Keywords

Pregnancy Glomerular filtration rate (GFR) Angiotensin-converting enzyme 2 (ACE2) Transmembrane protease serine 2 (TMPRSS2) Severe coronavirus disease 2019 (COVID-19)

Abstract

Background/Aims:

Pregnancy is associated with changes in renal hemodynamics, such as increases in renal blood flow and the glomerular filtration rate (GFR). Angiotensin-converting enzyme 2 (ACE2), a transmembrane glycoprotein involved in vasodilation, also acts as a receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during coronavirus disease 2019 (COVID-19).

Methods:

Using rats on pregnancy day 16 and postpartum day 5, we examined the histopathological changes in rat kidneys during and after pregnancy. The expressional changes in renal angiotensin-converting enzyme 2 (ACE2) and angiotensin (1-7) (Ang (1-7)) were examined, together with those of transmembrane protease serine 2 (TMPRSS2).

Results:

In pregnant rats, the renal arterioles and venules as well as the glomerular capillaries were markedly dilated, indicating renal vasodilation. Immunohistochemistry demonstrated increased ACE2 and Ang (1-7) expression within the proximal renal tubules during pregnancy, which then returned to the virgin levels in the postpartum period. Additionally, the proximal tubular expression of ACE2 and TMPRSS2 was similarly enhanced during pregnancy.

Conclusion:

As ACE2 and Ang (1-7) exert vasodilatory properties, they were considered responsible for renal vasodilation and the subsequent increase in GFR. Further, the similar distribution and enhanced expression of ACE2 and TMPRSS2 in the proximal renal tubules during pregnancy suggested their roles in the development of acute kidney injury (AKI) following COVID-19 in pregnancy. This study highlights the physiological and pathological significance of ACE2 during pregnancy.

Introduction

Pregnancy is associated with changes in systemic hemodynamics, such as an increase in plasma volume and cardiac output but a decrease in blood pressure as a result of peripheral vasodilation [1]. Peripheral vasodilation during pregnancy is primarily caused by sympathetic nerve-related tonus changes in systemic vascular resistance and the influence of multiple vasodilatory factors, including nitric oxide, relaxin and progesterone [1-3]. During pregnancy, renal hemodynamic parameters, such as renal blood flow and glomerular filtration rate (GFR), also increase because of plasma volume expansion [4, 5]. However, the gestational increase in the GFR is usually much greater than that in the plasma volume or cardiac output [6, 7], suggesting the involvement of additional mechanisms underlying the renal hemodynamic changes during pregnancy. In our previous study, renal accumulation of prostaglandin E₂ (PGE₂) during pregnancy was found to directly dilate the afferent arterioles in the glomeruli, thus increasing the GFR [8]. Additionally, independent of the systemic renin-angiotensin system (RAS), which is responsible for systemic hemodynamic changes [9], a local or intrarenal RAS, directly affects renal function or causes renal damage under certain pathological conditions [10]. In the RAS, angiotensin converting-enzyme 2 (ACE2), a transmembrane glycoprotein, converts vasoconstrictive angiotensin II (Ang II) into vasodilative angiotensin (1-7) (Ang (1-7)), thereby exerting vasodilatory properties [11]. ACE2 and Ang (1-7) are within the interactive network of vasodilator factors in pregnancy, participating in the systemic and local hemodynamic adaptations [12].

On the other hand, during coronavirus disease 2019 (COVID-19), ACE2 acts as a host cell surface receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [13]. Further, in diseases such as heart failure, chronic kidney disease and chronic obstructive pulmonary disease, enhanced ACE2 expression in damaged organs is strongly associated with the development of severe COVID-19 [14-16]. Since pregnancy is a risk factor for COVID-19 and is associated with the development of kidney injury after infection [17-19], renal ACE2 expression may be involved in the pathogenesis. In the present study, we elucidated the physiological and pathological significance of ACE2 during pregnancy by examining the histopathological features of the kidneys in pregnant and postpartum rats.

Materials and Methods

Animal Preparation
Eleven- to 12-week-old female Wistar rats (Japan SLC Inc., Shizuoka, Japan) were mated with fertile male rats. The day when vaginal plugs were observed was designated as pregnancy day 0. Pregnant rats and those prior to becoming pregnant had free access to standard rat chow and water throughout the experiment and were maintained in a humidity- and temperature-controlled room on a 12-hour light-dark cycle. On pregnancy days 16 and postpartum days 5, the rats were deeply anaesthetized with isoflurane, and then killed by cervical dislocation (n=3, respectively). In rats, since GFR increases most prominently at the later stage of pregnancy and restores by postpartum day 5 [6, 8], we examined the rats on these gestational days. Age-matched virgin female rats were used as controls (n=6). Kidneys were harvested for histological examination. All experimental protocols described in the present study were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Miyagi University. Animal protocols were approved by the Animal Care and Use Committee of Miyagi University (No. 2024-03). The animal study was reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

Histological Analyses
Renal cross sections were fixed in 4% paraformaldehyde, embedded in paraffin, deparaffinized in xylene, and then 3-μm sections were stained with hematoxylin-eosin (H&E). Staining for CD31 was conducted at the laboratory of Morpho Technology Co. Ltd (Sapporo, Japan).

Immunohistochemistry
The 3-μm paraffin sections of 4% paraformaldehyde-fixed kidneys were placed in citrate-buffered solution (pH 6.0) and then boiled for 30 min for antigen retrieval. Endogenous peroxidase was blocked with 3% hydrogen peroxide, and nonspecific binding was blocked with 10% BSA. Primary antibodies were as follows: Mouse anti-ACE2 (1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, U.S.A.), anti- transmembrane protease serine 2 (TMPRSS2) (1:50; Santa Cruz Biotechnology, Inc) and rabbit anti-Ang (1-7) (1:50; Cloud-Clone Corp., Katy, TX, U.S.A.). Diaminobenzidine substrate (Sigma Chemical Co., St. Louis, MO, USA) was used for the color reaction. At the end of the staining, the sections were counterstained with hematoxylin. The secondary antibody alone was consistently negative on all sections. As we described in our previous studies [20, 21], bright-field images were acquired from randomly selected, nonoverlapping high-power fields of view (more than 10 views from six virgin rats, three pregnant rats and three postpartum rats, respectively). The ACE2, Ang (1-7) or TMPRSS2 deposition, expressed as percentages of each protein-positive areas relative to the total areas, was averaged and quantified.

Results

Histological features of the kidneys in pregnant rats
Previous studies have revealed an increase in the glomerular volume and capillary wall surface area during pregnancy [22, 23]. However, these studies did not provide clear morphological evidence for these changes. In the present study, to reveal the influence of pregnancy on renal morphology, we carefully examined the histological features of rat kidneys on pregnancy day 16 and compared them with those in virgin rats and on postpartum day 5 (Fig. 1). Sections of kidneys from virgin rats showed normal vascular structures such as arterioles and venules, which were localized in parallel within the cortical tubulointerstitium (Fig. 1Aa). However, in rats on pregnancy day 16, both the arterioles and venules were markedly dilated within the intact tubulointerstitium (Fig. 1Ab). On postpartum day 5, the dilated blood vessels had returned to a size comparable to that in the virgin rats (Fig. 1Abc). Further, compared with the glomeruli from the kidneys of virgin or postpartum rats (Fig. 1Ad and f), those from pregnant rats were mostly enlarged (Fig. 1Ae). In these glomeruli, the capillaries were markedly dilated and the Bowman’s capsule was enlarged, indicating renal vasodilation and a subsequent increase in GFR. However, immunohistochemistry for CD31, which is a marker for endothelial cells, demonstrated an intact glomerular endothelium in the kidneys of pregnant rats (Fig. 1Bb vs. a, c), indicating that renal vasodilation during pregnancy was reversible without any accompanying structural damages.

ACE2 and Ang (1-7) expression in the kidneys of pregnant rats
As ACE2 and Ang (1-7) exert vasodilatory properties [11], we examined the renal expression of these proteins in pregnant and postpartum rats (Fig. 2 and 3). In the kidneys of virgin rats, consistent with previous findings [16, 24, 25], immunohistochemistry for ACE2 demonstrated positive expression in the brush border or apical membrane of the proximal tubules (Fig. 2Aa). In the kidneys on pregnancy day 16, ACE2 protein expression was also observed in the cytoplasm of proximal tubular cells (Fig. 2Ab). By postpartum day 5, the protein expression returned to a localization comparable to that in the kidneys of virgin rats (Fig. 2Ac vs. a). Quantitatively, the percentages of ACE2-positive areas relative to the total areas significantly increased during pregnancy (virgin, 12.0 ± 0.79 % vs. pregnancy, 26.6 ± 0.95 %; n=12, p-value = 1.29326022005154E-11 <0.05), which returned to the virgin value by postpartum day 5 (Fig. 2B). Further, as previously demonstrated [26-28], the expression of Ang (1-7) was widely but weakly distributed within the cytoplasm of proximal tubular cells in the kidneys of virgin rats (Fig. 3Aa). However, in the kidneys on pregnancy day 16, cytoplasmic expression became more intense (Fig. 3Ab), which by postpartum day 5, returned to a level similar to that in the virgin rats (Fig. 3Ac). There were statistically significant differences in the Ang (1-7) expression during pregnancy and postpartum (pregnancy, 34.3 ± 0.87 % vs. postpartum, 15.8 ± 0.85 %; n=15, p-value = 2.29238286339473E-15 <0.05) (Fig. 3B).

Renal TMPRSS2 expression in pregnant rats
In COVID-19, ACE2 acts as a host cell surface receptor for SARS-CoV-2 [13]. When the virus enters host cells, its spike protein binds to ACE2, which is predominantly expressed in the respiratory tract, lungs, heart, and kidneys [29]. One of the transmembrane proteases of host cells, TMPRSS2, cleaves viral spike proteins, thus activating and facilitating viral entry [29]. In the present study, consistent with our previous findings [16], immunohistochemistry for TMPRSS2 demonstrated positive staining in the brush border or apical membrane of proximal tubules of the kidneys from virgin rats (Fig. 4Aa), showing localization pattern similar to that of ACE2 (Fig. 2Aa). In the kidneys of pregnant rats, TMPRSS2 expression was observed in the cytoplasm of proximal tubular cells (Fig. 4Ab). However, in the kidneys of postpartum rats, protein localization returned to the brush border (Fig. 4Ac), showing an expression pattern almost identical to that of ACE2 (Fig. 4A vs. 2A). This was also quantitatively confirmed, as the percentages of TMPRSS2-positive areas relative to the total areas significantly increased during pregnancy (virgin, 15.3 ± 1.56 % vs. pregnancy 26.7 ± 0.68 %; n=14, p-value = 2.53090205408338E-6 <0.05) and returned to the virgin value by postpartum day 5 (Fig. 4B).

Discussion

In normal pregnancy, increased activities of the systemic RAS and sympathetic nerves result in sodium and water retention throughout the body, leading to plasma volume expansion [1]. In the RAS, ACE2 degrades and converts vasoconstrictive Ang II into vasodilatory Ang (1-7), thus exerting vasodilatory properties [11]. According to recent human and animal studies, in addition to serum concentrations [30], placental and renal concentrations of ACE2 are also elevated during pregnancy [27, 31], suggesting their direct effects on organ function. Previous studies have shown the increased renal expression of ACE2 and Ang (1-7) during pregnancy [26, 27, 32]. In this context, the present study further revealed that these protein expression during pregnancy specifically increased within the proximal tubular cells, which then returned to the virgin levels in the postpartum period (Fig. 2 and 3). Additionally, our study actually revealed that these changes were well synchronized with those in vascular tone observed during pregnancy (Fig. 1). Based on these results, the increased renal expression of ACE2 and Ang (1-7) in pregnancy was thought to be responsible for renal vasodilation, subsequently causing an increase in renal blood flow and the GFR (Fig. 5). Besides, as Ang (1-7) directly dilates the afferent arterioles in the glomeruli [33], it would bring about an additional increase in the GFR during pregnancy. In previous animal studies using rodents, such as rats or mice, short term or chronic infusion of Ang II decreased the renal expression of ACE2 [34-36]. In conditions such as renal hypoperfusion, Ang II more preferentially constricts the efferent arterioles than afferent arterioles in the glomeruli and thus maintains the GFR [37, 38]. In this context, the decreased ACE2 caused by Ang II infusion may be a negative feedback mechanism to modulate the excessive increase in GFR by reducing the renal blood flow.

Patients with severe COVID-19 sometimes develop acute kidney injury (AKI) [39, 40]. Recent studies have reported the involvement of direct viral invasion, renal medullary hypoxia due to hypoperfusion, rhabdomyolysis, and microangiopathy secondary to a cytokine storm in this pathogenesis. Thus, the pathological features of AKI following COVID-19 are typically characterized by proximal tubular damage due to acute tubular necrosis or interstitial inflammation [39-41]. Pregnancy is a risk factor for severe COVID-19 [17], and pregnant women are prone to develop AKI following COVID-19 [18, 19]. Our results showed that the proximal tubular expression of ACE2 and TMPRSS2 was similarly enhanced during pregnancy, which then returned to the virgin levels in the postpartum period (Fig. 2 and 4); notably, their distribution was almost overlapped with that of AKI lesions following COVID-19 [39-41]. ACE2 and TMPRSS2 are transmembrane proteins that facilitate SARS-CoV-2 entry into cells, allowing the virus to stimulate pro-inflammatory cytokine production and induce organ injury [29]. In chronic diseases, such as heart failure, chronic kidney disease, and chronic obstructive pulmonary disease, enhanced expression of these proteins in damaged organs results in the development of severe COVID-19 [14-16]. Therefore, enhanced proximal tubular expression of ACE2 and TMPRSS2 during pregnancy may be responsible for the development of AKI following COVID-19 in this condition (Fig. 5). Given this pathogenesis, the use of ACE2 inhibitors or soluble forms of ACE2 protein, which directly block the cellular entry of SARS-CoV-2 [42-44], as well as the use of natural products that directly or indirectly modulate ACE2 activity [45] would be beneficial in preventing AKI in pregnant women. However, since our observations are still hypothesis-generating, functional validation would additionally be required to confirm the causality. The possible approaches to address this issue in the future would include ACE2 inhibition studies, SARS-CoV-2 spike protein challenges or using SARS-CoV-2 infection models in pregnant animals. Because of pregnancy-related hemodynamic changes involving the kidneys, pregnant women are vulnerable to develop AKI independently from COVID-19 [46]. Although the histopathology of kidneys in pregnant animals with AKI has not been examined, previous studies clearly demonstrated the decreased renal tubular expression of ACE2 in animal models with ischemic AKI [47-49]. Therefore, in cases of AKI following COVID-19, ACE2 expression may become downregulated after ACE2 mediates SARS-CoV-2 entry into cells [47]. This can be the phylactic mechanism against the further progression of AKI during pregnancy by halting the additional entry of SARS-CoV-2.

Limitations of our study include the semi-quantitative nature of immunohistochemistry and the lack of biochemical validation, such as protein quantification by Western blotting or enzyme-linked immunosorbent assay (ELISA). Additionally, the sample size was relatively small in our study, with n=3 for the pregnant and postpartum group of rats. To overcome these limitations, we carefully executed immunohistochemical analyses and confirmed the consistency and reproducibility of our results in each group of kidney samples. Besides, the percentages of each protein-positive areas, which were averaged from randomly selected, nonoverlapping high-power fields of view (more than 10 views from six virgin rats, three pregnant rats and three postpartum rats, respectively), demonstrated a small statistical variability in each group. However, more quantitative analyses of ACE2, Ang (1-7) and TMPRSS2 expression would be required in the future to strengthen the significance of our findings. Additionally, since sex hormones, such as estrogen, progesterone and androgen [50-53], and systemic inflammatory mediators, such as interleukins, interferons and tumor necrosis factor-a (TNF-a) [54-56], positively or negatively influence ACE2 or TMPRSS2 expression in the serum, lung and placenta during pregnancy, these factors may also be quantitatively analyzed in pregnant kidneys.

Conclusion

In the kidneys of pregnant rats, in addition to renal arterioles and venules, glomerular capillaries are markedly dilated and the Bowman’s capsule is enlarged, indicating renal vasodilation. Immunohistochemistry demonstrated increased ACE2 and Ang (1-7) expression in the proximal renal tubules of pregnant rats. As these proteins exert vasodilatory properties, they are considered responsible for renal vasodilation and the subsequent increase in GFR during pregnancy.

Acknowledgements

We thank the people at Morpho Technology Co. Ltd (Sapporo, Japan) for their technical support.

Author contributions
YK performed the experiments and analyzed the data. IK designed the experiments, interpreted the results, and wrote the manuscript. All the authors have read and approved the final version of the manuscript.

Funding
This study was supported by the Tojuro Iijima Foundation for Food Science and Technology, No. 2023-46, and Miyagi Kidney Foundation Grant of IK.

Availability of data and materials
The data used to support the findings of this study are available from the corresponding author upon request.

Ethics approval and consent to participate
This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of Miyagi University, which included ethical considerations.

Consent for publication
Not applicable.

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