BACKGROUND: Increasing evidence suggests that superoxide ions produced by NOX (nicotinamide adenine dinucleotide phosphate oxidases) mediate vascular effects of Ang II (angiotensin II) evoked by atherogenic diets. Here, we analyzed the mechanism by which NOX2 contributes to Ang II–induced ET-1 (endothelin 1) production in human microvascular endothelial cells. METHODS: The effects of high-fat diet were compared between WT (wild type) and Nox2 ( mouse NOX2 gene )-deficient mice. ET-1 production and NOX2 expression by human microvascular endothelial cells in vitro were analyzed by ELISA, reverse transcription quantitative polymerase chain reaction, electrophoretic mobility shift assay, promoter deletions, RNA interference, and pharmacological inhibition. Production of superoxide anions was visualized by fluorescent cell labeling. RESULTS: Feeding mice high-fat diet for 10 weeks increased cardiac expression and plasma levels of Ang II and ET-1 in WT but not in Nox2 -deficient animals. Exposure of human microvascular endothelial cells to Ang II resulted in increased ET-1 production, which could be blocked by silencing NOX2 ( human NOX2 gene ). Ang II promoted NOX2 expression through induction of the Oct-1 (human/mouse octamer binding transcription factor 1 protein) and activation of the NOX2 promoter region containing Oct-1–binding sites. Stimulation of NOX2 expression by Ang II was associated with increased production of superoxide anions. Inhibition of Oct-1 by small interfering RNA reduced Ang II–induced NOX2 expression and superoxide anion production, and neutralization of superoxide by SOD (superoxide dismutase) abolished Ang II–stimulated ET1 ( human ET-1 gene ) promoter activity, ET1 mRNA expression, and ET-1 release. CONCLUSIONS: Ang II may promote ET-1 production in the endothelium in response to atherogenic diets through a mechanism that involves the transcription factor Oct-1 and the increased formation of superoxide anions by NOX2.

Aim: Dilatory microvascular function is impaired in ischemia/reperfusion injury in the kidney. Nitric oxide independent activators of soluble guanylyl cyclase (sGC) provide renal protection by dilating microvessels and preserving perfusion, but their efficacy declines after severe hypoxia. This study explores whether lipocalin‐2 (Lcn2), a key iron‐transporting protein, can restore the sGC‐mediated dilation in mouse afferent arterioles (AAs) after hypoxia/reoxygenation (H/R) and kidney transplantation.Methods: Dilation of isolated, angiotensin II (Ang II) pre‐constricted, AAs was induced by application of sGC activator cinaciguat after pre‐constriction with Ang II following H/R (H: 30 min, R: 10 min ± holo‐rLcn2, apo‐rLcn2, deferoxamine) and syngeneic kidney transplantation (cold ischemia: 30 min or 5.5 h, reperfusion: 20 h ± holo‐rLcn2) in C57BL/6 mice. To corroborate the dilatory function at the organ level, vascular relaxation was assessed using an isolated mouse kidney perfusion system.Results: Dilation of AAs was impaired following H/R. Pretreatment with holo‐rLcn2 (iron‐bound) preserved dilation, whereas apo‐rLcn2 (iron‐free) had no effect. The reversal of holo‐rLcn2's effect by deferoxamine confirmed the role of iron. AAs from kidney transplants showed reduced dilation compared to sham‐operated controls, with greater impairment following prolonged ischemia. Treatment with holo‐rLcn2 significantly improved dilatory function after extended cold ischemia (5.5 h), restoring it to levels seen with shorter ischemia (30 min). Ex vivo perfusion of the isolated mouse kidney with holo‐rLcn2 enhanced cinaciguat‐induced vascular relaxation, confirming its beneficial effect at the organ level.Conclusion: This study identifies a novel role for holo‐rLcn2 in preserving renal vascular function post‐H/R and kidney transplantation, apparently by upholding iron levels in vascular cells.

T-cell–mediated rejection (TCMR) remains a major cause of kidney transplant failure, despite being considered treatable. Its impact reflects a limited understanding of the underlying molecular mechanisms and their clinical consequences. To address this, we induced acute TCMR in mouse kidney transplants and profiled molecular changes using single-nucleus RNA sequencing (snRNA-seq), spatial transcriptomics and immunofluorescence. Results were compared with human snRNA-seq data from TCMR and stable allografts, as well as single-cell deconvolution analysis of bulk transcriptomic data from kidney transplant biopsies. Here we show that TCMR induces injured epithelial cell states in mouse kidney allografts, particularly in proximal tubules and thick ascending limbs. Spatial transcriptomics of these injured epithelial states demonstrated heterogeneous localization, interactions with immune cells and cellular microenvironments. Cross-species analysis confirmed similar severely injured epithelial states in human samples, whose abundances correlated with transplant survival and persisted despite TCMR resolution. Collectively, our results identify epithelial injury cell states as a determinant of outcome after TCMR.

Endothelin-1 is a key regulator of vascular tone and blood pressure in health and disease. We have recently found that ET-1 production in human microvascular endothelial cells (HMECs) can be promoted by angiotensin II (Ang II) through a novel mechanism involving octamer-binding transcription factor-1 (Oct-1), NADPH oxidase-2 (NOX2), and superoxide anions. As the formation of bioactive ET-1 also depends on endothelin-converting enzyme-1 (ECE-1), we investigated the transcriptional regulation of the ECE1 gene. We found that exposure of HMECs to Ang II resulted in a concentration- and time-dependent increase in ECE1 mRNA expression. Pharmacological inhibition of ECE-1 reduced Ang II-stimulated ET-1 release to baseline values. The effect of Ang II on ECE1 mRNA expression was associated with Oct-1 binding to the ECE1 promoter, resulting in its increased activity. Consequently, the Ang II-stimulated increase in ECE1 mRNA expression could be prevented by siRNA-mediated Oct-1 inhibition. It could also be abolished by silencing the NOX2 gene and neutralizing superoxide anions with superoxide dismutase. In mice fed a high-fat diet, cardiac expression of Ece1 mRNA increased in wild-type mice but not in Nox2-deficient animals. It can be concluded that Ang II engages Oct-1, NOX2, and superoxide anions to stimulate ECE1 expression in the endothelium.

T-cell mediated rejection (TCMR) remains a significant challenge after kidney transplantation and is associated with reduced allograft outcome. Previous research highlighted the critical role of TCMR-induced renal epithelial injury. Yet, the detailed cellular origin of these injury responses and the associated clinical implications remain poorly understood. To induce acute TCMR, we used mouse models of allogeneic (C57BL/6 to BALB/c and BALB/c to C57BL/6) kidney transplantation and syngeneic controls (C57BL/6 to C57BL/6 and BALB/c to BALB/c). Molecular changes were analyzed 7 days post-transplant using single-nucleus RNA sequencing and spatial transcriptomics. Results were compared with snRNA-seq data from three human TCMR biopsies and three stable allografts without rejection. The clinical impact of TCMR-induced epithelial injury was evaluated using marker gene sets on bulk transcriptomic data from 1292 kidney allografts, including 95 TCMR samples, with allograft outcome. Mouse kidneys from allogeneic transplants exhibited all hallmark histological features of TCMR. Single-nucleus RNA sequencing revealed TCMR-induced injured cell states and significant gene expression changes particularly in proximal tubules (PT) and thick ascending limbs (TAL). Spatial transcriptomics showed a heterogeneous spatial distribution of these injured cell states and proximity to leukocytes. Cross-species analysis confirmed similar injured PT and TAL cell states in human TCMR. Kidney allograft outcomes strongly correlated with TCMR-induced injured epithelial cell states. Distinct from other transplant biopsies, severe TAL injury emerged as a key factor for allograft survival after TCMR and was associated with reduced leukocyte proximity, suggesting potential non-immune mechanisms of epithelial damage.