Figures
Abstract
Background
Angiotensin-converting enzyme 2 (ACE2) has been implicated in the pathogenesis of experimental kidney disease. ACE2 is on the X chromosome, and in mice, deletion of ACE2 leads to the development of focal segmental glomerulosclerosis (FSGS). The relationship between sex and renal ACE2 expression in humans with kidney disease is a gap in current knowledge.
Methods
We studied renal tubulointerstitial microarray data and clinical variables from subjects with FSGS enrolled in the Nephrotic Syndrome Study Network (NEPTUNE) study. We compared relationships between ACE2 expression and age, estimated glomerular filtration rate (eGFR), urinary albumin to creatinine ratio (UACR), interstitial fibrosis, tubular atrophy, and genes implicated in inflammation and fibrosis in male and female subjects.
Results
ACE2 mRNA expression was lower in the tubulointerstitium of males compared to females (P = 0.0026). Multiple linear regression analysis showed that ACE2 expression was related to sex and eGFR but not to age or treatment with renin angiotensin system blockade. ACE2 expression is also related to interstitial fibrosis, and tubular atrophy, in males but not in females. Genes involved in inflammation (CCL2 and TNF) correlated with ACE2 expression in males (TNF: r = -0.65, P < 0.0001; CCL2: r = -0.60, P < 0.0001) but not in females. TGFB1, a gene implicated in fibrosis correlated with ACE2 in both sexes.
Citation: Maksimowski NA, Scholey JW, Williams VR, Nephrotic Syndrome Study Network (NEPTUNE) (2021) Sex and kidney ACE2 expression in primary focal segmental glomerulosclerosis: A NEPTUNE study. PLoS ONE 16(6): e0252758. https://doi.org/10.1371/journal.pone.0252758
Editor: Jaap A. Joles, University Medical Center Utrecht, NETHERLANDS
Received: February 18, 2021; Accepted: May 22, 2021; Published: June 7, 2021
Copyright: © 2021 Maksimowski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: The Nephrotic Syndrome Study Network Consortium (NEPTUNE), U54‐DK‐083912, is a part of the National Institutes of Health (NIH) Rare Disease Clinical Research Network (RDCRN), supported through a collaboration between the Office of Rare Diseases Research, National Center for Advancing Translational Sciences and the National Institute of Diabetes, Digestive, and Kidney Diseases. Additional funding and/or programmatic support for this project has also been provided by the University of Michigan, the NephCure Kidney International and the Halpin Foundation.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The renin angiotensin system (RAS) plays a key role in the progression of chronic kidney disease (CKD), and blockade of the RAS is a centerpiece of the current clinical approach to the treatment, especially in CKD associated with proteinuria. Our understanding of the RAS and angiotensin peptide processing is evolving. For example, angiotensin-converting enzyme (ACE) 2, expressed in the kidney mainly proximal tubules and podocytes [1–4], is not targeted by ACE inhibition. ACE2 metabolizes angiotensin (Ang) II to Ang-(1–7), whereas ACE metabolizes Ang I to Ang II [5, 6].
ACE2 plays a role in the pathogenesis of experimental kidney disease. Deletion of the gene for ACE2 leads to the development of proteinuria and focal segmental glomerulosclerosis (FSGS) in mice [7] and exacerbates diabetic kidney injury [8]. Treatment with recombinant ACE2 attenuates diabetic nephropathy [9] and kidney injury in mice with experimental Alport syndrome, a model of progressive proteinuria, kidney inflammation and fibrosis [10]. Interestingly, ACE2 is the cellular receptor for SARS-CoV-2, and this finding has renewed interest in ACE2 expression because it may be a determinant of viral localization and organ injury [11, 12]. There are relatively few studies characterizing ACE2 in human kidneys [2, 13–17].
Sex is an important determinant of kidney outcomes [18–20] and tubulointerstitial injury correlates inversely with glomerular filtration rate (GFR) in primary glomerular diseases like FSGS [21, 22]. We have recently reported that kidney ACE2 expression is lower in males than females in a diverse group of subjects with CKD [23]. Here, we focus on tubulointerstitial expression of ACE2 in female and male subjects with FSGS from the Nephrotic Syndrome Study Network (NEPTUNE) consortium. We compared men and women across a broad set of clinical and laboratory variables including kidney pathology (age, eGFR, UACR, interstitial fibrosis, and tubular atrophy).
Materials and methods
Data collection and study cohort
Percutaneous kidney biopsies were obtained from patients after informed consent and with approval of the local ethics committees at each of the participating kidney centers. Written consent and assent were obtained. This covers all aspects of the study including clinical data, biospecimens and any derivatives. Clinical and gene expression information from patients are accessible in a non-identifiable manner. University of Michigan institutional review board in the Department of Medicine (UMich IRBMED) is the institutional review board of record [24].
Biopsies from 111 subjects (45 females and 66 males) with FSGS were microdissected into glomerular and tubulointerstitial components (Tables 1 and 2). Renal biopsy tissue was manually micro-dissected to separate the tubulointerstitial compartment from the glomerular compartment. Total RNA was isolated, reverse transcribed, linearly amplified and hybridized on an Affymetrix 2.1 ST platform as described previously [25, 26]. Gene expression was normalized, quantified, and annotated at the Entrez Gene level.
Visual assessment was performed according to the Nephrotic Syndrome Study Network Digital Pathology Scoring System (NDPSS), on de-identified whole slide images of renal biopsies according to the NEPTUNE digital pathology protocol (NDPP) [24]. Visual quantitative assessment of IF and TA was reported as 0–100%. Pathological assessment of IF and TA was performed according.
Statistical analysis
Analyses were performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA). Data are presented as the mean ± SD, unless otherwise stated. Statistical significance was defined as a P value of less than 0.05 for the Spearman correlation coefficient analysis and for the multiple regression analysis. Tubulointerstitial median-centered log2 mRNA expression of ACE2 from renal biopsy samples from subjects with FSGS were compared in male and female subgroups. ACE2 expression levels were correlated against age, estimated GFR (eGFR), urine protein to creatinine ratio (UPCR), interstitial fibrosis percentage, and tubular atrophy percentage.
ACE2 expression was also correlated against expression of tumor necrosis factor alpha (TNF), monocyte chemoattractant protein 1 (MCP-1 or CCL2), transforming growth factor beta 1 (TGFB1), collagen, type I, alpha 1 (COL1A1), and actin alpha 2, smooth muscle (ACTA2 or α-SMA) in subjects with FSGS. For comparisons between two groups, two-tailed P values were determined by χ2 tests for categorical variables and unpaired Student’s t tests for continuous variables. Pearson’s correlation coefficient (r) with two-tailed P values were calculated. Linear regression was used to generate the line of best fit with 95% confidence intervals.
Results
Patient characteristics
There were 111 subjects in the FSGS cohort: 66 males and 45 females (Table 1). Although there were missing values for clinical and laboratory parameters (Table 2), we did not input missing values for our analyses. The average age of the group was 32.6 ± 20.6 years with a mean eGFR of 72.9 ± 34.1 ml/min/1.73 m2. There was no difference in age or BMI between the male and female subjects. Mean systolic blood pressure and hematocrit values were higher in male subjects compared to female subjects. In terms of kidney function, mean values for eGFR, UPCR, and urine albumin to creatinine ratios (UACR) were similar in males and females. Morphometric measures of kidney interstitial fibrosis and tubular atrophy were also similar (Table 1).
Correlation of ACE2 mRNA expression with clinical variables in males and females with FSGS
We compared ACE2 mRNA expression in the tubulointerstitium based on sex. Tubulointerstitial ACE2 mRNA expression was greater in females with FSGS compared to males with FSGS (P = 0.0026; Fig 1). In male subjects, ACE2 expression declined with age (r = -0.30, P = 0.016; Fig 2A), but this relationship was not observed in female subjects (r = 0.11, P = 0.50; Fig 2B). There was a relationship between ACE2 mRNA expression and eGFR in the tubulointerstitium in male subjects (r = 0.56, P < 0.0001; Fig 3A) but not in female subjects (r = -0.11, P = 0.49; Fig 3B). There were no relationships between the centrally measured timed UPCR values and ACE2 expression in either male (Fig 4A) or female subjects (Fig 4B). There was a relationship between sitting systolic blood pressure and ACE2 expression in males (r = -0.32, P = 0.010; Fig 5A).
Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) in male and female subjects with FSGS. Values are the mean ± SD (grey lines). Significance was defined as a P value of less than 0.05.
(A, B) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with age in (A) male and (B) female subjects with FSGS. Pearson’s correlation coefficient (r) with two-tailed P values were calculated. Linear regression was used to generate the line of best fit (solid lines) with 95% confidence intervals (dotted lines). Significance was defined as a P value of less than 0.05.
(A, B) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with eGFR in (A) male and (B) female subjects with FSGS. Pearson’s correlation coefficient (r) with two-tailed P values were calculated. Linear regression was used to generate the line of best fit (solid lines) with 95% confidence intervals (dotted lines). Significance was defined as a P value of less than 0.05.
(A, B) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with centrally measured timed UPCR in (A) male and (B) female subjects with FSGS. Pearson’s correlation coefficient (r) with two-tailed P values were calculated. Linear regression was used to generate the line of best fit (solid lines) with 95% confidence intervals (dotted lines). Significance was defined as a P value of less than 0.05.
(A, B) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with sitting systolic blood pressure in (A) male and (B) female subjects with FSGS. (C, D) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with sitting diastolic blood pressure in (C) male and (D) female subjects with FSGS. Pearson’s correlation coefficient (r) with two-tailed P values were calculated. Linear regression was used to generate the line of best fit (solid lines) with 95% confidence intervals (dotted lines). Significance was defined as a P value of less than 0.05.
A summary of the Spearman correlation coefficients and P values for ACE2 expression and clinical variables are shown in Table 3. The majority of males and females were treated with either angiotensin receptor blockers or angiotensin converting enzyme inhibitors at the time of kidney biopsy (Table 1). There was no effect of treatment on ACE2 expression in either male or female subjects (Fig 6). Multiple linear regression analysis showed that ACE2 expression was significantly related to sex and eGFR but not to age or treatment with renin angiotensin system blockade (Table 4). A one-unit increase in eGFR resulted in a 0.006 unit increase in ACE2 expression outcome. Women had on average 0.52 units higher ACE2 expression.
(A, B) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) in (A) male and (B) female subjects with FSGS who were either on or not taking RAAS blockade drugs prior to the baseline visit. Values are the mean ± SD (grey lines). Significance was defined as a P value of less than 0.05.
Correlation of ACE2 mRNA expression with kidney interstitial fibrosis and tubular atrophy in males and females with FSGS
We next compared the relationship between ACE2 expression and pathology measures of percent interstitial fibrosis and percent tubular atrophy in male and female subjects. In male subjects, ACE2 expression levels declined as values for percent interstitial fibrosis rose (r = -0.47, P = 0.0004; Fig 7A), but this relationship was not observed in female subjects (r = -0.10, P = 0.47; Fig 7B). There was also a relationship between ACE2 mRNA expression and percent tubular atrophy in male subjects (r = -0.46, P = 0.0005; Fig 7C) but not in female subjects (r = 0.05, P = 0.77; Fig 7D).
(A, B) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with interstitial fibrosis percentage (IF %) in (A) male and (B) female subjects with FSGS. (C, D) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with tubular atrophy percentage (TA %) in (C) male and (D) female subjects with FSGS. Pearson’s correlation coefficient (r) with two-tailed P values were calculated. Linear regression was used to generate the line of best fit (solid lines) with 95% confidence intervals (dotted lines). Significance was defined as a P value of less than 0.05.
Correlation of ACE2 mRNA expression with genes implicated in inflammation and fibrosis in males and females with FSGS
Experimental studies have shown that ACE2 can regulate the expression of genes implicated in inflammation and fibrosis. Therefore, we compared the relationship between ACE2 expression and TNF and CCL2 mRNA in males and females. TNF mRNA levels were strongly associated with ACE2 mRNA levels (r = -0.65, P < 0.0001; Fig 8A) in men, but the relationship was not significant in females (r = -0.29, P = 0.08; Fig 8B). Our analysis showed a similar difference between males and females in the relationship between ACE2 expression and CCL2 expression (Fig 8C and 8D). We did not see any sexual dimorphism in the relationship between ACE2 expression and the expression levels of genes implicated in kidney fibrosis. TGFB was negatively associated with ACE2 expression in both males (r = -0.29, P = 0.02; Fig 9A) and females (r = -0.53, P = 0.0002; Fig 9B). There were no relationships between either ACE2 expression and COL1A1 or ACTA2 expression (Figs 10 and 11). A summary of the Spearman correlation coefficients and P values for ACE2 and the expression of genes implicated in inflammation and fibrosis is also shown in Table 5.
(A, B) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with tumor necrosis factor (TNF) expression in (A) male and (B) female subjects with FSGS. (C, D) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with monocyte chemoattractant protein 1 (MCP-1 or CCL2) expression in (C) male and (D) female subjects with FSGS. Pearson’s correlation coefficient (r) with two-tailed P values were calculated. Linear regression was used to generate the line of best fit (solid lines) with 95% confidence intervals (dotted lines). Significance was defined as a P value of less than 0.05.
(A, B) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with TGFB1 in (A) male and (B) female subjects with FSGS. Pearson’s correlation coefficient (r) with two-tailed P values were calculated. Linear regression was used to generate the line of best fit (solid lines) with 95% confidence intervals (dotted lines). Significance was defined as a P value of less than 0.05.
(A, B) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with COL1A1 in (A) male and (B) female subjects with FSGS. Pearson’s correlation coefficient (r) with two-tailed P values were calculated. Linear regression was used to generate the line of best fit (solid lines) with 95% confidence intervals (dotted lines). Significance was defined as a P value of less than 0.05.
(A, B) Tubular ACE2 mRNA expression (median-centered log2 expression by microarray analysis) correlated with ACTA2 in (A) male and (B) female subjects with FSGS. Pearson’s correlation coefficient (r) with two-tailed P values were calculated. Linear regression was used to generate the line of best fit (solid lines) with 95% confidence intervals (dotted lines). Significance was defined as a P value of less than 0.05.
Discussion
The rationale for the current study is twofold. First, the RAS plays a key role in the pathogenesis of CKD associated with proteinuria, and blockade of the RAS limits CKD progression [27, 28]. The complexity of the RAS is evolving, and ACE2, an enzyme that processes Ang II, regulates Ang II-induced inflammation and fibrosis in the kidney [29]. Of current interest, ACE2 also functions as a cell membrane receptor for SARS-CoV and SARS-CoV-2. Notwithstanding these important functions, our knowledge of the determinants of ACE2 expression in the kidney is incomplete. Second, sex is an important determinant of kidney disease outcomes [18–20]. While the gene for ACE2 is on the X chromosome [30], studies of sex-based differences in kidney ACE2 expression and the relationships between ACE2 expression and kidney injury are limited. Accordingly, our goal was to study ACE2 expression in the NEPTUNE cohort [31] of FSGS and to compare relationships between ACE2 expression, clinical variables, and pathology in males and females.
We studied a well-characterized cohort of male and female subjects with FSGS [31]. We chose to study FSGS because clinical outcomes are dependent on sex: outcomes are better in females than males [23, 32]. For example, Troyanov and coworkers studied a cohort of patients with FSGS and found that female sex was associated with a sustained decrease in proteinuria, an important determinant of the rate of loss of kidney function [32]. In another study, kidney function declined more rapidly in men with FSGS than women [21]. Moreover, we have reported that there are sex differences in the kidney response to RAS blockade in females and males that may be due to differences in renal expression of components of the RAS [33]. Finally, we focused on the micro-dissected tubulointerstitial compartment of the kidney for two reasons. First, ACE2 is mainly in cells of the S1, S2, and S3 segments of the kidney proximal tubule [1, 2, 34], as shown by Wysocki and coworkers in a recent analysis of single cell transcriptomic data, and second, changes in the tubulointerstitium, in particular interstitial fibrosis and tubular atrophy, predict long term clinical outcomes in CKD [35, 36].
The FSGS cohort exhibited significant differences in systolic blood pressure, hematocrit values, and timed urine creatinine between males and females. The differences were expected based on population studies in females and males [37]. Our first observation was that ACE2 expression in the kidney tubulointerstitial compartment was lower in males than females (Fig 1). Interestingly, ACE2 is on the X chromosome [30]. Although the presence of two X chromosomes (and therefore two ACE2 genes) might cause a two-fold increase in ACE2 expression in females relative to males, X chromosome inactivation will lessen this difference. Genes on one of the X chromosomes from either parent are marked for inactivation. This leads to the silencing of genes on that chromosome leading to a gene dosage more comparable to males. In reality, incomplete inactivation occurs in human female tissues. Genes that escape inactivation may have higher levels of expression, but likely less than a two-fold increase when compared to males [38]. Tukiainen and coworkers profiled “escape genes” in multiple tissues in humans [39]. Interestingly they found that ACE2 expression was higher in males for most of the tissues profiled. However, they did not study kidney expression. These findings suggest that we should not expect to see a large difference in kidney ACE2 expression based on sex chromosomes alone. Alternatively, sex hormones (testosterone and 17β-estradiol) may regulate the expression of ACE2 [39, 40]. For example testosterone may upregulate ACE2 expression, and reduced testosterone levels with aging might account, at least in part, for the relationship between ACE2 expression and age in males [41, 42]. In addition, estradiol may also impact ACE2 activity in the kidney [43]. We also found that ACE2 expression was lower in men compared to women in a recent study in a cohort of subjects with chronic kidney disease due to a variety of pathologies [23]. ACE2 expression in the kidney declined in age but only in men [23].
Our next observation was that there was a relationship between ACE2 expression and eGFR in males but not in females, even though the distribution of eGFR values was similar in both groups (Fig 3). As values for eGFR decline, there is a decrease in ACE2 expression. Taken together, our findings of dichotomies in the relationships between ACE2 expression and sex, age, and eGFR in males and females suggests that male kidneys may be more vulnerable to kidney disease associated with activation of the RAS. Ang II drives both inflammation and fibrosis in the kidney [10, 44]. ACE2 is responsible for the proteolytic degradation of Ang II to Ang-(1–7), and reduced ACE2 expression ought to lead to an increase in Ang II levels in the kidney [45]. Infusing Ang II into mice with a deletion in the gene for ACE2 is associated with more fibrosis and inflammation compared to infusion in a wildtype mouse [46]. The RAS is activated in kidney disease associated with proteinuria [47]. Indeed, Remuzzi and coworkers reported that the clinical benefit of RAS blockade was dependent on the level of proteinuria [48]. More recently, Matsusaka and coworkers showed that liver-derived angiotensinogen contributed to increased kidney Ang II levels when glomerular injury was associated with impaired permselectivity [49]. Any reduction in ACE2 expression should lead to further increases in tissue Ang II levels Sex and age differences in ACE2 expression may well contribute, at least in part, to clinical outcomes in FSGS.
RAAS blockade may also affect tissue ACE2 expression [50], Danser and coworkers recently reported that neither angiotensin converting enzyme inhibition (ACEI) nor angiotensin receptor blockers (ARB) consistently increased lung and kidney ACE2 expression, although the focus of this work was on potential susceptibility to coronavirus infection and the role of ACE2 as a receptor for cellular viral entry [51]. In addition, Cahoya and coworkers reported that RAS blockade did not change ACE2 expression in kidney allografts, albeit by immunohistochemistry in a small cohort of subjects [52]. In contrast, Wysocki and coworkers found that RAS treatment in mice decreased kidney ACE2 protein expression [53]. We therefore compared ACE2 mRNA expression in female and male subjects treated with RAAS blockade in our cohort of FSGS subjects. ACE2 expression was similar in untreated and treated males and females suggesting that RAAS blockade did not have a major effect on kidney ACE2 expression in the cohort.
Given the above findings, we studied the impact of eGFR, age, sex, and the use of RAAS blockade on ACE2 expression in our cohort, we performed a multiple linear regression analysis in which we related ACE2 expression to eGFR, age, sex, and the use of renin-angiotensin-aldosterone system blocking drugs prior to time of biopsy. Neither age nor RAAS blockade were associated with ACE2 expression outcome in the multiple linear regression analysis.
In our previous report, we were unable to relate ACE2 expression in the kidney to kidney fibrosis [23]. Accordingly, our next observation was the discovery that there was a significant relationship between ACE2 expression in the kidney and both interstitial fibrosis and tubular atrophy, but only in males and not in females (Fig 7). As ACE2 expression falls, interstitial fibrosis and tubular atrophy measures rise in males, in accord with the expected effect on Ang II levels. These relationships do not establish causality, although we have reported that treatment with recombinant ACE2 limits kidney fibrosis in an experimental murine model of Alport syndrome that is also associated with the development of FSGS [10]. Interestingly, 17β-estradiol increases ACE2 expression [54], and this may account for the failure to observe any relationship between ACE2 expression and interstitial fibrosis and tubular atrophy in the kidney of females with FSGS. Interestingly, there was a negative correlation between ACE2 expression and TGFB expression in both men and women, whereas there were no relationships, only trends, between ACE2 expression and COL1A1 and ACTA2 in both sexes.
Our next observations were the associations between ACE2 expression and gene expression of cytokines related to inflammation (Fig 8). There were strong negative correlations between ACE2 expression and TNF and CCL2 expression in males. Similar trends were observed in females although the relationships did not achieve statistical significance for either cytokine. The sexual dichotomy was marked by differences in the strength of the correlation, although this may be due in part to the number of data points. Taken together, the findings support the hypothesis that reduced ACE2 expression may contribute to increased kidney inflammation. This assertion is supported again by our observations of Ang II infusion in mice with a deletion in the gene for ACE2 [46] and by our experimental findings of ACE2 treatment in murine Alport syndrome [10, 27].
Studies of ACE2 are timely, especially in the context of its role as a cellular receptor for SARS-CoV-2 that facilitates viral cell entry [55]. CKD and male sex are risk factors for both SARS-CoV-2 infection and COVID-19 disease severity [42, 56–58]. Although acute kidney injury is a common complication of severe COVID-19 [58], the role of direct viral infection of the kidney is not well understood [59, 60]. Much of the acute risk relates to a diffuse systemic inflammatory response and renal hypoperfusion. We did not study acute SARS-CoV-2 infection in humans with FSGS. The sex and age-dependent changes in kidney ACE2 expression that we found do not appear to account for the impact of these clinical factors on COVID-19 infection and disease severity, although there may be tissue-specific relationships that are important in other organs [61]. Nevertheless, we would argue that tissue depletion of ACE2 in the setting of viral infection is likely to promote inflammation and scarring, and this may be particularly important in both acute lung infection and chronic lung injury [62].
Our study has several strengths. The cohort is well characterized in terms of clinical variables, such as assessment of blood pressure, eGFR, proteinuria, and measures of IF and TA in kidney biopsy samples [31, 63]. This allowed us to study the relationships between several variables and ACE2 expression, perhaps most importantly, histological assessment of kidney fibrosis.
However, our study also has some important limitations. First, our analysis is limited to mRNA levels in micro-dissected kidney tubulointerstitial samples, and we did not perform in situ hybridization studies of biopsy tissue from our cohort. The localization of ACE2 expression is important, and in this regard, a recent analysis of publicly available single cell transcriptomic data by Wysocki and co-workers showed that ACE2 is mainly in kidney proximal tubule S1, S2, and S3 cells [53]. We have extended this analysis by performing an in-silico analysis of two publicly available single cell transcriptomic databases (S1 Fig) to show proximal tubule cell-specific localization of ACE2 in both adult and fetal kidney tissue. Although it is important to note that glomerular cells are relatively under-represented in these datasets, measures of mRNA levels in the micro-dissected tubulointerstitial datasets we analyzed likely reflect changes in the proximal tubule. We did not relate ACE2 mRNA levels to ACE2 protein expression by immunohistochemistry in our cohort. Studies have reported tissue ACE2 protein expression by immunohistochemistry in humans [13] and confirmed the localization to proximal tubule cells [13], but there are only a few case series of ACE2 immunohistochemistry in human kidney biopsy samples [2, 14–17].
Another major limitation of our work is that we did not relate kidney ACE2 mRNA to levels of plasma Ang II and Ang-(1–7). Measures of intra-renal RAS activity and systemic RAS activity may not always be concordant, as noted in studies by Brenner and coworkers [64]. We have also found inconsistencies between systemic and renal measures of the RAAS in our recent studies of youth with type 2 diabetes that limit interpretation [65]. Serfero et al have suggested that the production of Ang-(1–7) from Ang II in the circulation is independent of ACE2 [66]. In contrast to this observation, we have reported that kidney Ang II and Ang-(1–7) levels are dependent, at least in part, on renal ACE2 [10]. Taken together, these findings suggest that systemic (circulating) measures of circulating Ang II and Ang-(1–7) may not reliably reflect intra-renal levels. Nevertheless, measures of kidney Ang II and Ang-(1–7) would strengthen our work and should be the focus of future studies.
In conclusion, kidney ACE2 expression in the tubulointerstitium differs in males and females with FSGS. ACE2 expression is greater in females than in males. Multiple linear regression analysis confirmed that the effect of sex was independent of eGFR, age, and blockade of the RAAS. The associations between ACE2 expression and eGFR, interstitial fibrosis, and tubular atrophy in males may account, at least in part, for the differences in clinical outcomes between males and females with FSGS. Finally, lower ACE2 expression in males is associated with the higher expression of genes implicated in inflammation.
Supporting information
S1 Fig. Transcriptomic analysis of ACE2 expression.
Cell Clustering and ACE2 expression from the Kidney Interactive Transcriptomics (KIT), healthy human adult kidney (http://humphreyslab.com/SingleCell) and expression atlas, human fetal kidney (https://www.ebi.ac.uk/gxa/home).
https://doi.org/10.1371/journal.pone.0252758.s001
(TIF)
S1 Dataset. Clinical characteristics, RAAS patient drug information, and log2 median-centered microdissected tubulointerstitial biopsy microarray data.
This file includes all the data that was used for analysis in this manuscript.
https://doi.org/10.1371/journal.pone.0252758.s002
(XLSX)
Acknowledgments
Consortia Members of the Nephrotic Syndrome Study Network (NEPTUNE) are as follows: John Sedor, Katherine Dell, Marleen Schachere, Kevin Lemley, Lauren Whitted, Tarak Srivastava, Connie Haney, Christine Sethna, Kalliopi Grammatikopoulos, Gerald Appel, Michael Toledo, Laurence Greenbaum, Chia-shi Wang, Brian Lee, Sharon Adler, Cynthia Nast, Janine LaPage, Ambarish Athavale, Alicia Neu, Sara Boynton, Fernando Fervenza, Marie Hogan, John C. Lieske, Vladimir Chernitskiy, Frederick Kaskel, Neelja Kumar, Patricia Flynn, Jeffrey Kopp, Eveleyn Castro-Rubio, Jodi Blake, Howard Trachtman, Olga Zhdanova, Frank Modersitzki, Suzanne Vento, Richard Lafayette, Kshama Mehta, Crystal Gadegbeku, Duncan Johnstone, Daniel Cattran, Michelle Hladunewich, Heather Reich, Paul Ling, Martin Romano, Alessia Fornoni, Laura Barisoni, Carlos Bidot, Matthias Kretzler, Debbie Gipson, Amanda Williams, Renee Pitter, Patrick Nachman, Keisha Gibson, Sandra Grubbs, Anne Froment, Lawrence Holzman, Kevin Meyers, Krishna Kallem, Fumei Cerecino, Kamal Sambandam, Elizabeth Brown, Natalie Johnson, Ashley Jefferson, Sangeeta Hingorani, Kathleen Tuttle, Laura Curtin, S Dismuke, Ann Cooper, Barry Freedman, Jen Jar Lin, Stefanie Gray, Matthias Kretzler, Larua Barisoni, Crystal Gadegbeku, Brenda Gillespie, Debbie Gipson, Lawrence Holzman, Laura Mariani, Matthew G. Sampson, Peter Song, Johnathan Troost, Jarcy Zee, Emily Herreshoff, Colleen Kincaid, Chrysta Lienczewski, Tina Mainieri, Amanda Williams, Kevin Abbott, Cindy Roy, Tiina Urv, and John Brooks.
References
- 1. Hamming I, Timens W, Bulthuis M, Lely A, Navis G, van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004;203: 631–637. pmid:15141377
- 2. Lely A, Hamming I, van Goor H, Navis G. Renal ACE2 expression in human kidney disease. J Pathol. 2004;204: 587–593. pmid:15538735
- 3. Pei G, Zhang Z, Peng J, Liu L, Zhang C, Yu C, et al. Renal Involvement and Early Prognosis in Patients with COVID-19 Pneumonia. J Am Soc Nephrol. 2020;31: 1157–1165. pmid:32345702
- 4. Hirsch JS, Ng JH, Ross DW, Sharma P, Shah HH, Barnett RL, et al. Acute kidney injury in patients hospitalized with COVID-19. Kidney Int. 2020;98: 209–218. pmid:32416116
- 5. Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, et al. Hydrolysis of Biological Peptides by Human Angiotensin-converting Enzyme-related Carboxypeptidase. J Biol Chem. 2002;277: 14838–14843. pmid:11815627
- 6. Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, et al. A Novel Angiotensin-Converting Enzyme–Related Carboxypeptidase (ACE2) Converts Angiotensin I to Angiotensin 1–9. Circ Res. 2000;87. pmid:10969042
- 7. Oudit GY, Herzenberg AM, Kassiri Z, Wong D, Reich H, Khokha R, et al. Loss of Angiotensin-Converting Enzyme-2 Leads to the Late Development of Angiotensin II-Dependent Glomerulosclerosis. Am J Pathol. 2006;168: 1808–1820. pmid:16723697
- 8. Wong DW, Oudit GY, Reich H, Kassiri Z, Zhou J, Liu QC, et al. Loss of Angiotensin-Converting Enzyme-2 (Ace2) Accelerates Diabetic Kidney Injury. Am J Pathol. 2007;171: 438–451. pmid:17600118
- 9. Oudit GY, Liu GC, Zhong J, Basu R, Chow FL, Zhou J, et al. Human recombinant ACE2 reduces the progression of diabetic nephropathy. Diabetes. 2010;59: 529–38. pmid:19934006
- 10. Bae EH, Fang F, Williams VR, Konvalinka A, Zhou X, Patel VB, et al. Murine recombinant angiotensin-converting enzyme 2 attenuates kidney injury in experimental Alport syndrome. Kidney Int. 2017;91: 1347–1361. pmid:28249676
- 11. Ng JH, Hirsch JS, Hazzan A, Wanchoo R, Shah HH, Malieckal DA, et al. Outcomes Among Patients Hospitalized With COVID-19 and Acute Kidney Injury. Am J Kidney Dis. 2021;77: 204–215.e1. pmid:32961245
- 12. Puelles VG, Lütgehetmann M, Lindenmeyer MT, Sperhake JP, Wong MN, Allweiss L, et al. Multiorgan and Renal Tropism of SARS-CoV-2. N Engl J Med. 2020;383: 590–592. pmid:32402155
- 13. Hikmet F, Méar L, Edvinsson Å, Micke P, Uhlén M, Lindskog C. The protein expression profile of ACE2 in human tissues. Mol Syst Biol. 2020;16: e9610. pmid:32715618
- 14. Wang G, Lai FM-M, Kwan BCH, Lai K-B, Chow K-M, Li PKT, et al. Expression of ACE and ACE2 in patients with hypertensive nephrosclerosis. Kidney Blood Press Res. 2011;34: 141–9. pmid:21346373
- 15. Mizuiri S, Hemmi H, Arita M, Aoki T, Ohashi Y, Miyagi M, et al. Increased ACE and decreased ACE2 expression in kidneys from patients with IgA nephropathy. Nephron Clin Pract. 2011;117: c57–66. pmid:20689326
- 16. Reich HN, Oudit GY, Penninger JM, Scholey JW, Herzenberg AM. Decreased glomerular and tubular expression of ACE2 in patients with type 2 diabetes and kidney disease. Kidney Int. 2008;74: 1610–6. pmid:19034303
- 17. Mizuiri S, Hemmi H, Arita M, Ohashi Y, Tanaka Y, Miyagi M, et al. Expression of ACE and ACE2 in individuals with diabetic kidney disease and healthy controls. Am J Kidney Dis. 2008;51: 613–23. pmid:18371537
- 18. Klein SL, Morgan R. The impact of sex and gender on immunotherapy outcomes. Biol Sex Differ. 2020;11: 1–13. pmid:31900228
- 19. Carrero JJ, Hecking M, Chesnaye NC, Jager KJ. Sex and gender disparities in the epidemiology and outcomes of chronic kidney disease. Nat Rev Nephrol. 2018;14: 151–164. pmid:29355169
- 20. Forni Ogna V, Ogna A, Vuistiner P, Pruijm M, Ponte B, Ackermann D, et al. New anthropometry-based age- and sex-specific reference values for urinary 24-hour creatinine excretion based on the adult Swiss population. BMC Med. 2015;13: 40. pmid:25858764
- 21. Cattran DC, Reich HN, Beanlands HJ, Miller JA, Scholey JW, Troyanov S. The impact of sex in primary glomerulonephritis. Nephrol Dial Transplant. 2008;23: 2247–2253. pmid:18182409
- 22. Wehrmann M, Bohle A, Held H, Schumm G, Kendziorra H, Pressler H. Long-term prognosis of focal sclerosing glomerulonephritis. An analysis of 250 cases with particular regard to tubulointerstitial changes. Clin Nephrol. 1990;33: 115–22. Available: http://www.ncbi.nlm.nih.gov/pubmed/2323110 pmid:2323110
- 23. Maksimowski N, Williams VR, Scholey JW. Kidney ACE2 expression: Implications for chronic kidney disease. Shimosawa T, editor. PLoS One. 2020;15: e0241534. pmid:33125431
- 24. Barisoni L, Nast CC, Jennette JC, Hodgin JB, Herzenberg AM, Lemley K V., et al. Digital Pathology Evaluation in the Multicenter Nephrotic Syndrome Study Network (NEPTUNE). Clin J Am Soc Nephrol. 2013;8: 1449–1459. pmid:23393107
- 25. Lai JY, Luo J, O’Connor C, Jing X, Nair V, Ju W, et al. MicroRNA-21 in glomerular injury. J Am Soc Nephrol. 2015;26: 805–16. pmid:25145934
- 26. Schmid H, Boucherot A, Yasuda Y, Henger A, Brunner B, Eichinger F, et al. Modular activation of nuclear factor-kappaB transcriptional programs in human diabetic nephropathy. Diabetes. 2006;55: 2993–3003. pmid:17065335
- 27. Bae EH, Konvalinka A, Fang F, Zhou X, Williams V, Maksimowski N, et al. Characterization of the intrarenal renin-angiotensin system in experimental Alport syndrome. Am J Pathol. 2015;185. pmid:25777062
- 28. Alhenc-Gelas F, Drueke TB. Blockade of SARS-CoV-2 infection by recombinant soluble ACE2. Kidney Int. 2020;97: 1091–1093. pmid:32354636
- 29. Gheblawi M, Wang K, Viveiros A, Nguyen Q, Zhong J-C, Turner AJ, et al. Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System. Circ Res. 2020;126: 1456–1474. pmid:32264791
- 30. Komatsu T, Suzuki Y, Imai J, Sugano S, Hida M, Tanigami A, et al. Molecular Cloning, mRNA Expression and Chromosomal Localization of Mouse Angiotensin-converting Enzyme-related Carboxypeptidase (mACE2). DNA Seq. 2002;13: 217–220. pmid:12487024
- 31. Troost JP, Waldo A, Carlozzi NE, Murphy S, Modersitzki F, Trachtman H, et al. The longitudinal relationship between patient-reported outcomes and clinical characteristics among patients with focal segmental glomerulosclerosis in the Nephrotic Syndrome Study Network. Clin Kidney J. 2020;13: 597–606. pmid:32905199
- 32. Troyanov S, Wall CA, Miller JA, Scholey JW, Cattran DC, Toronto Glomerulonephritis Registry Group. Focal and segmental glomerulosclerosis: definition and relevance of a partial remission. J Am Soc Nephrol. 2005;16: 1061–8. pmid:15716334
- 33. Miller JA, Cherney DZ, Duncan JA, Lai V, Burns KD, Kennedy CRJ, et al. Gender Differences in the Renal Response to Renin-Angiotensin System Blockade. J Am Soc Nephrol. 2006;17: 2554–2560. pmid:16914541
- 34. Batlle D, Soler MJ, Sparks MA, Hiremath S, South AM, Welling PA, et al. Acute Kidney Injury in COVID-19: Emerging Evidence of a Distinct Pathophysiology. J Am Soc Nephrol. 2020;31: 1380–1383. pmid:32366514
- 35. Webster AC, Nagler E V, Morton RL, Masson P. Chronic Kidney Disease. Lancet. 2017;389: 1238–1252. pmid:27887750
- 36. Schelling JR. Tubular atrophy in the pathogenesis of chronic kidney disease progression. Pediatr Nephrol. 2016;31: 693–706. pmid:26208584
- 37. Barr DB, Wilder LC, Caudill SP, Gonzalez AJ, Needham LL, Pirkle JL. Urinary Creatinine Concentrations in the U.S. Population: Implications for Urinary Biologic Monitoring Measurements. Environ Health Perspect. 2005;113: 192–200. pmid:15687057
- 38. Gagliardi MC, Tieri P, Ortona E, Ruggieri A. ACE2 expression and sex disparity in COVID-19. Cell death Discov. 2020;6: 37. pmid:32499922
- 39. Tukiainen T, Villani A-C, Yen A, Rivas MA, Marshall JL, Satija R, et al. Landscape of X chromosome inactivation across human tissues. Nature. 2017;550: 244–248. pmid:29022598
- 40. Liu J, Ji H, Zheng W, Wu X, Zhu JJ, Arnold AP, et al. Sex differences in renal angiotensin converting enzyme 2 (ACE2) activity are 17β-oestradiol-dependent and sex chromosome-independent. Biol Sex Differ. 2010;1: 6. pmid:21208466
- 41. Hussain AN, Hussain F, Hashmi SK. Role of testosterone in COVID-19 patients–A double-edged sword? Med Hypotheses. 2020;144: 110287. pmid:33254589
- 42. Chanana N, Palmo T, Sharma K, Kumar R, Graham BB, Pasha Q. Sex-derived attributes contributing to SARS-CoV-2 mortality. Am J Physiol Metab. 2020;319: E562–E567. pmid:32726128
- 43. Yamaleyeva LM, Gilliam-Davis S, Almeida I, Brosnihan KB, Lindsey SH, Chappell MC. Differential regulation of circulating and renal ACE2 and ACE in hypertensive mRen2.Lewis rats with early-onset diabetes. Am J Physiol Renal Physiol. 2012;302: F1374–84. pmid:22378820
- 44. Simões e Silva AC, Teixeira MM. ACE inhibition, ACE2 and angiotensin-(1-7) axis in kidney and cardiac inflammation and fibrosis. Pharmacol Res. 2016;107: 154–162. pmid:26995300
- 45. Simões e Silva A, Silveira K, Ferreira A, Teixeira M. ACE2, angiotensin-(1–7) and Mas receptor axis in inflammation and fibrosis. Br J Pharmacol. 2013;169: 477–492. pmid:23488800
- 46. Zhong J, Guo D, Chen CB, Wang W, Schuster M, Loibner H, et al. Prevention of angiotensin II-mediated renal oxidative stress, inflammation, and fibrosis by angiotensin-converting enzyme 2. Hypertens (Dallas, Tex 1979). 2011;57: 314–22. pmid:21189404
- 47. Aoki T, Ohashi N, Isobe S, Ishigaki S, Matsuyama T, Sato T, et al. Chronotherapy with a Renin-angiotensin System Inhibitor Ameliorates Renal Damage by Suppressing Intrarenal Renin-angiotensin System Activation. Intern Med. 2020;59: 2237–2244. pmid:32938851
- 48. Ruggenenti P, Perna A, Gherardi G, Garini G, Zoccali C, Salvadori M, et al. Renoprotective properties of ACE-inhibition in non-diabetic nephropathies with non-nephrotic proteinuria. Lancet (London, England). 1999;354: 359–64. pmid:10437863
- 49. Matsusaka T, Niimura F, Shimizu A, Pastan I, Saito A, Kobori H, et al. Liver Angiotensinogen Is the Primary Source of Renal Angiotensin II. J Am Soc Nephrol. 2012;23: 1181–1189. pmid:22518004
- 50. Gallagher PE, Ferrario CM, Tallant EA. Regulation of ACE2 in cardiac myocytes and fibroblasts. Am J Physiol Heart Circ Physiol. 2008;295: H2373–9. pmid:18849338
- 51. Danser AHJ, Epstein M, Batlle D. Renin-Angiotensin System Blockers and the COVID-19 Pandemic: At Present There Is No Evidence to Abandon Renin-Angiotensin System Blockers. Hypertens (Dallas, Tex 1979). 2020;75: 1382–1385. pmid:32208987
- 52. Cahova M, Kveton M, Petr V, Funda D, Dankova H, Viklicky O, et al. Local Angiotensin-Converting Enzyme 2 Gene Expression in Kidney Allografts Is Not Affected by Renin-Angiotensin-Aldosterone Inhibitors. Kidney Blood Press Res. 2021;46: 245–249. pmid:33756485
- 53. Wysocki J, Lores E, Ye M, Soler MJ, Batlle D. Kidney and Lung ACE2 Expression after an ACE Inhibitor or an Ang II Receptor Blocker: Implications for COVID-19. J Am Soc Nephrol. 2020;31: 1941–1943. pmid:32669323
- 54. Ji H, Menini S, Zheng W, Pesce C, Wu X, Sandberg K. Role of angiotensin-converting enzyme 2 and angiotensin(1–7) in 17β-oestradiol regulation of renal pathology in renal wrap hypertension in rats. Exp Physiol. 2008;93: 648–657. pmid:18296494
- 55. Lan J, Ge J, Yu J, Shan S, Zhou H, Fan S, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020;581: 215–220. pmid:32225176
- 56. Viveiros A, Rasmuson J, Vu J, Mulvagh SL, Yip CYY, Norris CM, et al. Sex differences in COVID-19: candidate pathways, genetics of ACE2, and sex hormones. Am J Physiol Circ Physiol. 2021;320: H296–H304. pmid:33275517
- 57. Park MD. Sex differences in immune responses in COVID-19. Nat Rev Immunol. 2020;20: 461. pmid:32572247
- 58. Kant S, Menez SP, Hanouneh M, Fine DM, Crews DC, Brennan DC, et al. The COVID-19 nephrology compendium: AKI, CKD, ESKD and transplantation. BMC Nephrol. 2020;21: 449. pmid:33109103
- 59. Lynch MR, Tang J. COVID-19 and Kidney Injury. R I Med J (2013). 2020;103: 24–28. Available: http://www.ncbi.nlm.nih.gov/pubmed/32900008
- 60. Parmar MS. Acute kidney injury associated with COVID-19-Cumulative evidence and rationale supporting against direct kidney injury (infection). Nephrology (Carlton). 2020; e13814. pmid:33150674
- 61. Bourgonje AR, Abdulle AE, Timens W, Hillebrands J-L, Navis GJ, Gordijn SJ, et al. Angiotensin-converting enzyme 2 (ACE2), SARS-CoV-2 and the pathophysiology of coronavirus disease 2019 (COVID-19). J Pathol. 2020;251: 228–248. pmid:32418199
- 62. Gan R, Rosoman NP, Henshaw DJE, Noble EP, Georgius P, Sommerfeld N. COVID-19 as a viral functional ACE2 deficiency disorder with ACE2 related multi-organ disease. Med Hypotheses. 2020;144: 110024. pmid:32758871
- 63. Smith AR, Zee J, Ji N, Troost JP, Gillespie BW, Nair V, et al. Estimated GFR Trajectories in Pediatric and Adult Nephrotic Syndrome: Results From the Nephrotic Syndrome Study Network (NEPTUNE). Kidney Med. 2020;2: 407–417. pmid:32775980
- 64. Zatz R, Dunn BR, Meyer TW, Anderson S, Rennke HG, Brenner BM. Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. J Clin Invest. 1986;77: 1925–30. pmid:3011862
- 65. Dart AB, Wicklow B, Scholey J, Sellers EA, Dyck J, Mahmud F, et al. An evaluation of renin-angiotensin system markers in youth with type 2 diabetes and associations with renal outcomes. Pediatr Diabetes. 2020;21: 1102–1109. pmid:32657529
- 66. Serfozo P, Wysocki J, Gulua G, Schulze A, Ye M, Liu P, et al. Ang II (Angiotensin II) Conversion to Angiotensin-(1–7) in the Circulation Is POP (Prolyloligopeptidase)-Dependent and ACE2 (Angiotensin-Converting Enzyme 2)-Independent. Hypertens (Dallas, Tex 1979). 2020;75: 173–182. pmid:31786979