Midazolam Ameliorates Hyperglycemia-Induced Glomerular Endothelial Dysfunction by Inhibiting Transglutaminase 2 in Diabetes

Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca2+ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2−/− mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.

ZEB2 transduces HIF1α dependent regulation of Transglutaminase 2 in glomerular podocytes

Glomerular podocytes are instrumental in ensuring glomerular permselectivity and regulating the integrity of glomerular biology. However, podocytes are vulnerable to various noxious stimuli such as hypoxia, and podocyte injury presented with glomerulosclerosis and impaired kidney function. The mechanism of hypoxia-induced podocyte injury vis-a-vis glomerulosclerosis has remained enigmatic. Hypoxia inducible factor 1α (HIF1α) that transduces hypoxic adaptations, induces Transglutaminase 2 (TG2), a calcium dependent enzyme that catalyzes intramolecular ε-(γ-glutamyl) lysine cross-links of extracellular matrix (ECM) proteins. In this study, we investigated the mechanism of regulation of TG2 by HIF1α. Stabilization of HIF1⍺ by FG4592 (Roxadustat) and physiological hypoxia, resulted in elevated expression of ZEB2 (zinc-finger E-box-homeobox 2) and its downstream target TRPC6 (transient receptor potential channel 6). ZEB2 transcriptionally activates TG2 expression, whereas, via TRPC6, it induces calcium influx, inturn it increases the TG2 activity. Blocking the TRPC6 action or suppressing its expression only partially attenuated FG4592 induced TG2 activity, whereas suppression of ZEB2 expression significantly abolished TG2 activity. This study demonstrates that stabilization of HIF1α stimulates both TG2 expression and activity, whereas abrogation of HIF1⍺ by metformin prevented HIF1⍺ regulated TG2 and consequent glomerular injury.

Impaired Skeletal Muscle Development and Regeneration in Transglutaminase 2 Knockout Mice

Skeletal muscle regeneration is triggered by local inflammation and is accompanied by phagocytosis of dead cells at the injury site. Efferocytosis regulates the inflammatory program in macrophages by initiating the conversion of their inflammatory phenotype into the healing one. While pro-inflammatory cytokines induce satellite cell proliferation and differentiation into myoblasts, growth factors, such as GDF3, released by healing macrophages drive myoblast fusion and myotube growth. Therefore, improper efferocytosis may lead to impaired muscle regeneration. Transglutaminase 2 (TG2) is a versatile enzyme participating in efferocytosis. Here, we show that TG2 ablation did not alter the skeletal muscle weights or sizes but led to the generation of small size myofibers and to decreased grip force in TG2 null mice. Following cardiotoxin-induced injury, the size of regenerating fibers was smaller, and the myoblast fusion was delayed in the tibialis anterior muscle of TG2 null mice. Loss of TG2 did not affect the efferocytic capacity of muscle macrophages but delayed their conversion to Ly6C−CD206+, GDF3 expressing cells. Finally, TG2 promoted myoblast fusion in differentiating C2C12 myoblasts. These results indicate that TG2 expressed by both macrophages and myoblasts contributes to proper myoblast fusion, and its ablation leads to impaired muscle development and regeneration in mice.