Renal and cardiac effects of the PDE9 inhibitor BAY 73-6691 in 5/6 nephrectomized rats.

It has been suggested that the novel selective phosphodiesterase 9 (PDE9) inhibitor may improve cardiac and renal function by blocking 3',5'-cyclic guanosine monophosphate (cGMP) degradation. 5/6 nephrectomized (5/6Nx) rats were used to investigate the effects of the PDE9 inhibitor (BAY 73-6691) on the heart and kidney. Two doses of BAY 73-6691 (1 mg/kg/day and 5 mg/kg/day) were given for 95 days. The 5/6Nx rats developed albuminuria, a decrease in serum creatinine clearance (Ccr), and elevated serum troponin T levels. Echocardiographic data showed that 5/6 nephrectomy resulted in increased fractional shortening (FS), stroke volume (SV), and left ventricular ejection fraction (EF). However, 95 days of PDE9 inhibitor treatment did not improve any cardiac and renal functional parameter. Histopathologically, 5/6 nephrectomy resulted in severe kidney and heart damage, such as renal interstitial fibrosis, glomerulosclerosis, and enlarged cardiomyocytes. Telmisartan attenuated renal interstitial fibrosis and glomerulosclerosis as well as improved cardiomyocyte size. However, except for cardiomyocyte size and renal perivascular fibrosis, BAY 73-6691 had no effect on other cardiac and renal histologic parameters. Pathway enrichment analysis using RNA sequencing data of kidney and heart tissue identified chronic kidney disease pathways, such as phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, complement and coagulation cascades, and nuclear factor kappa B (NF-κB) signaling pathway. PDE9i did not affect any of these disease-related pathways. Two dosages of the PDE9 inhibitor BAY 73-6691 known to be effective in other rat models have only limited cardio-renal protective effects in 5/6 nephrectomized rats.

Renoprotective effects of empagliflozin are linked to activation of the tubuloglomerular feedback mechanism and blunting of the complement system.

The mechanisms of nephroprotection in nondiabetic chronic kidney disease (CKD) models by sodium-glucose cotransporter 2 (SGLT2) inhibitors are not well defined. Five groups were established: sham-operated rats, placebo-treated rats with 5/6 nephrectomy (5/6Nx), 5/6Nx + telmisartan (5 mg/kg/day), 5/6Nx + empagliflozin (3 mg/kg/day), and 5/6Nx + empagliflozin (15 mg/kg/day). Treatment duration was 95 days. Empagliflozin showed a dose-dependent beneficial effect on the change from baseline of creatinine clearance (Ccr). The urinary albumin-to-creatinine ratio likewise improved in a dose-dependent manner. Both dosages of empagliflozin improved morphological kidney damage parameters such as renal interstitial fibrosis and glomerulosclerosis. 5/6 nephrectomy led to a substantial reduction of urinary adenosine excretion, a surrogate parameter of the tubuloglomerular feedback (TGF) mechanism. Empagliflozin caused a dose-dependent increase in urinary adenosine excretion. The urinary adenosine excretion was negatively correlated with renal interstitial fibrosis and positively correlated with Ccr. Immunofluorescence analysis revealed that empagliflozin had no effect on CD8+ and CD4+ T cells as well as on CD68+ cells (macrophages). To further explore potential mechanisms, a nonhypothesis-driven approach was used. RNA sequencing followed by quantitative real-time polymerase chain reaction revealed that complement component 1Q subcomponent A chain (C1QA) as well as complement component 1Q subcomponent C chain (C1QC) gene expression were upregulated in the placebo-treated 5/6Nx rats and this upregulation was blunted by treatment with empagliflozin. In conclusion, empagliflozin-mediated nephroprotection in nondiabetic CKD is due to a dose-dependent activation of the TGF as well as empagliflozin-mediated effects on the complement system.

Neurorescuing effect of Cinacalcet against hypercalcemia-induced nerve injury in chronic kidney disease via TRAF2/cIAP1/KLF2/SERPINA3 signal axis.

Hypercalcemia is a common complication in chronic kidney disease (CKD) and unfortunately contributes to nerve injury. This study aims to investigate the potential role and underlying mechanisms of Cinacalcet (CIN) in hypercalcemia-driven nerve injury in CKD. A CKD mouse model was first established by adenine feeding to identify the therapeutic effects of CIN. Molecules related to CIN and CKD were predicted by bioinformatics analysis and their expression in the kidney tissues of CKD mice was measured by immunochemistry. Gain- and loss-of-functions assays were performed both in vitro and in vivo to evaluate their effects on nerve injury in CKD, as reflected by Scr and BUN, and brain calcium content as well as behavior tests. CIN ameliorated hypercalcemia-driven nerve injury in CKD mice. Interactions among TRAF2, an E3-ubiquitin ligase, KLF2, and SERPINA3 were bioinformatically predicted on CIN effect. CIN restricted the ubiquitin-mediated degradation of KLF2 by downregulating TRAF2. KLF2 targeted and inversely regulated SERPINA3 to repress hypercalcemia-driven nerve injury in CKD. CIN was substantiated in vivo to ameliorate hypercalcemia-driven nerve injury in CKD mice through the TRAF2/KLF2/SERPINA3 regulatory axis. Together, CIN suppresses SERPINA3 expression via TRAF2-mediated inhibition of the ubiquitin-dependent degradation of KLF2, thus repressing hypercalcemia-induced nerve injury in CKD mice.

Drp1 activates ROS/HIF-1α/EZH2 and triggers mitochondrial fragmentation to deteriorate hypercalcemia-associated neuronal injury in mouse model of chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD), characterized as renal dysfunction, is regarded as a major public health problem which carries a high risk of cardiovascular diseases. The purpose of this study is to evaluate the functional significance of Drp1 in hypercalcemia-associated neuronal damage following CKD and the associated mechanism. METHODS: Initially, the CKD mouse models were established. Next, RT-qPCR and Western blot analysis were performed to measure expression of Fis1 and Drp1 in CKD. Chromatin immunoprecipitation (ChIP) assay and dual-luciferase reporter gene assay were utilized to explore the relationship among Drp1, HIF-1α, EZH2, and ROS with primary cortical neurons isolated from neonatal mice. Next, CKD mice were subjected to calcitonin treatment or manipulation with adenovirus expressing sh-Drp1, so as to explore the effects of Drp1 on hypercalcemia-induced neuronal injury in CKD. TUNEL assay and immunofluorescence staining were performed to detect apoptosis and NeuN-positive cells (neurons) in prefrontal cortical tissues of CKD mice. RESULTS: It was found that hypercalcemia could induce neuronal injury in CKD mice. An increase of Fis1 and Drp1 expression in cerebral cortex of CKD mice correlated with mitochondrial fragmentation. Calcitonin suppressed Drp1/Fis1-mediated mitochondrial fragmentation to attenuate hypercalcemia-induced neuronal injury after CKD. Additionally, Drp1 could increase EZH2 expression through the binding of HIF-1α to EZH2 promoter via elevating ROS generation. Furthermore, Drp1 knockdown inhibited hypercalcemia-induced neuronal injury in CKD while overexpression of EZH2 could reverse this effect in vivo. CONCLUSION: Taken together, the key findings of the current study demonstrate the promotive role of Drp1 in mitochondrial fragmentation which contributes to hypercalcemia-induced neuronal injury in CKD.

GSK343 modulates macrophage M2 polarization through the EZH2/MST1/YAP1 signaling axis to mitigate neurological damage induced by hypercalcemia in CKD mice.

Chronic kidney disease (CKD) often culminates in hypercalcemia, instigating severe neurological injuries that are not yet fully understood. This study unveils a mechanism, where GSK343 ameliorates CKD-induced neural damage in mice by modulating macrophage polarization through the EZH2/MST1/YAP1 signaling axis. Specifically, GSK343 downregulated the expression of histone methyltransferase EZH2 and upregulated MST1, which suppressed YAP1, promoting M2 macrophage polarization and thereby, alleviating neural injury in hypercalcemia arising from renal failure. This molecular pathway introduced herein not only sheds light on the cellular machinations behind CKD-induced neurological harm but also paves the way for potential therapeutic interventions targeting the identified axis, especially considering the M2 macrophage polarization as a potential strategy to mitigate hypercalcemia-induced neural injuries.

Vitamin D status and its association with parathyroid hormone in 23,134 outpatients.

In vitro studies indicate that 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits the synthesis of parathyroid hormone (PTH). The degree of PTH inhibition in humans by circulating 25(OH)D and 1,25(OH)2D may be different. Moreover, age and sex as well as confounding factors like calcium and phosphate may likewise affect the relationship between vitamin D and PTH in humans. However, this was not done so far in adequately powered studies. We investigated the relationship between 25(OH)D as well as 1,25(OH)2D and intact parathyroid hormone (iPTH) in 23,134 outpatients (age mean: 59.81 years) from the Berlin-Brandenburg area of Germany with normal serum creatinine considering confounding factors like age, sex, calcium and phosphate. 25(OH)D and iPTH were inversely correlated (r = -0.17, p < 0.0001). The inverse linear correlation was observed over the entire spectrum of 25(OH)D concentrations - from low 25(OH)D concentrations to very high 25(OH)D concentrations. Multiple linear regression analysis revealed that this correlation was independent of age, sex, creatinine, calcium and phosphate (unstandardized coefficients B: -0.16, p < 0.0001). However, 1,25(OH)2D was only positively associated with iPTH in women (r = 0.05, p = 0.033) and in the subgroup of patients with lower 25(OH)D (25(OH)D< 40 ng/ml) (r = 0.09, p < 0.0001), which was also presented in multiple linear regression analysis (unstandardized coefficients B: 0.20, p = 0.001). Circulating 1,25(OH)2D does not contribute substantially to the regulation of PTH in middle aged and vitamin D sufficient outpatients from the Berlin-Brandenburg area of Germany with normal kidney function. Presumably, serum 25(OH)D that is converted to 1,25(OH)2D after uptake in the parathyroid chief cells plays the critical role.

Antifibrotic effects of low dose SGLT2 Inhibition with empagliflozin in comparison to Ang II receptor blockade with telmisartan in 5/6 nephrectomised rats on high salt diet.

To date, the lowest protective SGLT2 inhibitor dose is unknown. We initially performed a dose-response pilot study in normal rats. Based on the results of this pilot study we compared the cardio-renal effects of the SGLT-2 inhibitor empagliflozin, with placebo or telmisartan in rats with 5/6 nephrectomy (5/6 Nx) on a high salt diet (HSD). The experimental set up was as follows: Sham operation (Sham) with normal diet and placebo; 5/6 Nx with 2% HSD and placebo; 5/6 Nx with HSD and empagliflozin (0.6 mg/kg/day, bid); 5/6 Nx with HSD and telmisartan (5 mg/kg/day, qd). Empagliflozin treatment increased urinary glucose excretion, in parallel to empagliflozin plasma levels, in a dose-dependent manner starting at doses of 1 mg/kg in the pilot study. 5/6Nx rats on HSD treated with this low empagliflozin dose showed significantly reduced cardiac (-34.85%; P < 0.05) and renal (-33.68%; P < 0.05) fibrosis in comparison to 5/6Nx rats on HSD treated with placebo. These effects were comparable to the effects observed when implementing the standard dose (5 mg/kg/day) of telmisartan (cardiac fibrosis: -36.37%; P < 0.01; renal fibrosis; -43.96%; P < 0.01). RNA-sequencing followed by confirmatory qRT-PCR revealed that both telmisartan and empagliflozin exert their cardiac effects on genes involved in vascular cell stability and cardiac iron homeostasis, whereas in the kidneys expression of genes involved in endothelial function and oxidative stress were differentially expressed. Urinary adenosine excretion, a surrogate marker of the tubuloglomerular feedback (TGF) mechanism, was not affected. In conclusion, the antifibrotic properties of low dose empagliflozin were comparable to a standard dose of telmisartan. The underlying pathways appear to be TGF independent.

Silencing of O-linked N-acetylglucosamine transferase ameliorates hypercalcemia-induced neurotoxicity in renal failure by regulating EZH2/KLF2/CXCL1 axis.

Hypocalcemia, associated with Calcium neurotoxicity, has been reported to induce nerve dysfunction, which is a significant problem of renal failure. This study identifies a molecular mechanism of the O-linked N-acetylglucosamine transferase (OGT)-mediated enhancer of zeste homolog 2 (EZH2)/krüppel-like factor 2 (KLF2)/chemokine (C-X-C motif) ligand 1 (CXCL1) axis underlying the hypercalcemia-induced nerve injury in renal failure. Bioinformatics analyses were used to screen out the key factors in hypercalcemia-induced nerve injury in renal failure. Chronic kidney disease (CKD) was induced by an adenine diet in mice, followed by injection of adenovirus vector carrying short hairpin RNA targeting OGT, followed by behavioral tests and collection of the cerebral cortex for primary neurons. Calcium level in neurons was measured by Fluo-4-am and Perkin Elmer+ Operetta. Neuronal apoptosis and viability were detected by flow cytometry and the MTS method. The binding of EZH2 to KLF2 promoter was verified by chromatin immunoprecipitation assay. The concentration of Ca2+ in brain tissues of CKD model mice was increased, and nerve functions were obviously damaged. High expression of OGT occurred in kidney tissue of CKD model mice. Silencing OGT reduced the hypercalcemia-induced toxicity of neurons by inhibiting the expression of EZH2, which elevated the expression of CXCL1 in primary neurons by diminishing KLF2. Silencing OGT attenuated hypercalcemia-induced neurotoxicity by regulating the EZH2/KLF2/CXCL1 axis. In vivo experiments further confirmed that silencing OGT could reduce hypercalcemia-induced nerve injury in CKD mice. Taken together, silencing OGT downregulates EZH2, which increases the expression of KLF2 and then decreases the expression of CXCL1, thus alleviating hypercalcemia-induced nerve injury in renal failure.

Comprehensive Analysis of N6-Methyladenosine RNA Methylation Regulators Expression Identify Distinct Molecular Subtypes of Myocardial Infarction.

Background: Myocardial infarction (MI) is one of the leading threats to human health. N6-methyladenosine (m6A) modification, as a pivotal regulator of messenger RNA stability, protein expression, and cellular processes, exhibits important roles in the development of cardiac remodeling and cardiomyocyte contractile function. Methods: The expression levels of m6A regulators were analyzed using the GSE5406 database. We analyzed genome-wide association study data and single-cell sequencing data to confirm the functional importance of m6A regulators in MI. Three molecular subtypes with different clinical characteristics were established to tailor treatment strategies for patients with MI. We applied pathway analysis and differentially expressed gene (DEG) analysis to study the changes in gene expression and identified four common DEGs. Furthermore, we constructed the protein-protein interaction network and confirmed several hub genes in three clusters of MI. To lucubrate the potential functions, we performed a ClueGO analysis of these hub networks. Results: In this study, we identified that the levels of FTO, YTHDF3, ZC3H13, and WTAP were dramatically differently expressed in MI tissues compared with controls. Bioinformatics analysis showed that DEGs in MI were significantly related to modulating calcium signaling and chemokine signaling, and m6A regulators were related to regulating glucose measurement and elevated blood glucose levels. Furthermore, genome-wide association study data analysis showed that WTAP single-nucleotide polymorphism was significantly related to the progression of MI. In addition, single-cell sequencing found that WTAP is widely expressed in the heart tissues. Moreover, we conducted consensus clustering for MI in view of the dysregulated m6A regulators' expression in MI. According to the expression levels, we found MI patients could be clustered into three subtypes. Pathway analysis showed the DEGs among different clusters in MI were assigned to HIF-1, IL-17, MAPK, PI3K-Akt signaling pathways, etc. The module analysis detected several genes, including BAG2, BAG3, MMP2, etc. We also found that MI-related network was significantly related to positive and negative regulation of angiogenesis and response to heat. The hub networks in MI clusters were significantly related to antigen processing and ubiquitin-mediated proteolysis, RNA splicing, and stability, indicating that these processes may contribute to the development of MI. Conclusion: Collectively, our study could provide more information for understanding the roles of m6A in MI, which may provide a novel insight into identifying biomarkers for MI treatment and diagnosis.

Astragaloside IV attenuates renal fibrosis through repressing epithelial-to-mesenchymal transition by inhibiting microRNA-192 expression: in vivo and in vitro studies.

The aim of this study was to investigate the effect of Astragaloside IV (AS-IV) on renal fibrosis in vivo and in vitro, and further to explore the underlying mechanism. To investigate the effect of AS-IV treatment on renal fibrosis in vivo, mouse renal fibrosis model was established by performing unilateral ureteral occlusion (UUO). The mice in the intervention group of AS-IV were given AS-IV 20 mg/(kg/d) on the day after surgery for 7 consecutive days. Then renal sections were stained with hematoxylin and eosin (H&E) to evaluate the degree of fibrosis. For in vitro study, human kidney tubular epithelial cells induced by (TGF-ß1) were performed to research the protective role of AS-IV in anti-fibrosis. Results form the in vivo study showed that AS-IV treatment in UUO mice significantly reduced parenchymal loss and tubular atrophy, indicating that AS-IV treatment attenuated renal fibrosis caused by UUO. TGF-ß1 treatment significantly increased the expression of α-SMA, vimentin, collagen I, miR-192 and decreased E-cadherin expression in HK-2 cells, suggesting that TGF-ß1 stimulated renal tubulointerstitial fibrosis. Moreover, in TGF-ß1 stimulated HK-2 cells, AS-IV clearly inhibited the expression levels of α-SMA, vimentin, collagen I, and miR-192 in a dose-dependent fashion while increased the expression level of E-cadherin in the same manner, indicating that AS-IV functioned the inhibitory role in renal tubulointerstitial fibrosis. Interestingly, we noted that ZEB2 was a direct target of miR-192. The effects of AS-IV on the expression of α-SMA, vimentin, collagen I and E-cadherin were inhibited by miR-192 mimic and aggravated by miR-192 inhibitor. Taken together, our results provided evidence that AS-IV could effectively protect kidney against epithelial fibrosis, and this renoprotective effect involved miR-192. Therefore, AS-IV might be considered as a potential and promising candidate drug for the treatment of renal epithelial fibrosis.

Quercetin is able to alleviate TGF-ß-induced fibrosis in renal tubular epithelial cells by suppressing miR-21.

Patients with chronic kidney disease (CKD) are characterized by a gradual loss of kidney function over time. A number of studies have indicated that tubule interstitial fibrosis (TIF) is associated with the occurrence and development of CKD. The aim of the present study was to investigate the effect of quercetin treatment on the fibrosis of renal tubular epithelial cells and to determine whether the anti-fibrotic effects of quercetin are achieved via microRNA (miR)-21. Human tubular epithelial HK-2 cells were cultured with transforming growth factor (TGF)-ß to induce fibrosis and the expression of fibrotic markers collagen I, fibronectin, α-smooth muscle actin (SMA) and epithelial-cadherin were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Cells were treated with 7.5, 15 or 30 mg/ml quercetin, following which fibrosis and miR-21 expression were evaluated. Quercetin-treated cells were transfected with miR-21 mimics and the expression of fibrotic markers was examined using RT-qPCR. Finally, the expression of fibrosis-associated miR-21 target genes, phosphatase and tensin homolog (PTEN) and TIMP Metallopeptidase Inhibitor 3 (TIMP3), was measured in cells treated with quercetin with or without miR-21 mimics using RT-qPCR, western blotting and immunocytochemistry. The results revealed that TGF-ß treatment induced a significant increase in the expression of fibrotic markers in HK-2 cells, while quercetin treatment partially inhibited the fibrosis of HK-2 cells. Furthermore, quercetin treatment significantly inhibited TGF-ß-induced miR-21 upregulation and transfection with miR-21 mimics reversed the anti-fibrotic effects of quercetin. Quercetin treatment markedly upregulated PTEN and TIMP3 expression, whereas transfection with miR-21 mimics reversed this effect. The results of the present study suggest that quercetin is able to alleviate TGF-ß-induced fibrosis in HK-2 cells via suppressing the miR-21 and upregulating PTEN and TIMP3. Quercetin may have potential as an anti-fibrotic treatment for patients with renal fibrosis.