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ORIGINAL
Urinary angiotensin converting enzyme 2 and disease activity
in pediatric IgA nephropathy
Yuki Sakaki, Maki Urushihara, Takashi Nagai, Keisuke Fujioka, Shuji Kondo, Yukiko Kinoshita,
Tomoki Hattori, and Shoji Kagami
Department of Pediatrics, Institute of Biomedical Scicences, The Tokushima University Graduate School, Tokushima, Japan

Abstract : Background : Our previous studies demonstrated that the intrarenal renin-angiotensin system (RAS)
status was activated in pediatric patients with chronic glomerulonephritis. In the present study, we tested the
hypothesis that angiotensin-converting enzyme 2 (ACE2) expression in the kidney is associated with the development of pediatric IgA nephropathy. Methods : We analyzed urinary ACE2 levels and ACE2 expression in thekidney tissues of pediatric patients with IgA nephropathy treated with RAS blockade. Paired tests were used toanalyze changes from the first to the second biopsy. Results : Urinary ACE2 levels were significantly decreased
after RAS blockade treatment, accompanied by decreased ACE2 expression levels in kidney tissues, urinary
protein levels and mesangial hypercellularity scores. Urinary ACE2 levels at the first biopsy were positively correlated with the ACE2 expression levels. Conclusions : These data suggest that urinary ACE2 is associated with
ACE2 expression in the diseased kidney, which correlates with the pathogenesis of IgA nephropathy in pediatricpatients. J. Med. Invest. 68 : 292-296, August, 2021
Keywords : Angiotensin-converting enzyme 2, IgA nephropathy, renin-angiotensin system

INTRODUCTION
The renin-angiotensin system (RAS) is a pivotal system that
regulates blood pressure and controls the fluid / electrolyte balance (1). Angiotensin II is the most powerful biologically active
product of the RAS, which binds to the angiotensin II type 1(AT1) receptor (2). Local activation of the intrarenal RAS is
involved in the pathogenesis of hypertension and kidney injury(1). Recent reports have shown that progression of proteinuria
and kidney tissue damage is associated with intrarenal RAS
activation (3-6). Indeed, treatment with angiotensin-converting
enzyme (ACE) inhibitors and AT1 receptor blockers (ARBs) significantly decreases proteinuria in patients with chronic kidneydisease, which is independent of blood pressure changes (1, 5).
ACE2 is a homolog of ACE that counterbalances ACE activity(7). ACE2 cleaves angiotensin I into angiotensin 1-9 and angiotensin II into the vasodilator peptide angiotensin 1-7 (7). Angiotensin 1-7 is a biologically active peptide that binds the Mas
receptor to exert opposing effects on angiotensin II (8). Althoughmany studies have reported the protective role of angiotensin
1-7 in the prevention of angiotensin II, it has been shown thatangiotensin 1-7 stimulates growth factor expression and cell proliferation (9). Furthermore, the coronavirus disease 2019 (Covid19) interfaces with the RAS through ACE2, which functions as
a receptor for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) (10). The interaction between coronavirus and
ACE2 has been suggested as a potential factor in infectivity (10).
Recently, we demonstrated that ACE2 expression in thekidney was increased in pediatric IgA nephropathy and wasaccompanied by mesangial hypercellularity (11). Furthermore,
we clarified that intrarenal RAS activation was suppressed in
Received for publication April 22, 2021 ; accepted May 14, 2021.
Address correspondence and reprint requests to Maki Urushihara,
M.D., Ph.D., Assistant Professor, Department of Pediatrics, Instituteof Biomedical Sciences, The Tokushima University Graduate School,
Kuramoto-cho 3-18-15, Tokushima, Tokushima 770-8503, Japan andFax : +81-88-631-8697.

pediatric IgA nephropathy patients treated with RAS blockade(12). In the present study, we measured urinary ACE2 levels
following treatment with RAS blockade and evaluated theexpression of ACE2 in the kidney tissue to test the hypothesisthat ACE2 is associated with kidney damage in pediatric IgA
nephropathy.

PATIENTS AND METHODSPatients and samples

All procedures involving human participants were conductedin accordance with the ethical standards of the institutionaland / or national research committee. The study was performedin accordance with the 1964 Declaration of Helsinki and its later
amendments or comparable ethical standards. The Institutional
Review Board of Tokushima University approved the study protocol (IRB approval number 1085) and the use of tissue samples.
Informed consent was obtained from all enrolled children.
Pediatric patients with IgA nephropathy were recruited at
Tokushima University Hospital between April 1, 2011, andMarch 31, 2016. All study participants with other disorders,
including Henoch-Schonlein purpura, systemic lupus erythematosus, chronic liver disease, diabetes mellitus, malignancies,
and urinary tract infections, were excluded. At the time of
study enrollment, all patients were asked to undergo a second
kidney biopsy after 2 years following the end of RAS blockade(candesartan or benazepril). Clinical parameters, including sex,age, height, body weight, and blood pressure, and laboratory
parameters, including serum creatinine and urinary protein
and creatinine concentrations, were determined at the time ofbiopsy. Urine collections were combined to calculate the 24-h
creatinine clearance (ml / min), which was used as an index of
the glomerular filtration rate. Urinary concentrations of ACE2
were measured using a human ACE2 ELISA kit (Adipogen,
Liestal, Switzerland) in accordance with the manufacturer’s
instructions.

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The Journal of Medical Investigation Vol. 68 August 2021
Histological study
The biopsied tissues were fixed in 10% buffered formalin andembedded in paraffin according to routine clinical practice. Paraffin sections (3 μm) were stained with the periodic acid-Schiff
reagent. Kidney tissue sections at least ten glomeruli were scoredindependently by pathologist blinded to all clinical data ; discrepancies were resolved by a third investigator. Using modifications
from previous studies (12, 13), we scored histological changes asfollows : mesangial hypercellularity, 0 = none ; 1 = mild (threefour mesangial per peripheral lobule) ; 2 = severe segmental(more than four mesangial cells per peripheral lobule) ; and3 = severe global proliferation). Mesangial matrix score, 0 = none ;1 = mild ; 2 = marked segmental (width of mesangial interspace
between capillaries exceeding three mesangial cells) ; and 3 =marked global increase. Interstitial fibrosis, 0 = none ; 1 = < 25
% ; 2 = 26%-50% ; and 3 > 50% of the cortical area. Tubular atrophy, 0 = none ; 1 = < 25% ; 2 = 26%-50% ; and 3 = > 50% of thecortical tubules.

management and statistical analysis, were performed using
JMP software version 14 (SAS Institute, Cary, NC, USA).

RESULTS

Patient characteristics
Table 1 describes the patient’s characteristics and laboratory
data at the first biopsy. Kidney parameters and histological features at the first and second biopsies are shown in Table 2. The
urinary protein-creatinine ratio (UP / UCre) was significantly
lower at the second biopsy ; however, here was no significant
change in the creatinine clearance.

Table 1. Patient characteristics
Parameters

Data

Immunohistochemistry

Gender, F / M

16 / 15

Formalin-fixed, paraffin-embedded kidney sections were
stained with ACE2. Sections (3 μm) were incubated with anti-ACE2 antibody (ab15348 ; Abcam, Cambridge, MA, USA)overnight at 4°C, rinsed, and incubated with biotinylated secondary antibodies (Vector Labs, Burlingame, CA, USA). Afterrinsing, the sections were incubated with avidin-biotin-peroxidase complex (ABC Elite ; Vector Labs), followed by 3,3’-diaminobenzidine (Dojindo, Kumamoto, Japan). Each section
was counterstained with Mayer’s hematoxylin (Wako, Tokyo,
Japan), dehydrated, and cover-slipped. The fraction of glomeruli occupied by the immunoreactive area was determined using
EIS-Elements software (Nikon Corporation, Tokyo, Japan). For
each glomerulus, the immunoreactive area (brown) was automatically calculated by the software, and the affected area was,in turn, divided by the total area of the glomerulus. At least ten
equationally sectioned glomeruli were examined from each slide.

Age, Year

10.64 ± 0.74

Height, cm

139.77 ± 4.05

Body weight, kg

36.65 ± 2.90

Body mass index

17.70 ± 0.55

Serum creatinine, mg / dL

0.45 ± 0.02

Statistical analysis
Pearson and Spearman correlation coefficients were used forparametric and nonparametric data, respectively. The Wilcoxon signed-rank test was used to perform paired comparisonsat the first and second biopsies. All data are presented as themean ± standard error of the mean (SEM). Statistical significance was set at P < 0.05. All computations, including data

Histological findings and ACE2 in the urine and kidney
Scores for mesangial hypercellularity, mesangial matrix, andglomerular expression levels of ACE2 in kidney tissues werelower in the second biopsy than in the first biopsy (Table 2 andFigure 1a). In addition, the logarithmically transformed urinary
ACE2-creatinine ratio (log(UACE2 / UCre)) was significantly
lower in the second biopsy than in the first biopsy (Figure 1b).
Figure 2 demonstrated that the single-regression analyses forlog(UACE2 / UCre) levels were significantly positively correlated with UP / UCre, mesangial hypercellularity, and glomerular expression levels of ACE2 in the kidney tissue. However,log(UACE2 / UCre) levels were not correlated with age, height,
body weight, body mass index, creatinine clearance, mesangial
matrix, interstitial fibrosis, and tubular atrophy.

Table 2. Kidney parameters and histologic features at the time of kidney biopsy
Parameters

First biopsy, N = 31

Second biopsy, N = 31

Difference

P values

UP / UCre, g / g

1.45 ± 0.33

0.05 ± 0.01

-1.40 ± 0.33

< 0.0001*

CCr, mL / min108.61 ± 1.86

109.74 ± 2.91

1.14 ± 3.88

0.9760Mesangial hypercellularity

1.06 ± 0.07

0.41 ± 0.04

-0.65 ± 0.07

< 0.0001*

Mesangial matrix score1.06 ± 0.10

0.74 ± 0.07

-0.32 ± 0.11

0.0081*

Interstitial fibrosis

0.05 ± 0.04

0.08 ± 0.04

0.03 ± 0.02

0.5000Tubular atrophy

0.06 ± 0.03

0.10 ± 0.04

0.03 ± 0.05

0.7656

ACE2 expression

0.88 ± 0.08

0.22 ± 0.11

-0.66 ± 0.07

< 0.0001*

UP / UCre ; urinary protein-creatinine ratio, CCr ; creatinine clearance, * ; P < 0.05.

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Y. Sakaki, et al. Urinary ACE2 in pediatric IgA nephropathy

Figure 1. Representative images of angiotensin-converting
enzyme 2 (ACE2) expression in the first and second biopsies(a). ACE2 expression levels in the glomeruli were lower
in the second biopsy than in the first biopsy. Changes in
the logarithmically transformed urinary ACE2-creatinine
ratio (log (UACE2 / UCre)) in pediatric patients with IgA
nephropathy (b). Each marker and line represent an individual
study participant.

Figure 2. Single regression analyses for the logarithmicallytransformed urinary-angiotensin converting enzyme 2 (ACE2)creatinine ratio (log (UACE2 / UCre)) with the urinary proteincreatinine ratio (UP / UCre) (a), scores for mesangial hypercellularity(b), and ACE2 expression levels by immunohistochemical study(c). The log (UACE2 / UCre) levels showed a positive correlation
with UP / UCre, scores for mesangial hypercellularity, and ACE2
expression levels by immunohistochemical (IHC) study.

The Journal of Medical Investigation Vol. 68 August 2021
DISCUSSION
Activated intrarenal RAS is involved in the progression of
kidney injury (3, 4). Urinary angiotensinogen is a specific biomarker of intrarenal RAS activation in various kidney diseases(1, 5). Previously, we reported that ACE2 expression in thekidney was increased in pediatric IgA nephropathy and wasaccompanied by mesangial hypercellularity (11). However, urinary ACE2 excretion and kidney ACE2 expression associatedwith the pathophysiology of kidney injury have not been studied
extensively in humans. Our study is the first to demonstratethat urinary ACE2 and glomerular ACE2 expression levels at
the second biopsy were lower than those at the first biopsy inpediatric IgA nephropathy. We also found that urinary ACE2
levels positively correlated with urinary protein levels, scores formesangial hypercellularity, and expression levels of ACE2 in thekidney tissue.
Intrarenal RAS is independently regulated, and its inappropriate activation contributes to the pathogenesis of hypertension
and kidney disease (1, 4). The RAS in the kidney is unique
because all of the components necessary to generate intrarenal
angiotensin II are present along the nephron in both the interstitial and intratubular compartments (2). It is well known thatthe major fraction of angiotensin II present in kidney tissues isgenerated from angiotensinogen and then subsequently delivered to the kidney, as well as from angiotensinogen produced byproximal tubule cells (2). Renin is secreted by juxtaglomerularand proximal tubular cells (2). The brush border membrane ofthe proximal tubules also expresses abundant levels of ACE.
ACE was detected in proximal and distal tubular fluids, withhigher concentrations observed in the proximal tubule fluid,and catalyzes the transformation of angiotensin I to angiotensin
II (14). Therefore, all of the major components required to generate angiotensin II are expressed in the kidney (1, 5). In ourprevious study, cultured glomerular mesangial cells produced
ACE2 mRNA and protein (11). Furthermore, glomerular ACE2
expression and urinary ACE2 levels were found to be involvedin the pathogenesis of pediatric IgA nephropathy in the current
study. These findings suggest that ACE2 plays an important rolein the intrarenal RAS activation during the course of pediatricIgA nephropathy.
Previous studies have shown that urinary angiotensinogenlevels were increased not only in hypertensive subjects but alsoin patients with chronic kidney disease (5). Recently, urinary
angiotensinogen have been used as an index of intrarenal RAS
activation (1, 5). We previously reported that urinary angiotensinogen levels were increased in patients with chronic glomerulonephritis compared with control subjects and that treatmentswith RAS blockers suppressed these augmentations (15). In
addition, we found that urinary angiotensinogen levels andkidney angiotensinogen expression were decreased in patientswith pediatric IgA nephropathy treated with RAS blockade (12).
These findings suggest that urinary angiotensinogen could be anovel biomarker of intrarenal RAS status in pediatric patients.
In the present study, we demonstrated that elevated urinary
ACE2 levels were suppressed by RAS blockade in pediatric IgA
nephropathy. Furthermore, urinary ACE2 levels correlatedwith glomerular damage and ACE2 expression levels. Taken
together, urinary ACE2 could be associated with glomerular
ACE2 expression involved in intrarenal RAS activation in thedevelopment of kidney injury.
ACE is also a functional receptor for SARS-CoV-2, the virus
responsible for the Covid-19 pandemic (10). SARS-CoV-2 within
the host cell is a putative link between Covid-19 and hypertension, cardiovascular disease, and kidney disease (16). The
SARS-CoV-2 spike protein binds to membrane-bound ACE2295

on the surface of respiratory epithelial cells to gain cell entry(17, 18). SARS-CoV-2 infection can cause renal involvement,
and severe renal dysfunction is more common among patientswith chronic kidney disease (19). SARS-CoV-2 infection fundamentally initiates a mechanism of renal injury through highly
expressed ACE2 in renal tissue, suggesting that ACE2 signaling
pathway plays a key role in the development of the disease (19).
Furthermore, systemic effects such as host immune clearance
and immune tolerance disorders, endothelial cell injury, thrombus formation, glucose and lipid metabolism disorder, and hypoxia aggravate this renal injury (19). Endocytosis of SARS-CoV-2
combined with ACE2 contributes to decreased ACE2 expression
and activity in infected cells (20, 21). It has been suggested
that the loss of ACE2 functionality is critically important to
Covid-19 pathophysiology (16). Several studies have suggested
that RAS inhibitors may increase ACE2 expression, raisingconcerns regarding their safety in patients with Covid-19 (10).
However, sufficient data on human studies are not available,
and it is recommended to continue RAS inhibitors because theabrupt withdrawal of RAS inhibitors in high-risk patients may
result in clinical instability and adverse health outcomes (10).
In the future, further investigations to define risk factors anddetermine the pathophysiology of SARS-CoV-2 and Covid-19
are important. In fact, our current findings demonstrated that
ACE2 in the kidney is crucial in glomerular damage in pediatricIgA nephropathy ; however it is difficult to determine whether
ACE2 drives the activity of IgA nephropathy.
The relatively small number of subjects in this study is apotential limitation. However, our observations showed thatglomerular ACE2 expression and urinary ACE2 levels weresignificantly decreased by the treatment with RAS blockade.
In addition, urinary ACE2 levels were correlated with urinary
protein excretion, glomerular mesangial hypercellularity, andglomerular ACE2 expression levels.
These data strongly support the hypothesis that RAS plays
an important role in the pathogenesis of IgA nephropathy andis associated with increased ACE2 expression in the kidney.
We emphasize that measuring urinary ACE2 levels is a useful
tool to assess glomerular damage in pediatric IgA nephropathy.
However, we recognize that the future evaluation of urinary
ACE2 in prospective studies with a large number of patientsand an extended, long observation period is required. Subsequently, a prospective study was conducted to clarify the novel
mechanism of ACE2, which may provide useful information for
optimizing the approach for treating pediatric IgA nephropathy.

CONFLICT OF INTERESTS
The authors have declared that no conflict of interest exists.

ACKNOWLEDGEMENTS
The authors thank Ms. Naomi Okamoto and Ms. Chizuko
Yamamoto for their technical assistance. This study was supported by JSPS KAKENHI Grant Numbers 23591569 and
26461612.

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