Decreased expression of ADAM10 on monocytes is associated with chronic allograft dysfunction in kidney transplant recipients.

BACKGROUND: Chronic allograft dysfunction (CAD) is a common cause of allograft loss in kidney transplant recipients (KTRs). Our previous study found that elevated serum soluble T cell immunoglobulin mucin-3 (sTim-3) was positively associated with the severity of CAD in KTRs. sTim-3 was reported to be generated from ADAM10/ADAM17-mediated ectodomain shedding of membrane Tim-3 (mTim-3) in humans. However, whether mTim-3 shedding-related molecules participate in the progression of CAD remains unknown. Here, we explored the relationships between different forms of Tim-3, including mTim-3 on different peripheral blood cell subsets, serum and urine sTim-3, and ADAM10/17 expression and active status to investigate their roles in CAD. METHODS: 63 KTRs with stable grafts, 91 KTRs with CAD and 42 healthy controls (HCs) were enrolled. Total Tim-3, pADAM10/17 and mADAM10/17 proteins were semiquantified by western blot. Serum and urine sTim-3 concentrations were determined by ELISA. mTim-3 and ADAM10/17 expression on leukocyte subpopulations was determined by flow cytometry. RESULTS: The KTR groups displayed significantly higher levels of urine sTim-3 pg/µmol creatinine than the HC group, while no difference was found between the two KTR groups. KTRs with CAD presented reduced nonactive pADAM10 protein but unaltered active mADAM10 when compared to the Stable group; no difference was found between the KTR groups regarding total Tim-3 and p/m ADAM17 protein levels. In addition, the CAD group showed lower mTim-3 expression on BDCA3+ DC than the Stable group; no other difference was observed in its expression on B, T, NK, NKT, monocyte subsets and other DC subsets among groups. With the deterioration of allograft function, ADAM10 expression densities on classical, intermediate, and non-classical monocytes were significantly decreased. Correlation analyses revealed that eGFR and serum sTim-3 exhibited weak to modest correlations with ADAM10 on monocyte and DC subsets. CONCLUSIONS: Our data indicated that ADAM10, especially its decreased expression on monocytes, may play an important role in the progression of CAD in KTRs. However, whether there is an interaction between ADAM10 and mTim-3 in the pathogenesis of CAD in KTRs needs to be further studied.

Loss of Myostatin Alters Mitochondrial Oxidative Phosphorylation, TCA Cycle Activity, and ATP Production in Skeletal Muscle.

Myostatin (MSTN) is an important negative regulator of skeletal muscle growth in animals. A lack of MSTN promotes lipolysis and glucose metabolism but inhibits oxidative phosphorylation (OXPHOS). Here, we aimed to investigate the possible mechanism of MSTN regulating the mitochondrial energy homeostasis of skeletal muscle. To this end, MSTN knockout mice were generated by the CRISPR/Cas9 technique. Expectedly, the MSTN null (Mstn-/-) mouse has a hypermuscular phenotype. The muscle metabolism of the Mstn-/- mice was detected by an enzyme-linked immunosorbent assay, indirect calorimetry, ChIP-qPCR, and RT-qPCR. The resting metabolic rate and body temperature of the Mstn-/- mice were significantly reduced. The loss of MSTN not only significantly inhibited the production of ATP by OXPHOS and decreased the activity of respiratory chain complexes, but also inhibited key rate-limiting enzymes related to the TCA cycle and significantly reduced the ratio of NADH/NAD+ in the Mstn-/- mice, which then greatly reduced the total amount of ATP. Further ChIP-qPCR results confirmed that the lack of MSTN inhibited both the TCA cycle and OXPHOS, resulting in decreased ATP production. The reason may be that Smad2/3 is not sufficiently bound to the promoter region of the rate-limiting enzymes Idh2 and Idh3a of the TCA cycle, thus affecting their transcription.

Myostatin Knockout Affects Mitochondrial Function by Inhibiting the AMPK/SIRT1/PGC1α Pathway in Skeletal Muscle.

Myostatin (Mstn) is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes. The deletion of the Mstn gene in mice leads to reduced mitochondrial functions. However, the underlying regulatory mechanisms remain unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. Mstn-KO mice exhibited significantly larger skeletal muscles. Meanwhile, Mstn knockout regulated the organ weights of mice. Moreover, we found that Mstn knockout reduced the basal metabolic rate, muscle adenosine triphosphate (ATP) synthesis, activities of mitochondrial respiration chain complexes, tricarboxylic acid cycle (TCA) cycle, and thermogenesis. Mechanistically, expressions of silent information regulator 1 (SIRT1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were down-regulated, while peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) acetylation modification increased in the Mstn-KO mice. Skeletal muscle cells from Mstn-KO and WT were treated with AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR), and the AMPK inhibitor Compound C, respectively. Compared with the wild-type (WT) group, Compound C treatment further down-regulated the expression or activity of pAMPK, SIRT1, citrate synthase (CS), isocitrate dehydrogenase (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) in Mstn-KO mice, while Mstn knockout inhibited the AICAR activation effect. Therefore, Mstn knockout affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway. The present study reveals a new mechanism for Mstn knockout in regulating energy homeostasis.

Myostatin Deficiency Enhances Antioxidant Capacity of Bovine Muscle via the SMAD-AMPK-G6PD Pathway.

During exercise, the body's organs and skeletal muscles produce reactive oxygen species (ROS). Excessive ROS can destroy cellular lipids, sugars, proteins, and nucleotides and lead to cancer. The production of nicotinamide adenine dinucleotide phosphate (NADPH) by the pentose phosphate pathway (PPP) is an auxiliary process of the cellular antioxidant system that supplements the reducing power of glutathione (GSH) to eliminate ROS in the cell. Myostatin (MSTN) is mainly expressed in skeletal muscle and participates in the regulation of skeletal muscle growth and development. Loss of MSTN leads to muscular hypertrophy, and MSTN deficiency upregulates glycolysis. However, the effect of MSTN on the PPP has not been reported. This study investigated the effect of MSTN on muscle antioxidant capacity from a metabolic perspective. We found that reducing MSTN modulates AMP-activated protein kinase (AMPK), a key molecule in cellular energy metabolism that directly regulates glucose metabolism through phosphorylation. Downregulation of MSTN promotes tyrosine modification of glucose-6-phosphate-dehydrogenase (G6PD) by AMPK and is regulated by the Smad signaling pathway. The Smad2/3 complex acts as a transcription factor to inhibit the AMPK expression. These results suggest that reduced MSTN expression inhibits the Smad signaling pathway, promotes AMPK expression, enhances the activity of G6PD enzyme, and enhances the antioxidant capacity of nonenzymatic GSH.

The diagnostic role of PD-1+ CXCR5+ follicular helper CD8+ T cell in renal allograft dysfunction.

BACKGROUND: The roles of PD-1+ CXCR5+ follicular helper CD8+ T cell were reported in different disease conditions, but their roles in transplantation are unclear. In this study, the association between PD-1+ CXCR5+ follicular helper CD8+ T cell and renal allograft dysfunction in kidney transplant recipients (KTRs) was investigated. METHODS: 82 KTRs were enrolled in this study. 45 KTRs were included in the chronic allograft dysfunction (CAD) group, and 37 KTRs were included in the stable recipients group. Among the CAD group, 12 cases of antibody-mediated rejection (ABMR) and 4 cases of T cell-mediated rejection (TCMR) were diagnosed by biopsy. The percentage of CXCR5+ CD8+ T cells and the co-expression of signal transducers and activators of transcription 4 (STAT4), STAT5, and PD-1 in peripheral blood were determined by flow cytometry. RESULTS: The expression of CXCR5 on CD3+ CD8+ T cells and the percentage of STAT5+ CXCR5+ cells in the CD3+ CD8+ T-cell population were significantly lower in the CAD group (p < 0.05), while the expression of PD-1+ CXCR5+ CD8+ T cells was significantly higher (p < 0.05). Through logistic regression analysis, we concluded that the percentage of PD-1+ CXCR5+ CD8+ T cells was an independent risk factor for renal dysfunction. Grouping by pathological type, PD-1+ CXCR5+ CD8+ T cells showed relatively good diagnostic efficacy for ABMR by ROC analysis. CONCLUSIONS: Our results suggested that PD-1+ CXCR5+ CD8+ T cells were a promising biomarker for distinguishing renal allograft dysfunction and different allograft pathological types. Also, our findings may provide new ways of identifying and treating allograft rejection.

Iron deficiency exacerbates cisplatin- or rhabdomyolysis-induced acute kidney injury through promoting iron-catalyzed oxidative damage.

Iron deficiency is the most common micronutrient deficiency worldwide. While iron deficiency is known to suppress embryonic organogenesis, its effect on the adult organ in the context of clinically relevant damage has not been considered. Here we report that iron deficiency is a risk factor for nephrotoxic intrinsic acute kidney injury of the nephron (iAKI). Iron deficiency exacerbated cisplatin-induced iAKI by markedly increasing non-heme catalytic iron and Nox4 protein which together catalyze production of hydroxyl radicals followed by protein and DNA oxidation, apoptosis and ferroptosis. Crosstalk between non-heme catalytic iron/Nox4 and downstream oxidative damage generated a mutual amplification cycle that facilitated rapid progression of cisplatin-induced iAKI. Iron deficiency also exacerbated a second model of iAKI, rhabdomyolysis, via increasing catalytic heme-iron. Heme-iron induced lipid peroxidation and DNA oxidation by interacting with Nox4-independent mechanisms, promoting p53/p21 activity and cellular senescence. Our data suggests that correcting iron deficiency and/or targeting specific catalytic iron species are strategies to mitigate iAKI in a wide range of patients with diverse forms of kidney injury.

Proof-of-concept study investigating the role of S100P-RAGE in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an epithelial carcinoma that arises from the lining of the nasopharyngeal mucosa. The efficacy of radiation therapy is limited due to radiation resistance, particularly in the advanced stages of NPC. The S100P protein is a small isoform of the S100 protein family, which is involved in the regulation of various intracellular and extracellular processes, including proliferation, differentiation and intracellular signaling. The aim of the current study was to investigate the significance of the S100P-RAGE axis in NPC progression. The expression levels of S100P and receptor for activated glycation end-products (RAGE) in NPC specimens were determined by western blotting. In addition, the effect of the S100P-RAGE axis on NPC was evaluated in vitro by proliferation and migration assays using C666-1 cells treated with S100P or the RAGE inhibitor FPS-ZM1. The underlying mechanism was also investigated by western blotting. The expression of S100P and RAGE was detected in clinical specimens from 15 patients with NPC and 15 patients with benign nasopharyngeal inflammation, and was observed to be higher in NPC tissues compared with inflamed tissues. Furthermore, the interaction of S100P with RAGE increased the proliferation and migration potential of C666-1 cells, and activated mitogen-activated protein kinase and NF-κB signaling. These results indicate that the S100P-RAGE axis exerts a promoting effect on the progression of NPC. Therefore therapeutic strategies targeting S100P-RAGE merit further exploration for the treatment of NPC.

Metabolic syndrome components and acute pancreatitis: a case-control study in China.

BACKGROUND: Acute pancreatitis (AP) is a common inflammatory disorder of the pancreas. Recent evidence has shown that metabolic syndrome is positively correlated with the severity of AP. However, only a few studies have revealed the relationship between metabolic syndrome and the occurrence of AP. We therefore elucidated the association between metabolic syndrome and the occurrence of AP. METHODS: A hospital-based case-control study was conducted. A total of 705 patients admitted to our hospital from January 2016 to December 2018 were included in the study. Subjects were divided into case and control groups according to their diagnosis: (1) According to the revised Atlanta classification from 2012, patients diagnosed with AP were enrolled in the case group. (2) Patients without a history of AP or any disease related to metabolic syndrome were allocated into the control group. Controls were matched to cases individually by sex and age (control/case ratio = 1). RESULTS: The incidence rate of metabolic syndrome in AP patients was 30.9%, which was more frequent than that in controls (13.2%) (OR 2.837; 95% CI 1.873-4.298, p < 0.001). In the multivariate regression analysis, a history of smoking or alcohol consumption and biliary stones were significantly associated with AP (OR 2.441; 95% CI 1.865-5.172, p < 0.001; OR 1.777; 95% CI 1.060-2.977, p = 0.029; OR 28.995; 95% CI 13.253-63.435, p < 0.001). In addition, the occurrence of AP was significantly associated with total cholesterol (TC) (OR 1.992; 95% CI 1.246-3.183, p = 0.004), triglyceride (TG) (OR 2.134; 95% CI 1.403-3.245, p < 0.001), hyperglycaemia (OR 2.261; 95% CI 1.367-3.742, p = 0.001), and apolipoprotein A (Apo A) (OR 0.270; 95% CI 0.163-0.447, p < 0.001). CONCLUSIONS: Metabolic syndrome and its components were associated with AP occurrence.

3-deazaneplanocin A protects against cisplatin-induced renal tubular cell apoptosis and acute kidney injury by restoration of E-cadherin expression.

3-deazaneplanocin A (3-DZNeP) has been used as an inhibitor of enhancer of zeste homolog 2 (EZH2). Here, we explore the role and underlying mechanisms action of 3-DZNeP in abrogating cisplatin nephrotoxicity. Exposure of cultured mouse renal proximal tubular epithelial cells (mTECs) to cisplatin resulted in dose and time-dependent cleavage of caspase-3, decrease of cell viability, and increase of histone H3 lysine 27 trimethylation (H3K27me3), whereas expression levels of EZH2, a major methyltransferase of H3K27me3, were not affected. Treatment with 3-DZNeP significantly inhibited cisplatin-induced activation of caspase-3, apoptosis, loss of cell viability but did not alter levels of EZH2 and H3K27me3 in cultured mTECs. 3-DZNeP treatment did not affect activation of extracellular signal-regulated kinase (ERK) 1/2, p38 or c-Jun N-terminal kinases (JNK) 1/2, which contribute to renal epithelial cell death, but caused dose-dependent restoration of E-cadherin in mTECs exposed to cisplatin. Silencing of E-cadherin expression by siRNA abolished the cytoprotective effects of 3-DZNeP. In contrast, 3-DZNeP treatment potentiated the cytotoxic effect of cisplatin in H1299, a non-small cell lung cancer cell line that expresses lower E-cadherin levels. Finally, administration of 3-DZNeP attenuated renal dysfunction, morphological damage, and renal tubular cell death, which was accompanied by E-cadherin preservation, in a mouse model of cisplatin nephrotoxicity. Overall, these data indicate that 3-DZNeP suppresses cisplatin-induced tubular epithelial cell apoptosis and acute kidney injury via an E-cadherin-dependent mechanism, and suggest that combined application of 3-DZNeP with cisplatin would be a novel chemotherapeutic strategy that enhances the anti-tumor effect of cisplatin and reduces its nephrotoxicity.

Physiological functions of ferroportin in the regulation of renal iron recycling and ischemic acute kidney injury.

Renal iron recycling preserves filtered iron from urinary excretion. However, it remains debated whether ferroportin (FPN), the only known iron exporter, is functionally involved in renal iron recycling and whether renal iron recycling is required for systemic iron homeostasis. We deleted FPN in whole nephrons by use of a Nestin-Cre and in the distal nephrons and collecting ducts, using a Ksp-Cre, and investigated its impacts on renal iron recycling and systemic iron homeostasis. FPN deletion by Nestin-Cre, but not by Ksp-Cre, caused excess iron retention and increased ferritin heavy chain (FTH1) specifically in the proximal tubules and resulted in the reduction of serum and hepatic iron. The systemic iron redistribution was aggravated, resulting in anemia and the marked downregulation of hepatic hepcidin in elderly FPN knockout (KO)/Nestin-Cre mice. Similarly, in iron-deficient FPN KO/Nestin-Cre mice, the renal iron retention worsened anemia with the activation of the erythropoietin-erythroferrone-hepcidin pathway and the downregulation of hepatic hepcidin. Hence, FPN likely located at the basolateral membrane of the proximal tubules to export iron into the circulation and was required for renal iron recycling and systemic iron homeostasis particularly in elderly and iron-deficient mice. Moreover, FPN deletion in the proximal tubules alleviated ischemic acute kidney injury, possibly by upregulating FTH1 to limit catalytic iron and by priming antioxidant mechanisms, indicating that FPN could be deleterious in the pathophysiology of ischemic acute kidney injury (AKI) and thus may be a potential target for the prevention and mitigation of ischemic AKI.