1

Total Nephron Number and Single-Nephron Parameters in Patients with IgA

Nephropathy

Hirokazu Marumoto1, Nobuo Tsuboi

1, Vivette D. D’Agati

2, Takaya Sasaki

1, Yusuke

Okabayashi1, Kotaro Haruhara

1, Go Kanzaki

1, Kentaro Koike

1, Akira Shimizu

3, Tetsuya

Kawamura1, Andrew D. Rule

4, John F. Bertram

5 and Takashi Yokoo

1

1 Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei

University School of Medicine, Tokyo, Japan, 2 Department of Pathology and Cell Biology,

Columbia University Medical Center, New York, New York, USA, 3

Department of Analytic

Human Pathology, Nippon Medical School, Tokyo, Japan, 4 Division of Nephrology and

Hypertension, Mayo Clinic, Rochester, Minnesota, 5 Department of Anatomy and

Developmental Biology and Biomedical Discovery Institute, Monash University, Melbourne,

Australia.

Correspondence to: Nobuo Tsuboi, M.D., Ph.D.

Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei

University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo, Japan

Tel: 81-3-3433-1111, Fax: 81-3-3433-4297, E-mail: tsuboi-n@jikei.ac.jp

Kidney360 Publish Ahead of Print, published on March 10, 2021 as doi:10.34067/KID.0006972020

Copyright 2021 by American Society of Nephrology.

2

Key Points

The first study that estimated total nephron number and related single-nephron

parameters in patients with IgA nephropathy.

Associations of nephron level parameters with CKD stage and clinicopathologic

findings were cross-sectionally investigated.

This study illustrates the feasibility and usefulness of estimating single-nephron

dynamics in human glomerulonephritis.

Abstract

Background: Single nephron dynamics in progressive IgA nephropathy (IgAN) have not

been studied. We applied novel methodology to explore single nephron parameters in IgAN.

Methods: Non-globally sclerotic glomeruli (NSG) and globally sclerotic glomeruli (GSG)

per kidney were estimated using cortical volume assessment via unenhanced computed

tomography and biopsy-based stereology. Estimated single-nephron GFR (eSNGFR) and

urine protein excretion (SNUPE) were calculated by dividing eGFR and UPE by the number

of NSG. Associations with CKD stage and clinicopathologic findings were cross-sectionally

investigated.

Results: This study included 245 IgAN patients (mean age 43 y, 62% male, 45% on

renin-angiotensin aldosterone system [RAAS] inhibitors pre-biopsy) evaluated at kidney

biopsy. CKD stages were 10% CKD1, 43% CKD2, 19% CKD3a, 14% CKD3b and 14%

CKD4–5. With advancing CKD stage, NSG decreased from mean 992,000 to 300,000 per

kidney whereas GSG increased from median 64,000 to 202,000 per kidney. In multivariable

models, advancing CKD stage associated with lower numbers of NSG, higher numbers of

GSG, and lower numbers of GSG+NSG, indicating potential resorption of sclerosed

glomeruli. In contrast to the higher mean glomerular volume and markedly elevated SNUPE

3

in advanced CKD, the eSNGFR was largely unaffected by CKD stage. Lower SNGFR

associated with Oxford scores for endocapillary hypercellularity and crescents whereas

higher SNUPE associated with segmental glomerulosclerosis and tubulointerstitial scarring.

Conclusions: SNUPE emerged as a sensitive biomarker of advancing IgAN. The failure of

eSNGFR to increase in response to reduced number of functioning nephrons suggests limited

capacity for compensatory hyperfiltration by diseased glomeruli with intrinsic lesions.

4

Introduction

IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide.1

Approximately 30–40% of patients with IgAN will progress to end-stage kidney disease

(ESKD) within 20–30 years despite therapeutic interventions.2,3

Glomerulonephritis results

from the deposition of immunocomplexes containing galactose-deficient IgA1 in the

glomerular mesangium, reactive mesangial cell proliferation, variable peripheral capillary

wall deposits and endocapillary hypercellularity.4 Extracapillary lesions include inflammatory

crescents and podocytopathic changes associated with segmental glomerulosclerosis,

podocyte injury and depletion, and worsening proteinuria.4,5,6

Previous studies have

consistently identified impaired kidney function, hypertension, and heavy proteinuria at

biopsy as clinical factors predicting worse outcomes in IgAN.7,8,9

A proteinuria threshold of

more than 1g/day at biopsy has been linked to poor prognosis.8,10

Progressive glomerulosclerosis is a final common pathway to ESKD in chronic

nephropathies, in which the abnormalities in single-nephron dynamics are postulated to be

critically involved.11,12

Early studies used micropuncture to directly assess single nephron

filtration dynamics.11,13

Animal models of kidney ablation and/or high protein diet

demonstrated hyperfunction (hyperfiltration) in individual glomeruli, which was ameliorated

by angiotensin-converting inhibitor therapy.14,15,16

The renin-angiotensin aldosterone system

(RAAS) preferentially vasoconstricts the glomerular efferent arterioles, thereby increasing

single-nephron glomerular filtration rate (SNGFR).17

Findings from animal studies greatly

contributed to the development of RAAS inhibitors to delay progression in patients with

CKD, including IgAN.18

These studies implicated maladaptive intraglomerular

hemodynamics during disease progression, which has not yet been studied in human

glomerulonephritis due to the technical difficulty estimating SNGFR in patients.19

5

Denic et al. recently established a method for estimating the total number of

glomeruli in living humans using a combination of contrast-enhanced computed tomography

(CT) and biopsy-based stereology, allowing estimation of SNGFR.20

They showed that total

non-sclerotic glomerular number decreases with normal healthy aging, while SNGFR did not

vary significantly between different age groups under 70 years of age. To apply this

technology to patients with kidney disease, we established a regression equation to estimate

kidney cortical volume from measured kidney parenchymal volume (cortex and medulla) in

unenhanced CT images.21

An equation used for estimating cortical volume from parenchymal

volume was created based on a mixed cohort of healthy kidney donors and CKD patients

including those with CKD stages 4–5, who underwent enhanced and unenhanced CT imaging

at the same time. The primary objective of this study was to estimate total glomerular number

and single-nephron parameters in different CKD stages at the time of biopsy diagnosis of

IgAN. The association of clinical factors at biopsy (CKD stages, hypertension and

proteinuria) and histopathological lesions known to predict disease outcomes in IgAN were

then studied in relation to these nephron parameters.

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Methods

Patient selection

This cross-sectional study included all adult Japanese patients (≥18 years) who

underwent native kidney biopsies with diagnosis of primary IgAN at Jikei Hospital, Tokyo,

from 2007 to 2017. The sample size was set based on the number of kidney biopsies during

the period. Indications for biopsy were kidney functional decline (eGFR <60 mL/min) and/or

overt proteinuria (≥0.5 g/day) with or without gross or microscopic hematuria. Diagnosis of

IgAN was based on typical histopathological features of mesangial proliferative

glomerulonephritis, the presence of dominant or co-dominant glomerular IgA deposition by

immunohistochemistry or immunofluorescence, and the presence of electron dense mesangial

deposits by electron microscopy. Patients with other systemic diseases associated with

glomerular IgA deposition, including IgA vasculitis, liver cirrhosis, and systemic lupus

erythematosus were carefully excluded. At our institute, screening evaluation of kidney

morphology is routinely performed using unenhanced CT imaging prior to percutaneous

kidney biopsy. For this study, exclusion criteria were applied as follows: (i) patients whose

CT images were not available within 1 year before kidney biopsy, (ii) patients whose kidney

biopsy specimens contained < 5 nonsclerotic glomeruli on light microscopy or a cortical area

< 2 mm2. Consent was obtained by opting out for individual participants. All participants

were provided the opportunity to ask questions and discuss the study.

Definition

Hypertension was defined as a systolic blood pressure of >140 mmHg, a diastolic blood

pressure of >90 mmHg, or the use of antihypertensive medications. Patients who were

prescribed RAAS inhibitors for renoprotection despite having normal blood pressure were

not defined as having hypertension. Body surface area (BSA) was determined by the

following equation: BSA (m2) = Weight

0.425 (kg) × Height

0.725 (cm) ×71.84× 10

–4.22

The

7

estimated glomerular filtration rate (eGFR) was calculated from serum creatinine using a

modified equation for GFR based on Japanese individuals: eGFR = 194 × age–0.287

× (serum

creatinine) –1.094

(×0.739 if female).23

Chronic kidney disease (CKD) stages were defined

based on eGFR for Japanese individuals and were classified into 5 categories as follows:

CKD1: ≥90, 2: 60 to 89, 3a: 45 to 59, 3b: 30 to 44, and 4–5: <30 mL/min/1.73m2,

respectively. Creatinine clearance rate (CCr) was measured using serum and urine creatinine

concentrations in 24-hour urine collections. The eGFR and CCr values were calculated

without adjustment for BSA (units of mL/min) for the estimation of total GFR and eSNGFR.

Urinary protein excretion (UPE) was measured using 24-hour urine collection. Severe

proteinuria at biopsy was defined as UPE >1g/day.

Pathological analysis

All kidney tissue specimens were obtained by percutaneous needle biopsy. The tissues were

embedded in paraffin, cut into 3 µm sections, and stained with hematoxylin-eosin, periodic

acid-Schiff, Masson’s trichrome, and periodic acid silver-methenamine. All biopsy samples

were stained by immunohistochemistry or immunofluorescence for IgG, IgA, IgM, C3 and

C1q. Globally sclerosed glomeruli (GSG) was defined as the entire glomerulus involved by

sclerosis. Non-globally sclerotic glomerulus (NSG) was used when there was no sclerosis or

sclerosis only involved part of the glomerulus. Glomeruli containing segmental scars or

crescents were included among NSG. The cortical area with interstitial fibrosis/tubular

atrophy was semi-quantitatively scored to the nearest 10% and average values were estimated

across the entirety of each biopsy specimen. Arteriosclerotic lesions and arteriolar hyalinosis

were graded as previously described.24

The Oxford scores for mesangial hypercellularity (M),

endocapillary hypercellularity (E), segmental sclerosis or adhesion (S), interstitial fibrosis

and tubular atrophy (T) and crescents (C) were determined as described previously.24,25,26

Morphological measurements

8

The thickness of the obtained CT images was 5.0 mm. Kidney parenchymal volumes (PV)

were measured as previously described using software (ITK-SNAP version 3.6, University of

Pennsylvania, Philadelphia, PA, www.itksnap.org) to semi-automatically segment the

parenchymal images obtained from unenhanced CT images of both kidneys.27

Estimated

kidney cortical volumes were calculated using an equation as follows: estimated cortical

volume (cm3) = -1.3 (intercept) + 0.71 × parenchymal volume (cm

3).

21 An equation used for

estimating cortical volume from parenchymal volume is based on a mixed cohort of healthy

kidney donors and a variety of stages of CKD patients. Kidney biopsies were

semi-automatically analyzed to measure the individual areas of all glomerular capillary tufts

and the total area of the obtained renal cortex using image analysis software (Win roof 2017,

Mitani Corporation, Tokyo, Japan). Glomerular area was defined as an averaged area

described by outer capillary loops of the tuft. Mean glomerular volume was calculated from

the measured glomerular area as follows: Mean glomerular volume =

β

d ×(mean glomerular area)

3

2, where β is a dimensionless shape coefficient (β = 1.382), and d

is a size distribution coefficient (d = 1.01).28

The volumetric density of non-sclerotic

glomeruli (NSG) was determined using the Weibel-Gomez stereological method as follows:

NSG density = 1

β × √

(Total number of glomerulus

Area of cortex)

3

Total area of glomerulus

Area of cortex

2

, where β is a dimensionless shape coefficient (β =

1.38).28,29

The volumetric density of GSG was identically calculated: GSG density =

1

β × √

(Total number of sclerotic glomeruli

Area of cortex)

3

Total area of sclerotic glomeruli

Area of cortex

2

. The total number of all glomeruli was estimated based on the

sum of non-globally sclerotic and sclerotic glomeruli, as the sum of the non-sclerotic

glomerular density and sclerotic glomerular density. The total number of NSG per kidney

was calculated by multiplying the estimated cortical volume (mm3) and the volumetric

non-sclerotic glomerular density.30

The calculated value was divided by 2 for per kidney, by

9

1.43 for correcting tissue volume shrinkage due to paraffin embedding, and by 1.268 for

correcting volume shrinkage due to loss of tissue perfusion pressure. The single-nephron

glomerular filtration rate (eSNGFR) and single-nephron UPE (SNUPE) were calculated by

dividing total eGFR (mL/min) or total UPE by the total number of NSG in both kidneys.

Statistical analyses

Patients’ characteristics at baseline are presented as mean ± standard deviation (SD) or

median (25th

–75th

percentile) for continuous variables, and frequencies and proportions for

categorical variables. Data were log-transformed as appropriate. The Mann–Whitney U test

was used to compare continuous variables between two groups. For three or more groups,

trends were tested using linear regression or Jonckheere–Terpstra test. To analyze the

correlation with each factor, Spearman's rank correlation coefficient analysis was used. The

associations between clinical factors and glomerular number or single-nephron parameters

were analyzed using a linear regression model. To rule out the outliers, sensitivity analyses

were performed in subpopulations of the study participants. All reported p values were

two-sided. P values of <0.05 were considered to be statistically significant. All statistical

analyses were performed using EZR (Saitama Medical Center, Jichi Medical University), a

graphical user interface for R (R Foundation for Statistical Computing, version 3.5.2).31

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Results

Clinicopathological and morphometric findings

Total 245 IgAN patients were included in this study (Figure 1). Table 1 shows the

clinicopathological and morphometric findings of the patients at the time of biopsy diagnosis.

The biopsies included 239 initial kidney biopsies and six repeat biopsies. Overall, the mean

age was 42.6 years, 152 (62.0%) patients were males, and 52 (19.6%) had systemic

hypertension. One hundred-eleven patients (45.3%) had been administered RAAS inhibitors

before biopsy diagnosis. The initial indications of RAAS inhibitors were as follows; 81

(72.9%) patients were treated for hypertension and/or renoprotection in the presence of

hypertension and 30 (27.1%) patients were treated for renoprotection in the absence of

hypertension. The specific RAAS inhibitor therapies prescribed are listed in Supplemental

Table S1. eGFR was 61 ± 25 mL/min/1.73m2 and UPE was 835 (433–1536) mg/day. One

hundred-one patients (41.2%) had UPE ≥1g/day. Histopathology showed varying active and

chronic lesions as indicated by Oxford MESTC scores.

Clinicopathological characteristics of each CKD subgroup are separately shown in

Table 1. The distribution of CKD stage was 1 (9.8%), 2 (43.3%), 3a (19.2%), 3b (14.3%),

and 4–5 (13.5%). With advancing CKD stages, the characteristics of the patients shifted to

older ages, more systemic hypertension, heavier proteinuria and more chronic

histopathological lesions.

Comparisons of total glomerular number and single-nephron parameters among different

CKD stages

Total glomerular number and single-nephron parameters for the CKD stage subgroups are

shown in Figure 2. With advancing CKD stages, trend tests showed the number of NSG to

decrease, the number of GSG to increase, and the combined number of NSG and GSG to

decrease, respectively. Mean glomerular volume showed a trend to increase with advancing

11

CKD stages. The eSNGFR showed a slight change with CKD progression, but no statistically

significant trends between CKD stages, which was in sharp contrast to the profoundly

increased SNUPE levels identified with advancing CKD stages. Similar associations with

total glomerular number and single-nephron parameters were obtained in sub-analyses

restricted to patients who had not been administered RAAS inhibitors before biopsy

(Supplemental Figure S1, S2). Comparisons of total glomerular number and single-nephron

parameters among patients treated with or without RAAS inhibitors prior to biopsy diagnosis

are shown in Supplemental Figure S3. Patients treated with RAAS inhibitors had fewer

numbers of NSG and larger numbers of GSG at biopsy and showed larger glomeruli, higher

eSNGFR and higher SNUPE as compared to those without RAAS inhibitors.

Univariable and multivariable linear regression models for clinical factors associated with

total glomerular number and single-nephron parameters

Table 2 shows univariable and multivariable linear regression models that analyzed the

effects of clinical factors, including CKD stage, hypertension and proteinuria (UPE ≥1g/day)

at biopsy on total glomerular number and single-nephron parameters. In univariable models,

more advanced CKD was associated with lower numbers of NSG, higher numbers of GSG

and lower numbers of GSG + NSG, higher mean glomerular volume and higher SNUPE. All

of these variables associating with advancing CKD stages in univariate analyses were

significant in multivariable models adjusted for age, sex and BSA. Hypertension was

associated with lower numbers of NSG, lower numbers of GSG + NSG, higher eSNGFR and

higher SNUPE in univariable models and was associated with lower numbers of GSG + NSG

and higher eSNGFR in the multivariable models. Proteinuria was associated with lower

numbers of NSG, lower numbers of GSG + NSG, higher mean glomerular volume and higher

SNUPE in univariable models and was associated with higher SNUPE in multivariable

models. NSG, GGS and GSG + NSG were closely associated with eGFR and age in linear

12

regression models (Supplemental Table S2). In the sub-analyses restricted to patients not

receiving RAAS inhibitors, similar results were obtained (Supplemental Table S3). The

simple linear estimates of age-dependent reduction rate in the total numbers of NSG in the

present study of IgAN patients was greater than those for healthy donors (Supplemental

Figure S4).

Effects of hypertension on total glomerular number and single-nephron parameters in

relation to CKD stage at biopsy diagnosis

Figure 3 shows total glomerular number and single-nephron parameters in patients

categorized based on CKD stage (stage 1–2 vs. 3–5) and presence or absence of hypertension

at biopsy. Hypertension was associated with lower numbers of NSG and GSG + NSG than

normotension, higher eSNGFR levels in patients with CKD stage 1–2, and higher SNUPE

levels in patients with CKD stage 3–5. In the sub-analyses restricted to patients not receiving

RAAS inhibitors, similar results were obtained (Supplemental Figure S5). Comparisons in

patients categorized based on CKD stage and presence or absence of UPE ≥1g/day at biopsy

did not show any differences in total glomerular number and single-nephron parameters,

except that UPE ≥1g/day was associated with higher SNUPE in patients with CKD stage 3–5

(Supplemental Figure S6).

Comparisons of single-nephron parameters among patients with or without histopathological

lesions based on Oxford classification criteria

Figure 4 shows comparisons of single-nephron parameters in patients categorized based on

the presence or absence of lesions defined by Oxford MESTC scores. Mean glomerular

volumes were not different among patients with or without each MESTC component. The

eSNGFR was significantly lower in the presence of E and C lesions. The SNUPE was

significantly higher in the presence of S and T lesions. Comparisons of total single-nephron

parameters among patients with or without kidney functional decline or histopathological

13

lesions defined by Oxford MESTC scores are shown in Supplemental Figure S7. Univariate

linear regression models for the estimations of NSG and GGS per % or grade changes in each

renal histopathological variable are shown in Supplemental Table S4.

Sensitivity analyses

Sensitivity analyses were performed in patients whose number of NSG was within the 5–95th

percentile of the overall values (n = 221) or in patients whose biopsy specimens contained a

cortical area ≥4 mm2 (n = 233). Sensitivity analyses for eSNGFR were performed in patients

whose eSNGFR values were within the 5–95th percentile of the overall values (n = 221). The

results were similar to those of the original study population (Supplemental Table S5,

Supplemental Figure S8).

14

Discussion

In this study, we estimated the total number of glomeruli with and without global

glomerulosclerosis in patients with biopsy-proven IgAN. To estimate glomerular numbers,

we employed a newly established method that combines unenhanced CT scan images and

biopsy-based stereology. This approach has enabled us to estimate total glomerular numbers

in renal disease patients who are often not suitable candidates for contrast media.32,33

Our

results clearly show that the total numbers of NSG decreases and the total numbers of GSG

increases with advancing CKD stages in IgAN patients, as expected. Unexpectedly, the total

numbers of all glomeruli (the sum of GSG + NSG) showed a clear trend to decrease with

advancing CKD stages indicating that many glomeruli had disappeared without trace,

presumably through a process of resorption. This study further analyzed single-nephron

functional parameters in IgAN patients, providing important insights into the

pathophysiology of IgAN progression.

Kidney functional decline, hypertension and heavy proteinuria at biopsy diagnosis

are established predictors of worse outcomes in IgAN. 34,35,36

Among these factors, advanced

CKD stage and hypertension were both associated with lower numbers of NSG and NSG +

GSG. In addition, the numbers of NSG in patients with preserved kidney function (CKD

stage 1–2) were fewer in hypertensive than normotensive subjects, whereas there were no

differences in the numbers of GSG in these subjects. Given the evidence from

epidemiological and experimental studies showing a link between low nephron (glomerular)

endowment and essential hypertension, these results suggest that the presence of fewer

glomeruli in hypertensive IgAN patients may reflect lower nephron endowment.37

As

compared to kidney function or hypertension, proteinuria levels showed weaker association

with glomerular number. The preferential effects of CKD stage on total glomerular number

15

parameters are likely due to the fact that the number of glomeruli directly represents filtration

capacity.

Total glomerular number is known to decrease with normal aging.30,38,39,40,41

In this

study population of IgAN patients, the univariable linear estimate of decrease in NSG per

kidney was 11,504 per year (Supplemental Table S2), which is nearly double the rate of

nephron loss reported in aging subjects without kidney disease (6,200–6,700 per kidney per

year).30,42

In agreement with these observations, the simple linear estimates of age-dependent

reduction rate in the total numbers of NSG in the present study of IgAN patients was 1.8-fold

greater than those for healthy donors in our previous study using the same methodology

(Supplemental Figure S4).35

Of note, the total number of glomeruli in patients with CKD

stage 3b or more advanced was approximately 45% less than for patients in CKD stage 1.

In this study population, reliable surrogates for total glomerular number at birth,

such as birth weight, were not available. The requirement for biopsy diagnosis in IgAN may

produce a lead-time bias that could mask any substantial disease progression and loss of

glomeruli.43,44

Thus, we could not determine to what extent the low glomerular numbers

identified in advanced CKD stages in the present study were the cause or consequence of

disease progression of IgAN. The substantial decrease in the total glomerular number (GSG +

NSG) during aging as demonstrated in healthy living kidney donors suggests that some

globally sclerosed glomeruli are resorbed and disappear without trace.30

The same process of

decrease in total glomerular number suggesting resorption was observed in our IgAN patients

with advancing CKD stages, although to a more accelerated degree, as expected for a

progressive glomerulonephritis. Accordingly, chronic histopathological indices for glomerular,

vascular and tubulointerstitial injury were preferentially associated with lower numbers of

NSG (Supplemental Table S4).

16

A conceptual schematic that summarizes the factors involved in IgAN progression is

shown in Figure 5. An advantage of estimating total glomerular number in the clinical setting

is the capacity to estimate single-nephron parameters based on the corresponding clinical data.

Although values for eSNGFR did not differ between CKD stages, our results showed that

hypertension had an independent effect on higher SNGFR. In a study that examined

glomerular filtration in a rat model of nephrotoxic serum nephritis (NSN), SNGFR was kept

constant by increasing transcapillary hydraulic pressure difference (ΔP) while compensating

for a decrease in glomerular capillary ultrafiltration coefficient (Kf).45

These findings in NSN

rats are consistent with the findings in our present study of IgAN patients, where eSNGFR is

largely unaffected with advancing the CKD stages. On the other hand, the wide variation in

eSNGFR within each CKD stage suggests that additional factors other than systemic

hypertension such as aging and arteriosclerotic lesions of the kidney may affect intra-renal

plasma flow rate and thereby SNGFR. 46,47

Diversity in SNGFR among patients with the

same CKD stages may represent "biphasic" vulnerability of SNGFR (hyperfiltration or

hypofiltration). Thus, evaluating SNGFR in a clinical setting would allow us to detect

dynamic changes in filtration function at the single-nephron level rather than to simply count

the number of nephrons that appear to be functioning.

Despite an increase in mean glomerular volume, the SNGFR did not increase with

advancing CKD stage in response to reduced number of NSG. A discrepancy between

SNGFR and mean glomerular volume suggests a failure of compensatory glomerular

hyperfiltration. Abnormalities in single-nephron dynamics may be the effects of

histopathological lesions specific to IgAN, including segmental glomerular scarring,

compromise of Bowman’s space by crescents, and reduced glomerular capillary luminal

diameter caused by variable mesangial and/or endocapillary hypercellularity, increased

matrix and immune deposits. To address these possibilities, we compared total glomerular

17

number and single-nephron parameters among patients with and without Oxford M, E, S, T

and C lesions. We found eSNGFR was significantly lower in patients with E or C lesions,

supporting their contribution to reduced glomerular filtration at the single-nephron level in

IgAN (Figure 4, Supplemental Figure S7). Among the Oxford histopathological lesions, T

lesion is known to be closely correlated to global glomerulosclerosis and may influence the

eSNGFR values. However, we could not find any significant difference in eSNGFR values

among patients with or without T lesions. These results are consistent with the results that

single-nephron GFR is fairly unchanged with advancing CKD stages, which may be

explained by diversity in GFR values possibly due to the biphasic vulnerability

(hyperfiltration or hypofiltration) of individual glomeruli in patients with various degrees of

T lesions.

A novel biomarker used in this study, the SNUPE, may help elucidate the

pathophysiology of proteinuric glomerulopathies. Our results showed that the increase in

SNUPE was disproportionately much greater than for total UPE with advancing CKD stages.

Compared to the CKD stage 1 as reference, the stage 4–5 exhibited a 5-fold increase in total

UPE and a striking 19-fold increase in SNUPE. Interestingly, the SNUPE were significantly

higher in patients with S or T lesions, suggesting a pathogenetic link. Given the potential for

heavy proteinuria to reflect podocyte injury and loss of filtration barrier integrity, as well as

to cause tubulointerstitial injury via excessive protein trafficking, SNUPE may provide a

highly sensitive marker of and risk factor for disease progression.48

In this study, 111 (45.3%) of patients were treated with RAAS inhibitors prior to

biopsy diagnosis. According to renoprotective mechanisms, RAAS inhibition is expected to

attenuate glomerular hyperfiltration and thereby eSNGFR and SNUPE. However, patients

treated with RAAS inhibitors had fewer numbers of NSG and larger numbers of GSG at

biopsy as compared to those without RAAS inhibitors (Supplemental Figure S2), indicating

18

that these patients already had more advanced kidney injury, which probably influenced their

selection for treatment. Due to this treatment bias, it is difficult to determine whether RAAS

inhibitors effectively attenuated hyperfiltration and the associated proteinuria at the

single-nephron level in these patients.

This study has notable limitations. First, the method of estimating glomerular

number was based on needle biopsy specimens. The sensitivity analyses demonstrated that

our estimates were robust (Supplemental Table S5); however, this does not rule out the

possibility that differences in glomerular density between biopsy sites and SNGFR within a

kidney could cause sampling bias.49,50

Second, the single-nephron parameters are determined

as averaged values and do not always represent true ‘single’ values. Glomerular lesions in

IgAN are heterogeneous and not uniformly distributed throughout the kidney. The focality of

some glomerular lesions may cause divergence in eSNGFR and SNUPE within a kidney.

Third, like creatinine-based eGFR, creatinine-based estimation of SNGFR also reflects the

tubular secretion of creatinine.51

In this study cohort, we did not have measured GFR

available as it is not routinely obtained in clinical care of IgAN patients. Fourth, to determine

cortical volume (needed to estimate nephron number and SNGFR), total kidney volume was

measured on CT scans and the fraction of cortical volume was estimated from the total

kidney volume. Intravenous contrast is needed to separately measure cortical volume on CT

scans but is avoided in patients with CKD due to concerns for contrast nephropathy. Finally,

all patients included in this study are Japanese. Given the potential difference in total

glomerular number among races, validation studies among different races are required.

In conclusion, this study estimated total glomerular number and related

single-nephron parameters in 245 patients with IgAN. The findings support progressive

reduction in NSG and total glomeruli (GSG + NSG) with advancing CKD stage, indicating

that sclerosed glomeruli can be resorbed over time. The finding of lower numbers of NSG in

19

hypertensive than normotensive patients with preserved kidney function (CKD stage 1–2)

implicates a predisposing role for low nephron endowment. SNUPE emerged as a stronger

biomarker than SNGFR in advanced stage CKD, likely reflecting the limited ability of

diseased hypercellular glomeruli to respond by compensatory hyperfiltration. These findings

illustrate the feasibility and usefulness of estimating single-nephron dynamics in human

glomerulonephritis. Future studies are needed to determine the generalizability of these

findings to other forms of glomerulonephritis.

20

Disclosures

J. Bertram reports Scientific Advisor or Membership: Kidney International. A. Rule reports

Scientific Advisor or Membership: NIDDK - CKD Biomarker Consortium External Expert

Panel, JASN – Associate Editor, Mayo Clinic Proceedings - Section Editor; Other

Interests/Relationships: UpToDate. T. Yokoo reports Scientific Advisor or Membership:

Editorial Board Member of "HUMAN CELL". All remaining authors have nothing to

disclose.

Funding

This work was supported by JSPS KAKENHI grant numbers JP25461236 and JP16K0936

(to NT).

Acknowledgments

This study was approved by the ethics review board of the Jikei University School of

Medicine [30-385 (9406)] and conducted according to the Declaration of Helsinki. Parts of

this study were presented at the 62th Annual Meeting of The American Society of

Nephrology, Nov 5–10, 2019, Washington D.C., USA.

Authors’ Contributions

H Marumoto: Conceptualization; Data curation; Formal analysis; Investigation;

Methodology; Project administration; Validation; Visualization; Writing - original draft;

Writing - review and editing

N Tsuboi: Conceptualization; Formal analysis; Funding acquisition; Investigation;

Methodology; Supervision; Validation; Writing - original draft; Writing - review and editing

V D'Agati: Writing - review and editing

T Sasaki: Writing - review and editing

Y Okabayashi: Writing - review and editing

K Haruhara: Writing - review and editing

21

G Kanzaki: Writing - review and editing

K Koike: Writing - review and editing

A Shimizu: Writing - review and editing

T Kawamura: Writing - review and editing

A Rule: Writing - review and editing

J Bertram: Writing - original draft; Writing - review and editing

T Yokoo: Writing - review and editing

Each author contributed relevant intellectual content accepted accountability for the overall

work by ensuring that questions pertaining to the accuracy or integrity of any portion of the

work are appropriately investigated and resolved. The final version was approved by all

authors.

Supplemental Material

Supplemental Table S1. Specific RAAS inhibitor therapies prescribed prior to biopsy

diagnosis

Supplemental Table S2. Unadjusted estimates of total numbers of glomeruli depending on

clinical variables in linear regression models

Supplemental Table S3. The association of clinical characteristics with total glomerular

number per kidney and single-nephron parameters among IgAN patients who had not been

treated with RAAS inhibitors before biopsy

Supplemental Table S4. Renal histopathological variables associated with non-globally

sclerotic glomeruli and globally sclerotic glomeruli per kidney in univariate linear regression

models

Supplemental Table S5. Sensitivity analyses for clinical characteristics as predictors of

non-globally and globally sclerotic glomerular numbers in univariate linear regression

models

22

Supplemental Figure S1. Comparisons of total glomerular number and single-nephron

parameters among IgAN patients who had not been treated with RAAS inhibitors before

biopsy

Supplemental Figure S2. Comparisons of total glomerular number and single-nephron

parameters among IgAN patients who treated with RAAS inhibitors at biopsy

Supplemental Figure S3. Comparisons of total glomerular number and single-nephron

parameters among patients treated with or without RAAS inhibitors prior to biopsy diagnosis

Supplemental Figure S4. Correlations between age and non-sclerotic glomerular number or

total glomerular number

Supplemental Figure S5. Comparisons of total glomerular number and single-nephron

parameters among IgAN patients with and without kidney functional decline or hypertension:

Sub-group analyses of patients who had not been treated with RAAS inhibitors before biopsy

Supplemental Figure S6. Comparisons of total glomerular number and single-nephron

parameters among IgAN patients with and without kidney functional decline or presence and

absence of UPE ≥1g/day

Supplemental Figure S7. Comparisons of total single-nephron parameters among patients

with or without kidney functional decline or histopathological lesions defined by Oxford

MESTC scores.

Supplemental Figure S8. Comparison of single-nephron GFR within the 5–95th percentile of

the overall values

23

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29

Table 1. Baseline characteristics of patients in the different CKD stages

Overall (n=245) CKD stage

1 (n=24) 2 (n=106) 3a (n=47) 3b (n=35) 4–5 (n=33)

Clinical findings

Age (years) 42.6 ± 14.1 27.8 ± 5.7 38.0 ± 11.5 46.0 ± 10.8 53.9 ± 15.2 51.3 ± 13.4

Male (%) 62.0 54.2 55.7 74.5 62.9 69.7

BMI (kg/m2

) 23.0 ± 3.7 21.1 ± 4.3 22.8 ± 3.8 23.7 ± 3.10 23.9 ± 3.81 22.6 ± 3.4

BSA (m2

) 1.71 ± 0.20 1.63 ± 0.18 1.70 ± 0.20 1.76 ± 0.19 1.70 ± 0.22 1.71 ± 0.22

Patients with hypertension (%) 19.6 8.3 18.3 13.6 25.7 33.3

RAAS inhibitor use (%) 45.3 25 28.3 55 80 63.6

Laboratory findings

Serum albumin (g/dL) 3.8 ± 0.5 4.1 ± 0.3 3.9 ± 0.5 3.9 ± 0.4 3.6 ± 0.7 3.4 ± 0.6

Serum creatinine (mg/dL) 1.19 ± 0.63 0.67 ± 0.12 0.84 ± 0.15 1.13 ± 0.14 1.50 ± 0.25 2.45 ± 0.73

eGFR (mL/min) 60.0 ± 25.1 98.6 ± 27.3 73.0 ± 10.3 54.5 ± 7.2 36.1 ± 6.3 23.1 ± 6.2

24-hour Ccr (mL/min) 83 ± 34 119 ± 35 99 ± 24 85 ± 18 51 ± 14 37 ± 13

Serum uric acid (mg/dL) 6.5 ± 1.7 5.1 ± 1.3 5.9 ± 1.4 7.0 ± 1.1 7.7 ± 1.4 7.9 ± 1.8

Triglyceride (mg/dL) 144 ± 83 113 ± 76 136 ± 97 165 ± 84 152 ± 51 152 ± 53

HDL cholesterol (mg/dL) 62 ± 18 67 ± 17 64 ± 19 58 ± 17 62 ± 18 60 ± 19

LDL cholesterol (mg/dL) 121 ± 32 110 ± 34 121 ± 32 121 ± 30 122 ± 34 130 ± 35

HbA1c (%) 5.5 ± 0.5 5.4 ± 0.7 5.4 ± 0.4 5.5 ± 0.4 5.6 ± 0.6 5.5 ± 0.4

IgA (mg/dL) 324 ± 106 315 ± 107 307 ± 91 344 ± 106 370 ± 140 303 ± 95

C3 (mg/dL) 100 ± 19 93 ± 17 101 ± 16 103 ± 20 102 ± 25 96 ± 16

Urinary RBC count, grade 1–5 (%)* 77.0 66.7 81.0 87.2 74.3 60.6

UPE (mg/day) 835 (433–1,536) 419 (250–608) 633 (423–1129) 1,066 (432–1,392) 1,037 (610–2,333) 1,983 (1,263–

3,362)

Patients with UPE > 1g/day (%) 41.2 4.2 28.3 53.2 51.4 81.8

Histopathological findings

Total glomeruli identified in biopsy specimen 23.0 ± 11.0 25.0 ± 12.4 26.0 ± 11.4 21.3 ± 10.4 19.0 ± 8.5 18.8 ± 8.7

Glomeruli with global sclerosis (%) 16.9 ± 16.1 5.4 ± 7.4 10.7 ± 12.5 19.2 ± 14.6 24.1 ± 14.2 33.8 ± 17.8

Glomeruli with segmental sclerosis (%) 2.8 ± 4.8 1.1 ± 3.3 2.2 ± 3.2 2.2 ± 4.5 5.2 ± 6.1 4.0 ± 7.3

Glomeruli with cellular/fibrocellular crescents (%) 10.7 ± 27.6 7.1 ± 26.2 11.8 ± 32.7 7.8 ± 15.3 9.9 ± 29.4 14.7 ± 22.1

Glomeruli with fibrous crescents (%) 2.0 ± 6.8 2.0 ± 6.1 2.6 ± 7.4 0.0 ± 0.3 2.0 ± 4.8 2.7 ± 10.7

Interstitial fibrosis/tubular atrophy (%) 17.8 ± 17.0 8.1 ± 6.9 9.6 ± 7.2 16.2 ± 12.5 27.5 ± 17.1 43.0 ± 20.2

Arteriosclerotic lesion†

, grade 1–2 (%) 59.0 16.7 55.2 59.6 77.1 81.8

Arteriolar hyaline, grade†

1–3 (%) 34.7 16.7 30.2 36.2 48.6 45.5

Oxford Classification

Patients with M1 (%) 46.5 45.8 46.2 36.2 48.6 60.6

Patients with E1 (%) 12.2 8.3 13.2 8.5 5.7 24.2

Patients with S1 (%) 89.4 75.0 85.8 95.7 97.1 93.9

Patients with T1+2 (%) 24.1 4.2 3.8 19.1 54.3 78.8

Patients with C1+2 (%) 38.0 25.0 41.5 38.3 28.6 45.5

Morphological measurements

Renal parenchymal volume (cm3

/kidney) 131.9 ± 33.6 148.4 ± 29.8 142.2 ± 30.6 132.5 ± 23.5 112.0 ± 28.2 106.7 ± 40.4

Renal cortical volume (cm3

/kidney) 92.3 ± 23.9 104.1 ± 21.1 99.7 ± 21.7 92.7 ± 16.7 78.3 ± 20.0 74.5 ± 28.7

Globally sclerotic glomerular density (/mm3

) 2.54 (1.07–6.05) 1.06 (0.00–2.12) 1.52 (0.84–4.36) 3.34 (1.60–7.23) 3.72 (2.00–5.95) 5.40 (3.56–9.35)

Non-globally sclerotic glomerular density (/mm3

) 12.5 (7.97–17.0) 16.5 (11.2–21.5) 15.32 (12.1–18.8) 11.8 (7.75–14.2) 7.10 (5.79–9.34) 6.24 (4.76–10.0)

Values are presented as the means ± standard deviations or median (25th

–75th

percentile). *The urinary RBC count was graded as follows: grade 0, <5/high power field (HPF); grade 1,

5–9/HPF; grade 2, 10–19/HPF; grade 3, 20–49/HPF; grade 4, 50–99/HPF; and grade 5,

>99/HPF. †Arteriosclerotic lesions were defined as normal (grade 0), and less than 50%

(grade 1) or more than 50% of the thickness of media (grade 2). Arteriolar hyaline was

graded as the proportion of arterioles affected (grade 0, <1%; grade 1, 1–25%; grade 2, 26–

50%; grade 3, >50%). BMI, body mass index; BSA, body surface area; Ccr, Creatinine

clearance rate; CKD, chronic kidney disease; GFR, glomerular filtration rate; HDL, high

density lipoprotein; IgA, immunoglobulin A; LDL, low density lipoprotein; UPE, urinary

protein excretion; RAAS, renin-angiotensin aldosterone system; RBC, red blood cell.

30

Table 2. The association of clinical characteristics with total glomerular number per kidney and single-nephron parameters

*Adjusted for age, sex, and BSA in addition to CKD stage, hypertension, and UPE.

†Five categories of CKD stage were defined: eGFR levels ≥90, 60–89, 45–59, 30–44 and <30 mL/min/1.73m

2, respectively. BSA, body surface

area; CKD, chronic kidney disease; GFR, glomerular filtration rate; UPE, urinary protein excretion.

Non-globally sclerotic

glomeruli

(number per kidney)

Globally-sclerotic

glomeruli

(number per kidney)

Total glomeruli

(number per kidney)

Mean glomerular volume

(×106

μm3

)

Single-nephron GFR

(nL/min)

Single-nephron UPE

(μg/day)

Clinical variables Estimate P value Estimate P value Estimate P value Estimate P value Estimate P value Estimate P value

Univariable

CKD category† -196,284 <0.001 50,918 <0.001 -145,366 <0.001 0.327 <0.001 2.136 0.21 1.406 <0.001

Hypertension -154,544 0.02 -5,651 0.88 -160,195 0.02 0.403 0.06 10.507 0.047 1.432 <0.001

UPE ≥ 1 g/day -190,543 <0.001 56,199 0.053 -134,345 0.02 0.337 0.048 -2.034 0.63 2.949 <0.001

Multivariable*

CKD category† -172,720 <0.001 49,712 <0.001 -123,007 <0.001 0.311 <0.001 2.290 0.29 1.225 <0.001

Hypertension -93,575 0.08 -35,891 0.33 -129,466 0.046 0.186 0.36 11.420 0.04 0.600 0.15

UPE ≥ 1 g/day 20,944 0.66 -12,605 0.70 8,338 0.88 -0.265 0.14 -9.301 0.06 1.940 <0.001

31

Figures and legends

Figure 1. Patients selection

During the study period, we identified 324 Japanese patients with primary IgAN.

Seventy-nine patients were excluded from this study due to lack of CT data or inadequate

glomerular number or cortical area in biopsy specimens. CT, computed tomography; IgAN,

IgA nephropathy.

Figure 2. Comparisons of total glomerular number and single-nephron parameters

among IgAN patients at different CKD stages

Total number of non-globally sclerotic glomeruli (A), globally sclerotic glomeruli (B), total

glomeruli (C), mean glomerular volume (D), single-nephron GFR (E), single-nephron UPE

(F) were compared among patients with IgAN at different CKD stages at biopsy diagnosis.

Bold horizontal line and scale bar indicates each mean value and the 5–95 percentile,

respectively. Percent difference indicates change in mean values compared to mean value in

CKD stage 1 as a reference. CKD, chronic kidney disease; IgAN, IgA nephropathy.

Figure 3. Comparisons of total glomerular number and single-nephron parameters

among IgAN patients with and without kidney functional decline or hypertension

Patients were categorized based on CKD stage (stage 1–2 vs. stage 3–5) or presence or

absence of hypertension at biopsy diagnosis. The numbers of non-globally sclerotic glomeruli

(A), globally sclerotic glomeruli (B), total glomeruli (C), and values for mean glomerular

volume (D), single-nephron GFR (E), single-nephron UPE (F) were compared among the

groups. CKD, chronic kidney disease; GFR, glomerular filtration rate; HT, hypertension;

IgAN, IgA nephropathy; UPE, urinary protein excretion.

32

Figure 4. Comparisons of single-nephron parameters among patients with or without

histopathological lesions defined by Oxford MESTC scores

The mean glomerular volume (A, D, G, J, M), single-nephron GFR (B, E, H, K, N) and

single-nephron UPE (C, F, I, L, O) were compared between the groups categorized based on

the presence or absence of lesions defined by Oxford MESTC scores. Bold horizontal line

and scale bar indicates each mean value and the 5–95 percentile, respectively. GFR,

glomerular filtration rate; UPE, urinary protein excretion; M, mesangial hypercellularity; E,

endocapillary hypercellularity; S, segmental sclerosis or adhesion; T, interstitial fibrosis and

tubular atrophy; C, crescents.

Figure 5. Factors involved in IgAN progression to ESKD

Individual nephron endowment may determine the sensitivity to progressive nephron loss

when it encounters acquired kidney diseases such as chronic glomerulopathies including

IgAN. In 245 IgAN patients analyzed in this study, single-nephron GFR level was largely

unchanged among the different CKD stage groups, but varied significantly between

individuals in the same CKD stages. Increased UPE levels were more pronounced at the

single nephron level in advanced CKD stages. This study therefore indicates "biphasic"

vulnerability of single-nephron GFR (hyper- or hypo-glomerular filtration) is involved in the

progression of IgAN to ESKD. Some of histopathological findings specific to IgAN may

interfere with the ability to achieve compensatory hyperfiltration during progressive nephron

loss. CKD, chronic kidney disease; ESKD, end-stage kidney disease; GFR, glomerular

filtration rate; IgAN, immunoglobulin A nephropathy; Kf, glomerular capillary ultrafiltration

coefficient; ΔP, transcapillary hydraulic pressure difference; UPE, urinary protein excretion.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5