Regulation of SARS CoV-2 host factors in the kidney and heart in rats with 5/6 nephrectomy—effects of salt, ARB, DPP4 inhibitor and SGLT2 blocker

Cardiovascular and renal diseases are considered as risk factors for increased coronavirus disease 2019 (COVID-19) disease severity and worse outcomes, including higher mortality. During the COVID-19 pandemic, tight control of glucose levels and prevention of complications associated with diabetes might be crucial in patients with diabetes to lower the susceptibility and severe course of COVID-19. Recent studies suggest that drugs interfering with the renin–angiotensin–aldosterone system (RAAS) or dipeptidyl peptidase 4 (DPP4) inhibitors can be used safely in patients with diabetes mellitus and COVID-19 [1‐3]. In addition, the use of sodium-glucose cotransporter 2 (SGLT2) blockers seems to be a promising adjunct treatment option in patients with SARS-CoV2 infection and type 2 diabetes mellitus (T2DM) whereas an increased risk of protracted ketonemia and diabetic ketoacidosis was also reported [4].

ACE2 plays a central role in the regulation of RAAS and is involved in cardiac function, the development of hypertension and diabetes mellitus [5]. ACE2 exerts its protective effects by converting pro-inflammatory and pro-hypertensive AngII into anti-inflammatory and anti-hypertensive Ang1-7. ACE2 has been identified as a receptor for coronaviruses, including SARS-CoV-2. Once attached to ACE2 through the binding with the receptor binding domain in the viral spike protein, it is primed by the host TMPRSS2, which can enhance this endocytic way of entry but is not essential [6, 7]. An alternative route of viral entry is the direct fusion of the viral envelope and the cell membrane which is ACE2- and TMPRSS2-independent [8]. Increased ACE2 expression was observed as a response to inflammation, heart failure, lung injury and fibrosis [9‐12] which led to increased AngII level and might facilitate the viral entry. In contrast, AngII can induce the internalization and degradation of ACE2 in an AT1R-dependent manner [13]. In addition, MERS-CoV binds to human DPP4/CD26 to infect host cells [14] and a recent study predicts the structure of the SARS-CoV-2 spike glycoprotein and its glycan shield pattern suggests that DPP4/CD26 might be a receptor for SARS-CoV-2 [15] which needs further validation. The increased presence of ACE2 or DPP4 might contribute to increased disease severity of infected patients.

In experimental preclinical models, the effects of RAAS blocking drugs on cardiac and renal ACE2 mRNA and/or protein expression led to controversial results. Ace2 mRNA expression was increased in the left ventricle of normotensive rats after lisinopril or losartan treatment [16] whereas no increase in Ace2 mRNA level was observed after coronary artery ligation and treatment with valsartan, ramipril or both compared to control [17]. In kidneys, telmisartan treatment resulted in increased expression of renal Ace2 mRNA expression [18]. No effects on renal Ace2 and Tmprss2 mRNA expression after telmisartan treatment were previously verified in an independent study [19]. In a recent study, it was shown that captopril and telmisartan both decrease kidney ACE2 protein in kidney membranes without significantly affecting protein abundance in total kidney lysates. Captopril significantly reduced ACE2 protein in kidney membranes while cytosolic ACE2 was increased [20]. Importantly, mice with comorbid diabetes (aging, high fat diet and streptozotocin-induced diabetes) are characterized by increased renal Ace2 mRNA expression but not further affected after telmisartan treatment which led to the conclusion that the increased ACE2 level is a consequence of the comorbidity and not an effect after RAAS blockade [19].

Dietary salt intake is a known risk factor for hypertension and is associated with an imbalance of the RAAS. The high salt diet fed spontaneously hypertensive rats (SHR) showed slightly decreased cardiac Ace2 mRNA and protein expression [21] and renal expression was attenuated in uni-nephrectomized rats with subsequent high salt diet intake [22] but the effects of RAAS blocking drugs in a salt-induced experimental model have not been investigated yet. Recognizing that people with chronic kidney disease, who are often consuming a high-salt diet and commonly prescribed RAAS blocking drugs and/or DPP4-inhibitor and/or SGLT2 blocker, are at increased risk of severe COVID-19 outcomes, we studied the expression profiles of ACE2 and TMPRSS2 and other genes involved in the RAAS in the kidney and the heart in a rat model that mimics this phenotype (impaired kidney function combined with a high salt intake – most patients consume unfortunately several times more salt than they actually should control blood pressure). Here we used the rat 5/6 nephrectomy model, one of the most well-established experimental non-diabetic CKD model which is characterized by increased hypertension, inflammation and fibrosis.

We used the ARB telmisartan, the DPP-4 inhibitor linagliptin and the SGLT2 blocker empagliflozin, in doses where we found positive pharmacodynamic action on systolic and diastolic blood pressures for all drugs under high salt diet conditions in the well-established experimental non-diabetic rat 5/6 nephrectomy. Our study shows that the induced effects on renal and cardiac mRNA and protein expression of the two key host proteins for SARS CoV-2 viral host cell entry (ACE2 and TMPRSS2) do not provide any evidence about facilitating SARS CoV-2 virus infection via the above-mentioned host receptors. The renal and cardiac gene expression level of Ace2 was not affected either under disease conditions or under treatment conditions. Recently, it was demonstrated that the renal Ace2 expression was not significantly altered in a STZ/high fat diet induced diabetic mouse model even if the animals were treated with ramipril (ACE inhibitor) or telmisartan [19], whereas the ACE2 protein expression was increased in this diabetic model independent of any treatment regimen. Our experimental model showed no effects on ACE2 protein expression after 5/6 nephrectomy in both the kidney and heart. Interestingly, high salt conditions led to significantly lower ACE2 level in the kidney which was normalized by linagliptin treatment, whereas the cardiac levels were unaffected. Linagliptin treatment significantly increased renal ACE2 level whereas this expression level was similar to the control groups sham and 5/6 Nx normal diet-fed rats. A recent study demonstrated that the administration of linagliptin significantly increased the ACE2 expression, which is consistent with this finding[35]. In addition, our study also revealed no significant effects on TMPRSS2 level (mRNA and protein) in the kidney which is consistent with previous finding in the experimental diabetes model [19]. However, the cardiac TMPRSS2 protein expression was significantly increased in the heart after 5/6 Nx and all drug interventions led to normalized cardiac TMPRSS2 suggesting a beneficial effect with regards to lower viral entry targets.

The discrepancy between changes in ACE2 and TMPRSS2 mRNA and protein expression was previously described in mouse and human studies [19, 36‐38] indicating that the expression of ACE2 and TMPRSS2 is regulated at the post-transcriptional level. Recent studies demonstrated that post-transcriptional regulation of ACE2 can occur via microRNAs [39] or protein shedding [40]. Single-cell sequencing analysis revealed that Ace2 is predominantly expressed in proximal tubules, whereas Tmprss2 is predominantly expressed in the distal nephron [41, 42]. In heart tissue, Qi et al. showed that the cardiomyocytes contain 6% ACE2-expressing cells and 0.8% TMPRSS2-expressing cells [43] which might explain the absence of detectable cardiac Tmprss2 mRNA levels in our study.

Our study revealed that only the expression of renin was affected by a more than tenfold suppressed level in placebo treated 5/6 Nx rats which has previously been described [44]. Telmisartan normalized the Ren mRNA level compared to linagliptin treated 5/6 Nx rats as detailed recently [45]. Also, empagliflozin restored the renin levels in 5/6 Nx rats. In a sub‐study of a double‐blind, randomized, placebo‐controlled, multicentre study (EMPA‐RESPONSE‐AHF) empagliflozin treatment was associated with a significant increase in plasma renin compared to placebo treated patients [46]. In sham kidneys, abundant expression of Ren mRNA was noted in the juxtaglomerular apparatus and not in the tubular epithelium whereas subtotal nephrectomy (STNx) resulted in decreased renin level based on the loss of renal mass. Moreover, altered distribution of renin gene expression was detected in the kidney of nephrectomized rats resulted by de novo renin expression in renal tubular epithelial cells with minimal or absent expression in the juxtaglomerular apparatus [47]. In perindopril-treated STNx rats, areas distant from the infarct scar demonstrated a pattern of renin gene transcription similar to that of control animals which is in line with our findings observed in telmisartan and empagliflozin treated rats.

A recently conducted comprehensive meta-analysis reported that RAAS-blocking drugs are not associated with increased risk of severe outcomes in COVID-19 patients and may further decrease all-cause mortality in COVID-19 patients[1]. Furthermore, DPP4 plays a role in SARS-CoV-2 infection as a co-receptor, and sDPP4 levels are upregulated in obesity and T2DM, possibly complicating disease outcomes, if these patients acquire COVID-19. DPP-4 inhibitors are currently investigated as a therapeutic approach preventing cardiovascular complications in COVID-19 due to their anti-inflammatory effects at the vascular level. Several clinical studies are currently under investigation which use RAAS-blocking drugs (BRACE-CORONA (NCT04364893)), gliptins (SIDIACO (NCT04365517); linagliptin trials NCT04371978 & NCT04341935) and SGLT2 inhibitors (DARE-19 (NCT04350593) in COVID-19 patients.

Our study also has limitations. First, it must be shown that our data in a rat CKD model are transferable to humans. It is also important to investigate other animal models to verify whether our observations regarding the regulation of SARS CoV-2 host factors can also be found in other CKD animal models and thus be generalized. In particular, CKD animal models with diabetes would also be of interest.

Our study revealed that telmisartan, linagliptin and empagliflozin are not associated with a further increase in ACE2 and TMPRSS2 levels in kidney and heart tissue under high-salt condition compared to sham control and normal diet-fed 5/6 nephrectomy rats. The results obtained in a preclinical, experimental non-diabetic kidney failure model need confirmation in ongoing interventional clinical trials. Ongoing clinical trials with above mentioned drugs in the setting of COVID-19 will ultimately clarify their potential involvement.