Knockdown of TMEM30A in renal tubular epithelial cells leads to reduced glucose absorption.

The kidney reabsorbs large amounts of glucose through Na+-glucose cotransporter 2 (SGLT2). P4-ATPase acts together with the ß-subunit TMEM30A to mediate the asymmetric distribution of phosphatidylserine (PS), phosphatidylethanolamine (PE), and other amino phospholipids, promoting plasma membrane and internal vesicle fusion, and facilitating vesicle protein transport. We observed reduced TMEM30A expression in renal tubules of DKD and IgA patients, suggesting a potential role of TMEM30A in renal tubular cells. To investigate the role of TMEM30A in renal tubules, we constructed a TMEM30A knockdown cell model by transfecting mouse kidney tubular epithelium cells (TCMK-1) with TMEM30A shRNA. Knockdown of TMEM30A in TCMK-1 cells attenuated vesicle transporter protein synthesis, resulting in reduced transport and expression of SGLT2, which in turn reduced glucose absorption. These data suggested that TMEM30A plays a crucial role in renal tubules.

Inositol 1,4,5-trisphosphate receptor type 2 is associated with the bone-vessel axis in chronic kidney disease-mineral bone disorder.

Objective: The pathogenesis of renal osteopathy and cardiovascular disease suggests the disordered bone-vessel axis in chronic kidney disease-mineral bone disorder (CKD-MBD). However, the mechanism of the bone-vessel axis in CKD-MBD remains unclear.Methods: We established a CKD-MBD rat model to observe the pathophysiological phenotype of the bone-vessel axis and performed RNA sequencing of aortas to identify novel targets of the bone-vessel axis in CKD-MBD.Results: The microarchitecture of the femoral trabecular bone deteriorated and alveolar bone loss was aggravated in CKD-MBD rats. The intact parathyroid hormone and alkaline phosphatase levels increased, 1,25-dihydroxyvitamin D3 levels decreased, and intact fibroblast growth factor-23 levels did not increase in CKD-MBD rats at 16 weeks; other bone metabolic parameters in the serum demonstrated dynamic characteristics. With calcium deposition in the abdominal aortas of CKD-MBD rats, RNA sequencing of the aortas revealed a significant decrease in inositol 1,4,5-trisphosphate receptor type 2 (ITPR2) gene levels in CKD-MBD rats. A similar trend was observed in rat aortic smooth muscle cells. As a secretory protein, ITPR2 serum levels decreased at 4 weeks and slightly increased without statistical differences at 16 weeks in CKD-MBD rats. ITPR2 serum levels were significantly increased in patients with vascular calcification, negatively correlated with blood urea nitrogen levels, and positively correlated with serum tartrate-resistant acid phosphatase 5b levels.Conclusion: These findings provide preliminary insights into the role of ITPR2 in the bone-vessel axis in CKD-MBD. Thus, ITPR2 may be a potential target of the bone-vessel axis in CKD-MBD.

Ubiquitin-specific protease 47 is associated with vascular calcification in chronic kidney disease by regulating osteogenic transdifferentiation of vascular smooth muscle cells.

Chronic kidney disease (CKD) has recently become a serious health and social concern. Vascular calcification, a common complication of CKD, is a risk factor that increases the incidence and mortality of cardiovascular events in patients with CKD. However, there are currently no effective therapeutic targets that can facilitate treatment with fewer side effects for vascular calcification in CKD. To identify potential therapeutic targets, we performed label-free quantification (LFQ) analyses of protein samples from rat aortic vascular smooth muscle cells (RASMCs) after high-phosphorus treatment by nano-UPLC-MS/MS. We determined that ubiquitin-specific protease 47 (USP47) may be associated with CKD vascular calcification by regulating the osteogenic transdifferentiation of the vascular smooth muscle cell (VSMC) phenotype, thus suggesting a novel and potentially effective therapeutic target for CKD vascular calcification. USP47 knockdown significantly reduced the expression of ß-transducin repeat-containing protein (BTRC), serine/threonine-protein kinase akt-1 (AKT1), Klotho, fibroblast growth factor (FGF23), and matrix Gla protein (MGP) in RASMCs after high-phosphorus treatment. Consistent with the results of protein-protein interaction (PPI) analyses, USP47 may be involved in regulating osteogenic transdifferentiation markers, such as runt-related transcription factor 2 (RUNX2), Klotho, FGF23, and MGP through the BTRC/AKT1 pathway upon CKD vascular calcification. These data indicate that USP47 may be associated with vascular calcification in CKD by regulating osteogenic differentiation of VSMCs. USP47 may regulate osteogenic transdifferentiation in VSMCs upon CKD vascular calcification through a process involving the BTRC/AKT1 pathway. This study identified a novel potential therapeutic target for the treatment of vascular calcification in CKD.

Sclerostin is involved in osteogenic transdifferentiation of vascular smooth muscle cells in chronic kidney disease-associated vascular calcification with non-canonical Wnt signaling.

Vascular calcification is prominent in patients with chronic kidney disease (CKD) and is a strong predictor of cardiovascular mortality in the CKD population. However, the mechanism underlying CKD-associated vascular calcification remains unclear. To identify potential therapeutic targets, a 5/6 nephrectomy rat model was established by feeding of a high-phosphorous diet as the CKD group and compared with sham group rats at 4 and 16 weeks. Sequencing analyses of the rat aorta revealed 643 upregulated and 1023 downregulated genes at 4 weeks, as well as 899 upregulated and 1185 downregulated genes at 16 weeks in the CKD group compared to the sham group. Bioinformatics analyses suggested that SOST (which encodes sclerostin) and Wnt signaling are involved in CKD-associated vascular calcification. Furthermore, protein-protein interactions analysis revealed interactions between SOST, WNT5A, and WNT5B, that involved runt-related transcription factor 2 (RUNX2) and transgelin (TAGLN). SOST was increased in CKD-associated vascular calcification following reduction of the Wnt signaling, including WNT5A and WNT5B, both in vivo and in vitro. TargetScan was used to predict the microRNAs (miRNAs) targeting WNT5A and WNT5B. The expression levels of miR-542-3p, miR-298-3p, miR-376b-5p, and miR-3568 were significantly reduced, whereas that of miR-742-3p was significantly increased in calcified rat aortic vascular smooth muscle cells (VSMCs). In CKD rat aortas, the expression of miR-542-3p, miR-298-3p, miR-376b-5p, miR-3568, miR-742-3p, and miR-22-5p were significantly reduced at both 4 and 16 weeks. Altogether, owing to several assessments, potentially diagnostic and prognostic biomarkers for improving common CKD diagnostic tools were identified in this study. Abbreviations: BUN: blood urea nitrogen; CKD: chronic kidney disease; CKD-MBD: chronic kidney disease-mineral bone disorder; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GO: the Gene Ontology; HE: hematoxylin-eosin; HRP: horseradish peroxidase; KEGG: Kyoto Encyclopedia of Genes and Genomes; MiRNAs: microRNAs; PAS: periodic acid-Schiff; RUNX2: runt-related transcription factor 2; SCr: serum creatinine; STRING: the Search Tool for the Retrieval of Interacting Genes/Proteins; TAGLN: transgelin; VSMC: vascular smooth muscle cell.

M133S mutation possibly involve in the ER stress and mitophagy pathway in maintenance hemodialysis patients with occult hepatitis B infection.

Occult hepatitis B virus infection (OBI) is characterized by the presence of HBV DNA in the absence of detectable HBsAg. OBI is an important risk factor for cirrhosis and hepatocellular carcinoma, but its pathogenesis has not been fully elucidated. Mutations in the HBV preS/S genes can lead to impaired secretion of either HBsAg or S-protein resulting in the accumulation of defective viruses or S protein in cells. In our previous work, the M133S mutation was present in the HBV S gene of maintenance hemodialysis (MHD) patients with OBI. In this study, we investigated the potential role of amino acid substitutions in S proteins in S protein production and secretion through the construction of mutant S gene plasmids, structural prediction, transcriptome sequencing analysis, and in vitro functional studies. Protein structure prediction showed that the S protein M133S mutant exhibited hydrophilic modifications, with greater aggregation and accumulation of the entire structure within the membrane phospholipid bilayer. Differential gene enrichment analysis of transcriptome sequencing data showed that differentially expressed genes were mainly concentrated in protein processing in the endoplasmic reticulum (ER). The expression of heat shock family proteins and ER chaperone molecules was significantly increased in the wild-type and mutant groups, whereas the expression of mitochondria-associated proteins was decreased. Immunofluorescence staining and protein blotting showed that the endoplasmic reticulum-associated protein PDI, the autophagy marker LC3, and the lysosome-associated protein LAMP2 co-localized with the S proteins in the wild-type and mutant strains, and their expression was increased. The mitochondria-associated TOMM20 protein was also co-expressed with the S protein, but expression was significantly reduced in the mutant. The M133S mutation in the S gene is expressed as a defective and misfolded protein that accumulates in the endoplasmic reticulum causing secretion-impaired endoplasmic reticulum stress, which in turn triggers mitochondrial autophagy and recruits lysosomes to fuse with the autophagosome, leading to mitochondrial clearance. This study preliminarily demonstrated that the mutation of M133S in the S gene can cause OBI and is associated with disease progression, providing a theoretical basis for the diagnosis and treatment of OBI.

Tmem30a protects against podocyte injury through suppression of pyroptosis.

Podocytopathies, such as focal segmental glomerulosclerosis (FSGS), are characterized by podocyte injury and can easily progress to end-stage kidney disease. However, the mechanisms underlying podocyte injury remain unclear. We observed podocyte injury along with pyroptosis in patients with FSGS. Bioinformatic analysis of public datasets revealed that transmembrane protein 30a (Tmem30a) might be associated with FSGS. The expression of Temem30a and the podocyte-related protein, nephrin, were significantly downregulated in patients with FSGS, adriamycin (ADR)-induced mice, and podocyte-specific Tmem30a lox P /loxP ; NPHS2-Cre mice, whereas the expression of NLR family pyrin domain containing 3 (NLRP3) and ASC, two pyroptosis-related proteins, were significantly upregulated. Meanwhile, the pyroptosis inhibitor MCC950 and disulfiram (DSF) increased Tmem30a and podocyte-related proteins expression, and inhibited pyroptosis-related proteins expression in ADR-induced mouse podocytes and Tmem30a knockdown (KD) mouse podocytes. Therefore, Tmem30a might protect against podocyte injury by inhibiting pyroptosis, suggesting a potential therapeutic target for podocytopathies.

Urinary complement profile in IgA nephropathy and its correlation with the clinical and pathological characteristics.

Background and objectives: The activated complement profile in IgA nephropathy (IgAN) is still unclear. Our study investigated the profile of urinary complements in IgAN patients and its correlations with clinical and pathological characteristics. Methods: Urinary protein abundance was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in 50 IgAN, 50 membranous nephropathy (MN), and 68 healthy controls (HC). Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to identify differentially expressed proteins in IgAN patients. The differentially expressed complement proteins were screened in IgAN patients, and their correlations with laboratory or pathological parameters were analyzed. Thereafter, 7 complement components were validated by enzyme-linked immunosorbent assay (ELISA) in the urine samples of 45 IgAN patients. Results: There were 786 differentially expressed proteins between IgAN and HC. KEGG analysis showed that differentially expressed urinary proteins in IgAN were enriched with complement. Of these, 67% of urinary complement protein abundance was associated with the estimated glomerular filtration rate. The urinary complement-related protein collectin12 (colec12), complement H factor (CFH), complement H factor-related protein 2 (CFHR2), and complement B factor (CFB) were positively correlated with serum creatinine; colec12, CFHR2, CFB, and C8g were positively correlated with glomerulosclerosis; CFH, CFHR2, C8g, and C9 were positively correlated with tubular atrophy/interstitial fibrosis. Conclusion: Abnormally increased components of complement pathways significantly correlate with reduced renal function, proteinuria, and renal histological damage in IgAN. It could provide a potential biomarker panel for monitoring IgAN and provide clues for therapeutic choice targeting complement system of IgAN patients.

Embelin attenuates lipopolysaccharide-induced acute kidney injury through the inhibition of M1 macrophage activation and NF-κB signaling in mice.

Septic acute kidney injury (AKI) is commonly associated with renal dysfunction and high mortality in patients. Owing to the rapid and violent occurrence of septic AKI with inflammation, there are no effective therapies to clinically treat it. Embelin, a natural product, has a potential regulatory role in immunocytes. However, the role and mechanism of embelin in septic AKI remains unknown. This study aimed to elucidate the role of embelin in macrophage regulation in lipopolysaccharide (LPS)-induced septic AKI. Embelin was intraperitoneally administered to mice after LPS injection. And bone marrow-derived macrophages (BMDMs) were subsequently isolated from the mice to explore the immunomodulatory role of embelin in macrophages. We found that embelin attenuated renal dysfunction and pathological renal damage in the LPS-induced sepsis mouse model. Molecular docking predicted that embelin could bind to phosphorylated NF-κB p65 at the ser536 site. Embelin inhibited the translocation of NF-κB p65 via phosphorylation at ser536 in LPS-induced AKI. It also reduced the secretion of IL-1ß and IL-6 and increased the secretion of IL-10 and Arg-1 of BMDMs and mice after LPS stimulation, indicating that embelin suppressed macrophage M1 activation in LPS-induced AKI. Therefore, embelin attenuated LPS-induced septic AKI by suppressing NF-κB p65 at ser536 in activated macrophages. This study preclinically suggests a therapeutic role of embelin in septic AKI.