DataSheet_1_Comparison the effects of finerenone and SGLT2i on cardiovascular and renal outcomes in patients with type 2 diabetes mellitus: A network meta-analysis.docx

BackgroundFinerenone and sodium-glucose cotransporter 2 inhibitors (SGLT2i) have been shown to improve cardiovascular and renal outcomes in patients with type 2 diabetes mellitus (T2DM), while the relative efficacy has not been determined.MethodsThe databases of PubMed, Embase and Cochrane were searched for relevant cardiovascular or renal outcome trials of SGLT2i or finerenone. The end points were major adverse cardiovascular events (MACE), nonfatal stroke (NS), myocardial infarction (MI), hospitalization for heart failure (HHF), cardiovascular death (CVD), and renal composite outcome (RCO). Network meta-analysis was performed using Bayesian networks to obtain pooled hazard ratios (HR) and 95% confidence intervals (CI). The probability values for ranking active and placebo interventions were calculated using cumulative ranking curves.Results1024 articles were searched, and only 9 studies were screened and included in this meta-analysis with 71793 randomized participants. Sotagliflozin (HR 0.72 95%CI 0.59-0.88, SUCAR=0.93) and canagliflozin (HR 0.80 95%CI 0.67-0.97, SUCAR=0.73) can significantly reduce the risk of MACE compared with placebo. Canagliflozin (HR 0.64 95%CI 0.48-0.86, SUCAR=0.73), sotagliflozin (HR 0.66 95%CI 0.50-0.87, SUCAR=0.69) and empagliflozin (HR 0.65 95%CI 0.43-0.98, SUCAR=0.68) can significantly reduce the risk of HHF compared with placebo. Empagliflozin (HR 0.62 95%CI 0.43-0.89, SUCAR=0.96) can significantly reduce the risk of CVD compared with placebo. Empagliflozin (HR 0.61 95%CI 0.39-0.96, SUCAR=0.74), canagliflozin (HR 0.66 95%CI 0.46-0.92, SUCAR=0.63), and dapagliflozin (HR 0.53 95%CI 0.32-0.85, SUCAR=0.88) can significantly reduce the risk of RCO compared with placebo. Finerenone has reduced the risk of MACE, MI, HHF, CVD and RCO to varying degrees, but they do not show significant difference from placebo and each SGLT2i.ConclusionBoth SGLT2i and finerenone could reduce the risk of MACE, HHF, MI, CVD, RCO. Finerenone has no obvious advantage than SGLT2i on the effects of cardiovascular and renal protective.Systematic review registrationhttps://www.crd.york.ac.uk/PROSPERO/, identifier CRD42022375092.</p

<i>In vitro</i> OCG with CD109 knockdown cell lines.

<p>RANKL-induced OCG <i>in vitro</i> experiments showed decreased fusion efficiency in CD109 KD cells when compared to the negative shRNA control. (n = 3, <i>P<0.05</i>).</p

RT-qPCR Primer sequences.

<p>RT-qPCR Primer sequences.</p

Schematic model of CD109 down-regulation of TGF-β.

<p>CD109 down-regulation of TGF-β signaling and inhibition Smad3 signaling, would cause a decrease in the TRAF6-TAB1-TAK1 complex formation resulting in a decrease in osteoclast formation. Up-regulation of CD109 showed an increase in OC formation suggesting that CD109 plays a role in OCG and it might be independent of TGF-β in osteoclasts.</p

Osteoclast formation potential of H10 cells <i>vs.</i> C8 cells.

<p>Photomicrograph of TRACP and DAPI stained osteoclasts derived from H10 and C8 cells after 4 days in culture with sRANKL. H10 cells were able to fuse formed large TRACP positive multinucleated cells (FI>60%) while C8 cells did not fuse at all (FI = 0%).</p

CD109 Protein expression over the days of OCG.

<p>Western blot analysis showing that CD109 protein expression is increased in the critical days of osteoclastogenesis in H10 cells, RAW264.7 cells and BMMs (n = 3, <i>P<0.05</i>). Band intensity of CD109 expression was normalized with β-actin. Band intensity of C8 cells was used as the baseline for 4A. Day 1 for 4B, and Day 0 for 4C.</p

CD109 Knockdown cell lines.

<p>Western blot analysis was used to reveal the percentage of protein knockdown per construct used. β-actin was used as the internal loading control. CD109 protein expression in cells expressing a scrambled shRNA (shRNA construct 13) was used as the control. Quantification of the Western blot showed that the higher knockdown efficiency was observed in constructs 97 and 99 when compared to scramble control. Note that 07 is RAW264.7 cells stably transfected with the empty shRNA vector pGFP-V-RS, which was used to harbor gene specific shRNA to knockdown CD109.</p

Heatmap of microarray analysis.

<p>Total RNA was extracted from H10 and C8 cells undergoing day 2 of RANKL-induced OCG. Microarray analysis was performed with an Affymetrix array and genes with a FDR of 0.01 and fold change over 5 were selected. Heatmap showing 8 randomly selected genes for RT-qPCR confirmation, that have not been reported to be involved in OCG.</p

The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

<div><p>Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion.</p></div

Adseverin expression is up-regulated during OCG in response to sRANKL.

<p><b>A</b>) Quantitative real-time PCR analysis was used to quantify Ads gene expression in osteoclast cultures derived from BMMs (black diamonds) and RAW macrophages (black circles). Results are expressed as fold expression versus Day 0 and are normalized against GADPH ± SEM. In BMM-derived osteoclasts, Ads expression was significantly up-regulated by 10-fold after 2 days and 23-fold after 4 days. No difference was noted between Day 4 and Day 6 cultures. In RAW-derived osteoclasts, Ads gene expression was significantly up-regulated by 24-fold after 2 days and 32-fold after 4 days. <b>B</b>) Immunoblot analysis was used to quantify protein expression in BMMs and RAW macrophage-derived osteoclast cultures. Results are expressed as fold expression versus Day 0 and normalized against ß-actin ± SD. In BMM-derived osteoclasts, Ads protein expression was significantly up-regulated by 6-fold in Day 2 cultures. 5-fold and 4-fold increases in expression were noted in Day 4 and 6 cultures, respectively. The decrease in expression in days 4 and Day 6 was not found to be statistically significant. Gelsolin expression was not altered during OCG in response to sRANKL. Cathepsin K expression was significantly increased in response to sRANKL after a 4 day stimulation with sRANKL. <b>D</b>) Similarly in RAW macrophage-derived osteoclast cultures, Ads was up-regulated 17-fold and 23-fold in Day 3 and Day 4 cultures respectively. Gelsolin expression was not altered during OCG, and Cathepsin K expression was significantly increased in Day 3 and 4 cultures. <b>C & E</b>) Quantification of immunoblots (* p<0.05, ** p<0.01, *** p<0.001, n = 3).</p