Chronic NOS inhibition actuates endothelial-mesenchymal transformation

Abstract

Chronic kidney diseases are accompanied by the accumulation of substances like asymmetric dimethylarginine, phenylacetic acid, homocysteine, and advanced glycation end products, known to either inhibit endothelial nitric oxide synthase (eNOS) or uncouple it, consequently limiting the amount of available nitric oxide (NO). Reduced bioavailability of NO induces endothelial dysfunction. An early loss of peritubular capillaries in tubulointerstitial fibrotic areas and injury to endothelial cells have been linked to progressive renal disease. Screening endothelial genes in cells treated with NOS inhibitors showed upregulation of collagen XVIII, a precursor of a potent antiangiogenic substance, endostatin. This finding was confirmed at the level of mRNA and protein expression. Tie-2 promoter-driven green fluorescent protein mice treated with nonhypertensinogenic doses of a NOS inhibitor exhibited upregulation of collagen XVIII/endostatin and rarefaction of capillary profiles. This was accompanied by the increased expression of transforming growth factor-beta and connective tissue growth factor in the kidney. Occasional endothelial cells expressed both the marker of endothelial lineage (green fluorescent protein) and mesenchymal marker (alpha-smooth muscle actin or calponin). In vitro studies of endothelial cells treated with asymmetric dimethylarginine showed decreased expression of eNOS and Flk-1 and enhanced expression of calponin and fibronectin, additional markers of smooth muscle and mesenchymal cells. These cells overexpressed transforming growth factor-beta and connective tissue growth factor, as well as endostatin. In conclusion, data presented here 1) ascribe to NO deficiency in endothelial cells the function of a profibrotic stimulus associated with the expression of an antiangiogenic fragment of collagen XVIII (endostatin) and 2) provide evidence of endothelial-mesenchymal transdifferentiation in the course of inhibition of NOS by a pathophysiologically important antagonist, asymmetric dimethylarginine. Both mechanisms may account for microvascular rarefaction.

Curtailing Endothelial TGF-β Signaling Is Sufficient to Reduce Endothelial-Mesenchymal Transition and Fibrosis in CKD

“…In light of these issues, our observations that curtailing endothelial TGF-b signaling is sufficient to blunt progressive scarring of injured kidneys is supported by the fact that myofibroblasts can be derived from the capillary endothelium through EndoMT. 16,35 Participation of EndoMT in renal fibrosis has been shown in three mouse models of CKD (including Alport's syndrome, diabetic nephropathy, and chronic nitric oxide synthase inhibition 16,35,36 ) and our findings add to this list that mice with curtailed endothelial TGF-b signaling have significantly blunted EndoMT during chronic renal injury. Data also imply that EndoMT contributes to renal fibrosis after prolonged, but not transient, injury.…”

Fluorescence Microangiography for Quantitative Assessment of Peritubular Capillary Changes after AKI in Mice

“…9,10 Capillary rarefaction induces focal hypoxia, activating an injury cascade with inflammation and ultimately interstitial fibrosis. 2,11 It has been described in a variety of rodent AKI models, 7,[12][13][14][15] and in human patients with CKD the loss of capillary density correlates with the severity of fibrosis. [16][17][18] Despite the established association between AKI and peritubular capillary loss, the precise nature of microvascular changes occurring after AKI are poorly defined because high-resolution techniques have not been used to examine this question.…”

The Krebs Cycle and Mitochondrial Mass Are Early Victims of Endothelial Dysfunction

“…9 Endothelial nitric oxide synthase (NOS)-deficient mouse model represents an ultimate lack of bioavailable NO, but it also lacks the concomitant generation of superoxide by the uncoupled enzyme. The strategy we elected was to chronically inhibit NO generation by subpressor doses of N G -monomethyl-L-arginine (L-NMMA), 10 and to analyze the differential proteome of isolated microvasculature. By using difference gel electrophoresis (DIGE), we found a cluster of differentially expressed mitochondrial proteins, among others, that lead to the preclinical development of defective Krebs cycle and mitochondrial biogenesis.…”