ABSTRACT
Background: There is an ongoing controversy as to whether SARS CoV-2 can infect the kidney parenchyma directly. To date, the presence of SARS CoV-2 in the kidney has been described mainly post-mortem in autopsy studies of patients who died of or with COVID-19, but this has not been examined in an experimental model where the timing of SARS-CoV-2 infection can be defined. We used transgenic mice expressing human ACE2 (k18hACE2) susceptible to lethal SARS-CoV-2 infection to study this issue directly on kidney tissue taken at defined time points and using lung tissue as positive control. Method(s): Transgenic k18hACE2 mice were inoculated with 3x104 PFU SARSCoV-2 in a BSL-3 facility. Kidneys and lungs were removed from the animals sacrificed on days 5 to 7 and used for histology (PAS-staining), immunofluorescence (IF) of the S1 spike protein of SARS-CoV-2 and measurement of viral load by plaque assay. Kidney samples were additionally evaluated by IF using kidney injury markers NGAL and KIM-1. Result(s): Kidney tissue stained using an anti-S1-spike antibody showed negative results in all samples (n=15). By plaque assay, viral titers were also not detectable in any of the kidneys. By contrast, lungs from infected mice showed strong staining for the S1 spike protein in 13 of 14 cases and this was associated with positive viral titers in all lung samples. Despite severe lung disease, only mild and variable kidney damage was observed by histopathology. Positive staining for NGAL in the proximal tubules was consistently seen, while KIM-1 staining was rarely positive. Conclusion(s): In a transgenic mouse model with lethal SARS-CoV-2 infection and severe lung but mild kidney disease there is no evidence of S1 spike protein in the kidney, which is consistent with lack of detection of replicating virus by plaque assay.
ABSTRACT
Background: We have previously reported that ACE2 618-DDC-ABD, a soluble ACE2 protein with extended duration of action and increased binding affinity for SARSCoV-2, provides lung and kidney protection in a lethal mouse model of SARS-CoV-2 infection. Moreover, we showed that this protein also neutralizes the gamma and delta variant SARS-CoV-2 infection in Vero E6 cells. As omicron is most prevalent SARSCoV-2 variant we tested whether ACE2 618-DDC-ABD can also neutralize this variant and hypothesized that it is more sensitive to mouse ACE2 as well as human ACE2. Method(s): The omicron BA.1 SARS-CoV-2 strain was incubated with various concentrations of ACE2 618-DDC-ABD (0-180ug/ml) for 1 hour at 37degreeC. Human ACE2 1-740 and mouse ACE2 1-740 were used as controls at the same concentrations. These mixtures were then used to infect Vero E6 cells. Cells were allowed to grow for 3-4 days until a noticeable cytopathic effect was observed in control wells (0mug/ml soluble ACE2 proteins). Cell numbers were assessed by staining cells with crystal violet and reading absorbance of each well at 595 nm. Values were then normalized to the 0mug/ml control and expressed as a percentage of the mock (no virus) control wells. Result(s): ACE2 618-DDC-ABD (red) neutralized the omicron BA.1 variant at all concentrations tested and to a similar extent as native human soluble ACE2 1-740 (blue) used as control. Native mouse ACE2 1-740 (black) also neutralized infection completely at high concentrations while lower concentrations were less effective as compared to low concentrations of ACE2 618-DDC-ABD or human ACE2 1-740.
ABSTRACT
Background: We have previously shown that in the ischemia reperfusion model of AKI kidney ACE2 activity decreases and that the administration of a shorter soluble ACE2 variant markedly attenuates AKI in terms of GFR and kidney histology (Shirazi et al, ASN 2019). Here, we report the effect of a novel ACE2 variant designed to prevent/ treat SARS-CoV-2 in transgenic k18-hACE2 mice infected with a lethal viral dose. Methods: In a BSL-3 facility, transgenic k18-hACE2 mice were infected intranasally with 2×104 PFU SARS-CoV-2. ACE2 1-618-DDC-ABD was administered intranasally and intra-peritoneally 1 hour prior to viral challenge as well as 24 and 48 hours afterwards for a total of 3 doses. Infected control animals received PBS at the same time-points. Kidneys were removed from all animals and examined by light microscopy (LM) histologically and for apoptosis, using PAS and TUNEL staining, respectively. Results: In mice infected with SARS-CoV-2, variable degrees of AKI were found by LM with the following features seen in the few most severe cases: proximal tubule brush border loss (black arrows, figure 1A and B), cytolysis (red arrow, figure 1A), tubular basement membrane disruption (blue arrows, figure 1A and B) and apoptosis (white arrows, figure 1A, B, D and E). In animals treated with ACE2 1-618-DDC-ABD, survival was near 100% and proximal tubular kidney injury was absent or markedly attenuated with less proximal tubule injury (figure 1C) and minimal apoptosis (figure 1F). Glomeruli appeared ischemic (figure 1B, green arrow) but otherwise normal without evidence of thrombosis. Conclusions: Kidneys from a transgenic mouse susceptible to SARS-CoV-2 infection, like patients with COVID-19, displays variable degrees of proximal tubular injury suggesting that this model can be useful to study AKI in COVID-19. Mice that received soluble ACE2 1-618-DDC-ABD protein were essentially protected from AKI suggesting a potential preventative/therapeutic role for soluble ACE2 in this otherwise pharmacologically untreatable devastating disease.
ABSTRACT
Background: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2) as the main receptor to enter the target cells. A novel soluble ACE2 protein with increased duration of action and binding capacity to exert a decoy effect as a way to intercept SARS-CoV-2 from binding to membrane-bound ACE2 was generated. The protein was administered to a lethal mouse model of COVID-19 to examine its efficacy. Methods: A human soluble ACE2 variant fused with a 5kD albumin binding domain (ABD) was linked via a dimerization motif hinge-like 4-cysteine dodecapeptide to improve binding capacity to the SARS-CoV-2. This novel protein (ACE2 1-618-DDCABD) was administered intranasally and intraperitoneally prior to viral inoculation and on the two following consecutive days. Infected animals were observed for weight, clinical score and mortality in a BSL-3 facility. Upon sacrifice, lung histopathology was evaluated, and viral loads were measured by plaque assay. Results: Infected mice that received ACE2-1-618-DDC-ABD developed only moderate disease assessed by a clinical score, modest weight loss and lung histology. At 6 days, mortality was totally prevented in the treated group (figure), lung histopathology was markedly improved and viral lung and brain titers reduced or non-detectable. By contrast, in untreated animals, lung histology revealed extensive pulmonary alveolar hemorrhage and mononuclear infiltrates, and they all became severely ill and had to be euthanized by day 6/7 (figure). Conclusions: This study demonstrates for the first time in vivo the preventative/ therapeutic efficacy of a soluble ACE2 protein in a preclinical animal model.
ABSTRACT
Background: Studies at the single cell level have revealed that the localization of TMPRSS2 is in the distal nephron whereas ACE2 is in the proximal tubule. Since TMPRSS2 is a serine protease necessary for activation of the SARS-CoV-2 S spike protein after it binds to ACE2, this spatial separation would make it difficult to explain how SARS-CoV-2 can infect the kidney. The purpose of this study was to examine the localization of these proteins by immunofluorescence in the kidneys of patients who died from COVID-19. Methods: Human kidney slides from a Northwestern COVID-19 repository were used after IRB approval. Slides from paraffin-embedded blocks were probed with different antibodies (ACE2, TMPRSS2, ACE, NBC-1, Aquaporin 2) for immunofluorescence studies. Mouse kidneys were also examined as additional controls. Results: In mouse kidneys, TMPRSS2 was found in the brush border of proximal tubules and co-localized strongly with ACE2. Similarly, in human kidneys from patients who died from COVID-19, with or without AKI and from non-COVID-19 subjects, ACE2 and TMPRSS2 co-localized in the proximal tubule. TMPRSS2 and ACE2 also co-localized with ACE, a marker of the apical proximal tubule and to a lesser extent with NBC-1, a marker of the basolateral proximal tubule membrane. By contrast, TMPRSS2 and ACE2 did not co-localize with Aquaporin 2, a marker of principal cells in the collecting tubule. Conclusions: In both mouse and human kidneys, ACE2 and TMPRSS2 co-localize in the proximal tubule. In kidneys from patients with COVID-19 with or without AKI obtained at autopsy, both proteins co-localized in the proximal tubule but not in the collecting tubule. Contrary to what was suggested from single-cell mRNA analysis?, the co-localization of both proteins in the proximal tubule would make it possible for the SARS-CoV-2-ACE2 complex to be activated when coronavirus reaches the kidney.
ABSTRACT
Background: There have been concerns that ACE inhibitors and Ang II receptor blockers may cause an increase in full length (FL) membrane bound ACE2, the main receptor for SARs-CoV-2, that could enhance the risk and worsen the clinical course of COVID -19. Information on the impact of ACE deficiency and AT1 blockade on ACE2 expression at target sites is required to understand this issue. Methods: Kidneys from two genetic models of kidney ACE ablation and mice treated with captopril or telmisartan were used to examine ACE2 in isolated kidney and lung membranes. Results: In global ACE KO mice, ACE2 protein abundance in kidney membranes was reduced to 42 % of wild type, p < 0.05. In ACE 8/8 mice that over-express cardiac ACE protein but has no kidney ACE expression, ACE2 protein in kidney membranes wasalso decreased (38 % of the WT, p<0.01). In kidney membranes from mice that received captopril or telmisartan for 2 weeks there was a reduction in ACE2 protein to the level of 37%, p<0.01 and 76%, p <0.05 of that of vehicle control mice, respectively. In lung membranes the expression of ACE2 was very low and not detected by western blotting but no significant differences in terms of ACE2 activity could be detected in mice treated with captopril (118% of control) or telmisartan (93% of control). Conclusions: Genetic kidney ACE deficiency, suppressed ACE enzyme activity by Captopril or blockade of the AT1 receptor with telmisartan are all associated with a decrease in ACE2 expression in kidney membranes. These findings altogether suggest that ACE2 protein abundance at two potential target sites for SARS-CoV-2 infection is decreased or unaffected by RAS blockers. Since these medications do not increase ACE2 expression in lung or kidney epithelia, we conclude that they likely would not pose a risk for increased susceptibility to COVID -19.