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Background: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2) as a main receptor to enter target cells. The goal of this study was to demonstrate the preclinical efficacy of a novel soluble ACE2 protein with increased duration of action and binding capacity in a lethal mouse model of COVID-19. Methods: A human soluble ACE2 variant fused with an albumin binding domain (ABD) was linked via a dimerization motif hinge-like 4-cysteine dodecapeptide (DDC) to improve binding capacity to SARS-CoV-2. This novel soluble ACE2 protein (ACE2 1-618-DDC-ABD) was then administered intranasally and intraperitoneally to mice prior to intranasal inoculation of SARS-CoV-2 and then for two additional days post viral inoculation. Results: Untreated animals became severely ill and all had to be humanely euthanized by day 6/7 and had pulmonary alveolar hemorrhage with mononuclear infiltrates. In contrast, all but one mouse infected with a lethal dose of SARS-CoV-2 that received ACE2-1-618-DDC-ABD survived. In the animals inoculated with SARS-CoV-2 that were untreated, viral titers were high in the lungs and brain but virus was absent in the kidneys. However, some untreated animals had variable degrees of kidney proximal tubular injury with increased NGAL and TUNEL staining indicating attenuation of the proximal tubular brush border. In contrast, viral titers in the lung and brain were reduced or non-detectable in mice that received ACE2 1-618 DDCABD, and the animals developed only moderate disease as assessed by a near-normal clinical score, minimal weight loss, and improved lung and kidney injury Conclusions: This study demonstrates the preclinical efficacy of a novel soluble ACE2 protein, termed ACE2 1- 618-DDC-ABD, in a lethal mouse model of SARS-CoV-2 infection that causes severe lung injury as well as variable degrees of moderate proximal tubular injury.

Overexpression of the proximal tubular enzyme -inositol oxygenase (MIOX) induces oxidant stress However, the relevance of MIOX to tubular pathobiology remains enigmatic. To investigate the role of MIOX in cisplatin-induced tubular AKI, we generated conditional MIOX-overexpressing transgenic (MIOX-TG) mice and MIOX-knockout (MIOX) mice with tubule-specific MIOX overexpression or knockout, respectively. Compared with cisplatin-treated wild-type (WT) mice, cisplatin-treated MIOX-TG mice had even greater increases in urea, creatinine, and KIM-1 levels and more tubular injury and apoptosis, but these effects were attenuated in cisplatin-treated MIOX mice. Similarly, MIOX-TG mice had the highest and MIOX mice had the lowest renal levels of Bax, cleaved caspase-3, and NADPH oxidase-4 expression and reactive oxygen species (ROS) generation after cisplatin treatment. , cisplatin dose-dependently increased ROS generation in LLC-PK1 cells. Furthermore, MIOX overexpression in these cells accentuated cisplatin-induced ROS generation and perturbations in the ratio of GSH to oxidized GSH, whereas MIOX-siRNA or-acetyl cysteine treatment attenuated these effects. Additionally, the cisplatin-induced enhancement of p53 activation, NF-B binding to DNA, and NF-B nuclear translocation in WT mice was exacerbated in MIOX-TG mice but absent in MIOX mice. , MIOX-siRNA or NAC treatment reduced the dose-dependent increase in p53 expression induced by cisplatin. We also observed a remarkable influx of inflammatory cells and upregulation of cytokines in kidneys of cisplatin-treated MIOX-TG mice. Finally, analysis of genomic DNA in WT mice revealed cisplatin-induced hypomethylation of the MIOX promoter. These data suggest that MIOX overexpression exacerbates, whereas MIOX gene disruption protects against, cisplatin-induced AKI.

Conceivably, upregulation of myo -inositol oxygenase (MIOX) is associated with altered cellular redox. Its promoter includes oxidant-response elements, and we also discovered binding sites for XBP1, a transcription factor of endoplasmic reticulum (ER) stress response. Previous studies indicate that MIOX’s upregulation in acute tubular injury is mediated by oxidant and ER stress. Here, we investigated whether hyperglycemia leads to accentuation of oxidant and ER stress while these boost each other’s activities, thereby augmenting tubulointerstitial injury/fibrosis. We generated MIOX-overexpressing transgenic (MIOX-TG) and MIOX knockout (MIOX-KO) mice. A diabetic state was induced by streptozotocin administration. Also, MIOX-KO were crossbred with Ins2 Akit a to generate Ins2 Akita /KO mice. MIOX-TG mice had worsening renal functions with kidneys having increased oxidant/ER stress, as reflected by DCF/dihydroethidium staining, perturbed NAD-to-NADH and glutathione-to-glutathione disulfide ratios, increased NOX4 expression, apoptosis and its executionary molecules, accentuation of TGF-β signaling, Smads and XBP1 nuclear translocation, expression of GRP78 and XBP1 (ER stress markers), and accelerated tubulointerstitial fibrosis. These changes were not seen in MIOX-KO mice. Interestingly, such changes were remarkably reduced in Ins2 Akita /KO mice and, likewise, in vitro experiments with XBP1 siRNA. These findings suggest that MIOX expression accentuates, while its deficiency shields kidneys from, tubulointerstitial injury by dampening oxidant and ER stress, which mutually enhance each other’s activity.

Regulated cell death (RCD), distinct from accidental cell death, refers to a process ofwell-controlled programmed cell death with well-defined pathological mechanisms. In thepast few decades, various terms for RCDs were coined, and some of them have beenimplicated in the pathogenesis of various types of acute kidney injury (AKI). Cisplatin iswidely used as a chemotherapeutic drug for a broad spectrum of cancers, but its usagewas hampered because of being highly nephrotoxic. Cisplatin-induced AKI is commonlyseen clinically, and it also serves as a well-established prototypic model for laboratoryinvestigations relevant to acute nephropathy affecting especially the tubular compartment.Literature reports over a period of three decades indicate that there are multiple types ofRCDs, including apoptosis, necroptosis, pyroptosis, ferroptosis and mitochondrial permeability transition (MPT)-mediated necrosis, and some of them are pertinent to the pathogenesis of cisplatin-induced AKI. Interestingly, myo-inositol metabolism, a vital biological process that is largely restricted to the kidney, seems to be relevant to thepathogenesis of certain forms of RCDs. A comprehensive understanding of the RCDs in cisplatin-induced AKI and their relevance to myo-inositol homeostasis may yield novel therapeutic targets for the amelioration of cisplatin-related nephropathy.