Excessive TGF-b signaling in epithelial cells, pericytes, or fibroblasts has been implicated in CKD. This list has recently been joined by endothelial cells (ECs) undergoing mesenchymal transition. Although several studies focused on the effects of ablating epithelial or fibroblast TGF-b signaling on development of fibrosis, there is a lack of information on ablating TGF-b signaling in the endothelium because this ablation causes embryonic lethality. We generated endothelium-specific heterozygous TGF-b receptor knockout (TbRII endo+/2 ) mice to explore whether curtailed TGF-b signaling significantly modifies nephrosclerosis.These mice developed normally, but showed enhanced angiogenic potential compared with TbRII endo+/+ mice under basal conditions. After induction of folic acid nephropathy or unilateral ureteral obstruction, TbRII endo+/2 mice exhibited less tubulointerstitial fibrosis, enhanced preservation of renal microvasculature, improvement in renal blood flow, and less tissue hypoxia than TbRII endo+/+ counterparts. In addition, partial deletion of TbRII in the endothelium reduced endothelial-to-mesenchymal transition (EndoMT). TGF-b-induced canonical Smad2 signaling was reduced in TbRII +/2 ECs; however, activin receptor-like kinase 1 (ALK1)-mediated Smad1/5 phosphorylation in TbRII +/2 ECs remained unaffected. Furthermore, the S-endoglin/L-endoglin mRNA expression ratio was significantly lower in TbRII +/2 ECs compared with TbRII +/+ ECs. These observations support the hypothesis that EndoMT contributes to renal fibrosis and curtailing endothelial TGF-b signals favors Smad1/5 proangiogenic programs and dictates increased angiogenic responses. Our data implicate endothelial TGF-b signaling and EndoMT in regulating angiogenic and fibrotic responses to injury.

Stress-induced premature senescence (SIPS) of endothelial cells (ECs) has emerged as a contributor to global EC dysfunction. One of the cellular abnormalities mechanistically linked to SIPS is lysosomal dysfunction. In this study, we examined the impact of a range of cardiovascular risk factors on the expression of sirtuin 1 (SIRT1), SIPS, and apoptosis, and we documented the role of SIRT1 in reduced EC and endothelial progenitor cell (EPC) viability. These findings were confirmed in mice with selective endothelial SIRT1 knockout. The effects of stressors could be partially mimicked by inducing lysosomal membrane permeabilization or inhibiting autophagy, and were reversed by a cathepsin inhibitor. We provide evidence that SIRT1 is an important substrate of cysteine cathepsins B, S, and L. An antioxidant/peroxynitrite scavenger, ebselen, prevented stress-induced SIRT1 depletion and subversion of autophagy by mitigating lysosomal dysfunction. In conclusion, our data advance the concept of "stem cell aging" by establishing the critical role of lysosomal dysfunction in the development of SIPS through the cathepsin-induced proteolytic cleavage of SIRT1, a mechanism linking cell stress to apoptosis and SIPS. Ebselen potently protects lysosomal membrane integrity, preventing cathepsin-induced cleavage of SIRT 1 in EPCs and blunting SIPS and apoptotic cell death induced by relevant cardiovascular stressors. The proposed mechanism of SIRT1 depletion in stress has all of the attributes of being a paradigm of SIPS of EPCs.

High mobility group box 1 (HMGB1) undergoes acetylation, nuclear-to-cytoplasmic translocation and release from stressed kidneys, unleashing a signaling cascade of events leading to systemic inflammation. Here we tested whether the deacetylase activity of Sirtuin1 (SIRT1) participates in regulating nuclear retention of HMGB1 to ultimately modulate damage signaling initiated by HMGB1 secretion during stress. When immunoprecipitated acetylated HMGB1 was incubated with SIRT1, HMGB1 acetylation decreased by 57%. Proteomic analysis showed that SIRT1 deacetylates HMGB1 at four lysine residues (55, 88, 90 and 177) within the pro-inflammatory and nuclear localization signal domains of HMGB1. Genetic ablation or pharmacological inhibition of SIRT1 in endothelial cells increased HMGB1 acetylation and translocation. In vivo, deletion of SIRT1 reduced nuclear HMGB1 while increasing its acetylation and release into circulation during basal and ischemic conditions causing increased renal damage. Conversely, resveratrol pretreatment led to decreased HMGB1 acetylation, its nuclear retention, decreased systemic release and reduced tubular damage. Thus, a vicious cycle is set into motion in which the inflammation-induced repression of SIRT1 disables deacetylation of HMGB1, facilitates its nuclear-to-cytoplasmic translocation and systemic release, thereby maintaining inflammation.