectamine 2000, and the expression level was tested with reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. The gap junction channel function was explored by Lucifer Yellow scrape loading and dye transfer (SLDT), and the intracellular calcium concentrations ([Ca(2+)]i) were characterized by confocal microscopy on cells loaded with Fura-3/AM. RESULTS: There was an inverse correlation between Cx43 expression and MC proliferation (P<0.05). SLDT studies revealed that there was no difference in the gap junction channel function between the normal and Aldosterone (Aldo)-stimulated groups (P>0.05). Our data also showed that the mineralcorticoid receptor (MR) antagonist spironolactone, ERK1/2 inhibitor PD98059 and PKC inhibitor GF109203X could attenuate the down-regulation of Cx43 expression in Aldo-induced MC proliferation; however, the PI3K inhibitor LY294002 could block MC proliferation without affecting Cx43 expression at either the mRNA or protein level. In addition, Aldo promoted MC proliferation in parallel with increasing [Ca(2+)]i (P<0.05), suggesting that the classical PKC pathway might be activated. CONCLUSIONS: Our study provides preliminary evidence that Cx43 is an important regulator of Aldo-promoted MC proliferation. Furthermore, reduced Cx43 expression promoted MC proliferation independent of the gap junction channel function, and this process might be mediated through the ERK1/2- and PKC-dependent pathways.

[Bufalin inhibits PDGF-BB-induced mesangial cell proliferation via mediating gap junctional intercellular communication].

OBJECTIVE: To study the role and possible mechanisms of gap junctional intercellular communication (GJIC) involved in mesangial cell (MC) proliferation which could be inhibited by bufalin. METHODS: Rat mesangial cells were cultured in vitro. The effect of bufalin on platelet-derived growth factor-BB (PDGF-BB)-induced MC proliferation was evaluated by MTT assay. The function of GJIC was detected by Lucifer Yellow scrape loading and dye transfer (SLDT). mRNA levels of Cx43, Cx45 and Cx40 were measured by RT-PCR. Intracellular calcium concentrations ([Ca(2+)]i) were examined in laser scanning confocal microscopy after loading by Fura-3/AM. RESULTS: MTT indicated that bufalin could inhibited PDGF-BB-induced MC proliferation (P<0.01). Compared with the hormal control group, PDGF-BB inhibited GJIC function, increased the expression of Cx45 and Cx40 (P<0.01) without altering the Cx43 (P>0.05) in gene level and also increased [Ca(2+)]i. However, bufalin treatment enhanced GJIC function, decreased Cx45 mRNA and Cx40 mRNA expression (P<0.01), and reduced [Ca(2+)]i (P<0.01). CONCLUSIONS: Bufalin inhibits PDGF-BB-induced MC proliferation, and its possible mechanisms may be related to regulation of Cx45 and Cx40 expression in the gene level, reduction of [Ca(2+)]i and enhancement of GJIC function.

Attenuation of glomerular filtration barrier damage in adriamycin-induced nephropathic rats with bufalin: an antiproteinuric agent.

Proteinuria is an important risk factor for the progression and prognosis of chronic kidney disease. Bufalin, a cardiotonic steroid, has been shown to posses a variety of biological activities including cardiotonic, anaesthetic and antineoplastic activities, and regulate the immune response. This study investigated the effects of bufalin against proteinuria and glomerular filtration barrier damage in rats with adriamycin (ADR)-induced nephropathy. We compared the blood and urine biochemical indices and the histologic and ultrastructure of the glomerulus in ADR rats with and without the intervention of bufalin or prednisone. The transcription, expression and distribution of the podocyte-associated molecules were compared utilising RT-PCR, western blotting and immunohistochemical staining. We found that bufalin reduced the urinary protein excretion and optimised the lipidaemia of the ADR rats. Bufalin alleviated the removal of podocyte foot processes and attenuated the changes in nephrin, podocin and integrin-linked kinase (ILK) stainings in the glomerulus of the ADR rats. Bufalin notably decreased the expression of nephrin and ILK but inhibited the down-regulation of podocin in protein levels on the renal cortex of the ADR rats. Additionally, bufalin inhibited the up-regulation of podocin and ILK in mRNA levels but did not affect nephrin mRNA levels. These results suggest that bufalin could alleviate ADR-induced proteinuria by protecting the glomerular filtration barrier and may be a novel potential therapeutic agent for proteinuria-associated kidney disease.

Bufalin inhibits platelet-derived growth factor-BB-induced mesangial cell proliferation through mediating cell cycle progression.

Bufalin, a traditional Chinese medicine, has been reported as a protective factor in many tumors. We therefore investigated the effect of bufalin on platelet-derived growth factor (PDGF)-BB-induced proliferation of cultured rat mesangial cells. The effect of bufalin on cell proliferation and its underlying mechanisms were investigated in cultured rat mesangial cells (MCs) by the methylthiazoletetrazolium (MTT) assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and cyclin-dependent kinases (CDK)2 and CDK4 kinase assays. Bufalin inhibited 20 ng/ml PDGF-BB-induced MC proliferation in a dose-dependent manner. Similar results were observed in different concentrations of bufalin, which blocked PDGF-BB-induced progression through G0/G1 to S phase of the cell cycle. Furthermore, bufalin not only inhibited upregulation of cyclin D1 and CDK4, but also downregulation of p21 in both mRNA and protein levels. Although bufalin did not affect p27 and CDK2 mRNA expression, it reversed downregulation of p27 and upregulation of CDK2 in protein level. Activity of CDK2 and CDK4 was also inhibited by bufalin. However, both bufalin and PDGF-BB did not affect cyclin E mRNA or protein expression. These results suggest that bufalin could inhibit MC proliferation by modulating cell cycle progress, indicating that bufalin could be a potential therapeutic agent for the prevention of mesangial proliferative glomerulonephritis.