Evidences of histologic thrombotic microangiopathy and the impact in renal outcomes of patients with IgA nephropathy.

INTRODUCTION: IgA nephropathy (IgAN) is the most common primary glomerulopathy worldwide. According to the Oxford Classification, changes in the kidney vascular compartment are not related with worse outcomes. This paper aims to assess the impact of thrombotic microangiopathy (TMA) in the outcomes of Brazilian patients with IgAN.
MATERIALS AND METHODS: Analysis of clinical data and kidney biopsy findings from patients with IgAN to assess the impact of TMA on renal outcomes.
RESULTS: The majority of the 118 patients included were females (54.3%); mean age of 33 years (25;43); hypertension and hematuria were observed in 67.8% and 89.8%, respectively. Median creatinine: 1.45mg/dL; eGFR: 48.8ml/min/1.73m2; 24-hour proteinuria: 2.01g; low serum C3: 12.5%. Regarding to Oxford Classification: M1: 76.3%; E1: 35.6%; S1: 70.3%; T1/T2: 38.3%; C1/C2: 28.8%. Average follow-up: 65 months. Histologic evidence of TMA were detected in 21 (17.8%) patients and those ones presented more frequently hypertension (100% vs. 61%, p <0.0001), hematuria (100% vs 87.6%, p = 0.0001), worse creatinine levels (3.8 vs. 1.38 mg/dL, p = 0.0001), eGFR (18 vs. 60 ml/min/1.73m2), p = 0.0001), low serum C3 (28.5% vs. 10.4%, p = 0.003), lower hemoglobin levels (10.6 vs. 12.7g/dL, p<0.001) and platelet counts (207,000 vs. 267,000, p = 0.001). Biopsy findings of individuals with TMA revealed only greater proportions of E1 (68% vs. 32%, p = 0.002). Individuals with TMA were followed for less time (7 vs. 65 months, p<0.0001) since they progressed more frequently to chronic kidney disease (CKD) requiring kidney replacement therapy (KRT) (71.4% vs. 21,6%, p<0.0001). Male sex, T1/T2, and TMA were independently associated with progression to CKD-KRT.
CONCLUSIONS: In this study patients with TMA had worse clinical manifestations and outcomes. In terms of histologic evidence, E1 distinguished patients with TMA from other patients. Further studies are necessary to analyze the impact of vascular lesions on IgAN prognosis.

Introduction

IgA nephropathy (IgAN) is a highly prevalent condition worldwide and ranks as the most common primary glomerulopathy in some countries [1-3]. Given the high prevalence of the disease and the fact that about 30% of the patients with IgAN progress to chronic kidney disease (CKD) requiring kidney replacement therapy (KRT) [1,4], it is imperative to identify clinical and histology markers associated with worse renal outcomes.

The most widely accepted explanation for the IgAN pathogenesis is the 4-hit hypothesis, in which Hit 1 involves the production of hypoglycosylated IgA1; Hit 2 starts with the production of IgG antibodies that recognize hypoglycosylated IgA1; Hit 3 regards the formation of potentially nephritogenic IgG/IgA1 immune complexes; and in the Hit 4 there is deposition of formed complexes in the glomerular mesangium and capillaries, thereby activating the immune system and leading to the recruitment of inflammatory cells, cytokines, and the activation of the complement system [1,2,5].

The Oxford Classification (OC) [6,7] was first published in 2009 as an attempt to identify kidney biopsy alterations possibly associated with worse outcomes in patients with IgAN. Mesangial hypercellularity, segmental glomerulosclerosis, and interstitial fibrosis/tubular atrophy have been associated with progression to CKD-KRT, while endocapillary hypercellularity was first correlated with function decline in patients on immunosuppressant therapy and later with worse renal outcomes. An updated version of the Oxford Classification was published in 2017 [8], and cellular crescents were added as markers of worse renal outcomes. It should be mentioned that vascular alterations were not included in the Oxford Classification, since they were not associated with worse outcomes in patients with IgAN. However, recent studies [9-11] have looked into the role of vascular alterations and their ties with the outcomes of patients with IgAN, shedding light on a matter yet unresolved in the literature.

Thrombotic microangiopathy (TMA) is a histology finding of vascular involvement associated with some renal conditions–atypical and typical hemolytic-uremic syndrome, eclampsia, accelerated hypertension, thrombotic thrombocytopenic purpura–that may also be induced by certain drugs [12,13]. Histology and serum findings of TMA have been associated with other primary and secondary glomerulopathies–lupus nephritis, ANCA-associated vasculitis, focal segmental glomerulosclerosis, and IgA nephropathy–and correlated with worse renal outcomes in individuals with IgAN [9,10,14,15].

This study aimed to assess the impact of histologic findings of TMA on the renal outcomes of individuals with IgAN seen at a healthcare center in Brazil.

Materials and methods

Study design and population

This retrospective single-center study included patients diagnosed with IgA nephropathy based on kidney biopsy findings from 2000 to 2018. Patients with IgAN secondary to systemic conditions (Henoch-Schönlein purpura, liver disease, autoimmune disease, HIV infection) and individuals with insufficient follow-up or outcome data were excluded, along with patients with fewer than eight glomeruli for analysis via the Oxford Classification.

Among our patients there are no solid organs transplanted patients, bone marrow transplant, pregnant women or patients diagnosed with cancer. We have no description of possible medications associated with TMA. Serologies were performed for Hepatitis B, C and HIV as well as research for autoimmune diseases (ANA, ANCA, Rheumatoid Factor, lupus anticoagulant). No genetic tests have been performed.

The following clinical data were considered at the time of kidney biopsy: age; sex; serum creatinine (SCr); estimated glomerular filtration rate (e-GFR); 24-hour proteinuria and/or urine protein/creatinine (UPC) ratio; hematuria; hypertension; serum C3 level; serum IgA; hemoglobin; platelet count; lactate dehydrogenase (LDH); and indirect bilirubin. The glomerular filtration rate was estimated based on the CKD-EPI [16] equation. Hematuria was defined as ≥3 red blood cells/high-power field in a sample of urine. Hypertension was defined as a blood pressure ≥140 and/or 90mmHg [17]. The reference ranges laboratory tests were as follows: C3 (90-180mg/dL); IgA (69-382mg/dL); LDH (135-214U/L); indirect bilirubin (0.2; 0.8mg/dL); haptoglobin (30-200mg/dL). Anemia was defined as hemoglobin <12g/dL for females and 13g/dL for males [18]. Thrombocytopenia was defined as having a platelet count <150,000/mm3 [19]. Presence of schistocytes was verified via peripheral blood smear tests.

Patients were also analyzed for prescription of angiotensin-converting-enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs), corticosteroids, and other immunosuppressants. The end of follow-up was defined either by the last visit of the patient to the healthcare unit or by referral to dialysis or kidney transplantation.

Histopathology

Kidney biopsy specimens analyzed by light microscopy were stained by hematoxylin and eosin (H&E), Masson's trichrome, periodic acid–Schiff (PAS), and periodic acid silver methenamine stain (PAMS). Analysis by immunofluorescence microscopy included serum anti-IgA, IgG, IgM, C3, C1q, kappa and lambda light chains, and fibrinogen, with positive deposition defined when intensity ≥1. The biopsies were reviewed by a renal pathologist and classified based on the latest version of the Oxford Classification [8].

As previously described in the literature [20], acute TMA histologic findings were categorized based on renal compartments. In glomeruli: presence of thrombi, edema, or endothelial denudation, fragmented red blood cells, mesangiolysis, microaneurysms; in arterioles: thrombi, edema, or endothelial denudation, intramural fibrin, fragmented red blood cells, edema of the intima, myocyte necrosis; and in arteries: thrombi, myxoid intimal change, intramural fibrin, fragmented red blood cells. Chronic findings included in glomeruli: double contour in capillaries with mesangial interposition; in arterioles hyaline deposits; in arteries fibrous intimal thickening and concentric lamination resembling the bulb of an onion. Histologic findings of TMA were assessed only based on light microscopy examination.

CD68 and C4d immunohistochemistry

Formalin-fixed, paraffin-embedded tissue was sectioned at 2μm and stained with rabbit monoclonal anti-CD68 (Santa Cruz Biotechnology) and anti-C4d (Clone SP91, Spring-Bioscience) as the primary antibody. All cases were stained by hand using routine protocols, including deparaffinization, followed by antigen retrieval (tissue section was boiled in 1mM EDTA, pH 8.0 for 10 min followed by cooling at room temperature for 20 min), protein blocking (DPB-125S; Spring, Pleasanton, CA, USA), incubation for primary antibody at room temperature for 30 min (1:40) and for secondary goat anti-rabbit IgG (DHRR-999; Spring, Pleasanton, CA, USA) at 1:360. Detection was performed with streptavidin/horseradish peroxidase (SPB-125; Spring) and developed with Stable DAB (Spring). CD68-positive cells in the glomeruli and tubulointerstitium were quantified and the final count was expressed as number of cells/glomerulus and cells/field, respectively. C4d immunohistochemistry was considered positive if glomerular staining was observed.

Endpoint analysis

Progression to CKD-KRT was the primary endpoint assessed. Secondary endpoint included achieving an e-GFR ≤60ml/min/1.73m2.

Statistical analysis

The distribution of variables was assessed with the Shapiro-Wilk test. Qualitative variables were expressed as proportions and compared against each other via the chi-squared test or Fisher’s exact test. Variables following a parametric distribution were expressed as mean values ± standard error and compared against each other with Student’s t-test. Variables with non-parametric distributions were expressed as median values (first and third quartiles) and compared against each other with the Mann-Whitney U test. Logistic multinomial regression was performed in all patients and it was used in multivariate analysis to assess independent risk factors to and adjusted for sex, hypertension, creatinine >1.2mg/dL (upper normality range of the test), Oxford classification parameters (M, E, S, T, C) and TMA. Statistical significance was attributed to significance level alpha = 0.05. Survival free of dialysis in patients with and without evidence of TMA in kidney biopsy specimens was analyzed by Kaplan-Meier curve.

Results

Exclusion criteria accounted for the removal of 50 patients from the original population of 168 individuals. Of the remaining 118 patients, 65 were females (55%), 86 were whites (73%), 25 were black (21%), 7 were east-asian (6%); 80 (67.8%) presented with hypertension and 106 (89.8%) hematuria (89.8%). Low serum C3 was detected in 15 (12.5%) patients. Table 1 shows patient data and laboratory tests at the time of kidney biopsy. Histologic findings based on the Oxford Classification were as follows: M1 (79.6%), E1 (35.6%), S1 (70.3%), T1/T2 (38.3%), and C1/C2 (28.8%). The median follow-up time was 65 months, and 36 individuals progressed to CKD-KRT.

Table 1

Baseline clinical characteristics and kidney biopsy findings of patients with IgA Nephropathy.

	N = 118
Age (years)	33 (25;43)
Female sex (n/%)	65 / 55
Race (n/%)
White	86 / 73
Black	25 / 21
East-asian	7 / 6
Serum creatinine (mg/dL)	1.45 (0.99;2.6)
e-GFR by CKD-EPI (ml/min/1.73m2)b	48.8 (27.5;78.5)
24h-proteinuria (g)	2.01 (1.1;3.7)
Serum albumin (g/dl)	3.4 (2.9;3.8)
Hematuria (n/%)	106 / 89.8
Hypertension (n/%)	80 / 67.8
Low serum C3 levels (n/%)	15 / 12.5
Follow-up (months)	65 (27;115)
ΔeGFR (ml/min/1.73m2/year)	-1.25 (-7.11;0.91)
CKD-KRT (n/%)	36 / 30.5
Time to CKD-KRT (months)	9 (3;38)
Kidney Histology—Oxford Classification (n/%)
M1	94 / 79.6
E1	42 / 35.6
S1	83 / 70.3
T1/T2	45 / 38.3
C1/C2	34 / 28.8
Thrombotic Microangiopathy (n/%)	21 / 17.8

eGFR: Estimated glomerular filtration rate, CKD requiring RRT: Chronic Kidney Disease requiring Renal Replacement Therapy, M1: Mesangial hypercellularity, E1: Endocapillary hypercellularity, S1: Segmental glomerulosclerosis, T1/T2: Tubular atrophy or interstitial fibrosis, C1/C2: Cellular crescent.

Histologic evidences of TMA were seen in 21 (17.8%) patients and acute findings as well as chronic lesions predominate in arterioles over glomeruli. More frequent acute lesions in arteriolar compartment were intramural fibrin (33.3%) and myocyte necrosis (23.3%) while fibrous intimal thickening with concentric lamination hyaline deposits (100%) and hyaline deposits (90.5%) were the most common chronic lesions. Glomerular compartment showed more chronic lesions, such as double contour in capillaries with mesangial interposition in 38.1% of the patients, than acute findings such as edema or endothelial denudation (14.3%) and mesangiolysis (14.3%). The Table 2 illustrates histological findings of TMA on renal biopsy. Table 3 describes the presence of serum findings consistent with TMA in patients with and without histologic evidence of TMA. When compared to individuals without signs of TMA in kidney biopsy, patients with histologic evidence of TMA had more anemia (81.3% vs. 24.7%, p = 0.001), more schistocytes in peripheral blood (44.5% vs. 3.1%, p = 0.0007), and were more prone to developing thrombocytopenia, albeit not significantly (14.3% vs. 4.1%, p = 0.07).

Table 2

Histological findings of thrombotic mycroangiopathy in renal biopsies of IgAN patients.

	N = 21
Acute Findings (n/%)
Glomeruli
Glomeruli thrombi	2 / 9.5
Edema or endothelial denudation	3 / 14.3
Fragmented red blood cells	1 / 4.8
Mesangiolysis	3 / 14.3
Microaneurysms	0 / 0
Arterioles
Thrombi	3 / 14.3
Edema or endothelial denudation	4 / 19
Intramural fibrin	7 / 33.3
Fragmented red blood cells	1 / 4.8
Edema of the intima	0 / 0
Myocyte necrosis	5 / 23.8
Arteries
Thrombi	0 / 0
Myxoid intimal swelling	3 / 14.3
Intramural fibrin	0 / 0
Fragmented red blood cells	0 / 0
Chronic findings (n/%)
Glomeruli
Double contour in capillaries with mesangial interposition	8 / 38.1
Arterioles
Hyaline deposits	19 / 90.5
Arteries
Fibrous intimal thickening with concentric lamination	21 / 100

Table 3

Laboratory findings of thrombotic microangiopathy in patients with and without histologic evidence of TMA.

	TMA (n = 21)	No-TMA (n = 97)	p
Anemia* (n/%)	13 / 61.9	24 / 24.7	0.002
Low Platelet count (n/%)	3 / 14.3	4 / 4.1	0.07
High serum LDH level (n/%)	13 / 61.9	45 / 46.4	0.29
Low serum haptoglobin level (n/%)	2 / 9.5	2 / 2.6	0.29
High serum indirect bilirubin level (n/%)	0 / 0	4 / 4.12	0.77
Schistocytes on peripheral blood smear (n/%)	4 / 19	3 / 3.1	0.02

LDH: Lactate Dehydrogenase.

* Defined as hemoglobin <12g/l for females and <13g/l for males [20].

The analysis of patients with and without TMA on kidney biopsy (Table 4) revealed that the first group had no difference in prevalence or progression to ESKD regarding the race, had a greater proportion of hypertensive individuals (100 vs. 61, p<0.0001) and with hematuria (100 vs. 87.6, p = 0.0001). Patients with TMA had worse median serum creatinine levels (3.8 vs. 1.38 mg/dL, p = 0.0001), eGFR (18 vs. 60.2 ml/min/1.73m2, p = 0.0001), and more frequent low serum C3 (28.5 vs. 10.4, p = 0.003). There was no difference in the treatment prescribed to the two groups of patients. In regard to the histology parameters of the Oxford Classification, solely a higher proportion of individuals with E1 was observed (68% vs. 32%, p<0.002). Patients with histologic evidence TMA were followed for less time (7 vs. 65 months, p<0.0001), since a greater portion of them progressed to CKD-KRT (71.4% vs. 21.6%, p<0.0001) and in less time (3 vs. 16 months, p = 0.003) on account of the quicker eGFR decrease they experienced (-6.8 vs. -0.65 ml/min/1.73m2/year, p = 0.01).

Table 4

Analysis of clinical parameters and laboratory findings in IgAN patients with and without histologic evidence of TMA.

	TMA (n = 21)	No-TMA (n = 97)	p
Male (n/%)	10 / 47.6	43 / 44.3	0.62
Age (years)	32 (27;41)	33 (24;44)	0.83
Race (n/%)			0.653
White	14 / 16.3	72 / 83.7
Black	6 / 24	19 / 76
East-asian	1 / 14.3	6 / 85.7
Hypertension (n/%)	21 / 100	59 / 61	<0.0001
Hematuria (n/%)	21 / 100	85 / 87.6	0.0001
Serum creatinine (mg/dL)	3.8 (2.2;5.8)	1.38 (0.91;1.9)	0.0001
e-GFR by CKD-EPI (ml/min/1,73m2)a	18.3 (9.2;30.5)	60.2 (35.1;87.5)	0.0001
24h-proteinuria (g)	1.9 (0.9;3.96)	2 (1.3;3.6)	0.86
Serum albumin (g/dL)	3.2 (2.55;3.9)	3.5 (3.1;3.8)	0.26
Low serum C3 levels (n/%)	6 / 28.5	9 / 10.4	0.003
Treatment
ACE inhibitor or ARB (n/%)	19 / 89.4	78 / 80.4	0.37
Corticosteroids (n/%)	11 / 52.6	63 / 64.7	0.35
Other immunosuppressants (n/%)	8 / 36.8	34 / 35.2	0.9
Kidney Histology–Oxford Classification (n/%)
M1	19 / 89.7	75 / 77.3	0.23
E1	14 / 68	31 / 32	0.002
S1	16 / 78.9	70 / 72.1	0.54
T1/T2	12 / 57.9	35 / 36.1	0.07
C1/C2	8 / 32.1	26 / 26.8	0.57
Immunofluorescence positivity (n/%)
IgM	6 / 28.5	23 / 23.7	0.84
IgG	0 / 0	10 / 10.3	0.26
C3	18 / 85.7	75 / 77.3	0.57
C1q	0 / 0	7 / 7.2	0.44
CD68 Immunohistochemistry (cells/field)
Glomeruli	4.3 (3.02;6.0)	2.25 (1.56;5)	0.12
Tubulointerstitium	25.4 (14;34.7)	18.3 (9.8;28.3)	0.25
C4d glomerular staining (n/%)c	7 / 77.8	26 / 41.3	0.04
Follow-up (months)	7 (3;21)	65 (27;115)	<0.0001
ΔeGFR (ml/min/1,73m2/year)	-6.8 (-24;0)	-0.65 (-4.48;2.3)	0.01
CKD-KRT (n/%)	15 / 71.4	21 / 21.6	<0.0001
Time to CKD-KRT (months)	3 (3;7)	16 (4;64)	0.003

e-GFR estimated glomerular filtration rate, M1: Mesangial hypercellularity, E1: Endocapillary hypercellularity, S1: Segmental glomerulosclerosis, T1/T2: Tubular atrophy or interstitial fibrosis, C1/C2: Cellular crescent, CKD requiring RRT: Chronic Kidney Disease requiring Renal Replacement Therapy.

aAs determined by the Chronic Kidney Disease–Epidemiology Collaboration equation.

c Analysis of immunohistochemistry for C4d was possible in 72 patients, 9 from the group with TMA (43%) and 63 without TMA (64.9%).

The comparison of patients that progressed or not to CKD-KRT (Table 5) revealed that most of the individuals in the first group were males (63.8% vs. 36.5%, p = 0.01), younger (30 vs. 34 years of age, p = 0.04), had hypertension (86.1% vs. 56.1%, p = 0.0016), worse creatinine levels at the biopsy time (3 vs. 1.2mg/dL, p<0.0001), lower eGFR (22.5 vs. 64.8ml/min/1.73m2, p<0.0001), and more frequent low serum C3 (25.7% vs 7,3%, p = 0.01), without difference in proteinuria. Kidney biopsy findings pointed to a greater proportion of patients with T1/T2 (54.2% vs. 23,2%, p<0.0002) and a greater proportion of individuals with TMA (41.7% vs. 7,32%, p<0.0001). No difference was seen in the treatment prescribed to both patient groups. Patients progressing to CKD-KRT were followed for less time (7 vs. 69 months, p<0.0001) on account of more pronounced eGFR decreases (-8.17 vs. -0.21 ml/min/1.73m2/ano, p<0.0001).

Table 5

Clinical parameters and laboratory findings of IgAN patients progressing or not to CKD-KRT.

	CKD-KRT (n = 36)	No CKD-KRT (n = 82)	p
Age (years)	30 (24;40)	34 (26;50)	0.04
Male sex (n/%)	23 (63,8)	30 (36,5)	0.01
Creatinine (mg/dL)	3 (2.3;5.6)	1.2 (0.9;1.7)	<0.0001
eGFR (ml/min/1.73m2)	22.5 (9.6;36)	64.8 (40;91.7)	<0.0001
Proteinuria (g/day)	2.4 (1.3;4.1)	1.58 (1;2.98)	0.07
Albumin (g/dL)	3.4 (2.7;3.8)	3.5 (3;3.9)	0.61
Hematuria (n/%)	32 / 88.9	70 / 85.3	0.77
Hypertension (n/%)	31 / 86.1	46 / 56.1	0.0016
Consumption of C3 (n/%)	9 / 25.7	6 / 7.3	0.01
Follow-up (months)	7 (3;39)	69 (35;122)	<0.0001
ΔGFR (ml/min/1.73m2/year)	-8.17 (-31;2.46)	-0.21 (-2.1;2.7)	<0.0001
Kidney Histology–Oxford Classification (n/%)
M1	21 / 58.33	60 / 73.1	0.13
E1	16 / 44.4	24 / 29.2	0.13
S1	24 / 66.6	51 / 62.2	0.68
T1/T2	20 / 54.2	19 / 23.2	0.002
C1/C2	13 / 36.1	18 / 21.9	0.11
Immunofluorescence positivity (n/%)
IgM	11 / 30.5	18 / 21.9	0.44
IgG	3 / 8.3	7 / 8.53	0.74
C3	29 / 80.5	64 / 78	0.95
C1q	2 / 5.5	5 / 6.1	0.75
CD68 Immunohistochemistry (cells/field)
Glomeruli	3.35 (1.87;5.82)	2.24 (1.47;5.04)	0.38
Tubulointerstitium	32.5 (24.6;51.9)	15.6 (8.99;24.4)	<0.0001
TMA (n/%)	15 / 41.7	6 / 7.32	<0.0001
Treatment
ACE inhibitor or ARB (n/%)	17 / 47.2	54 / 65.8	0.06
Corticosteroids (n/%)	13 / 36.1	40 / 48.8	0.23
Other immunosuppressants (n/%)	8 / 22.2	16 / 19.6	0.81

eGFR estimated glomerular filtration rate. SD standard deviation. M1: Mesangial hypercellularity. E1: Endocapillary hypercellularity. S1: Segmental glomerulosclerosis. T1/T2: Tubular atrophy or interstitial fibrosis. C1/C2 cellular crescent. ACEi angiotensin-converting enzyme inhibitor. ARB angiotensin II receptor blocker CKD-RRT: Chronic Kidney Disease requiring Kidney Replacement Therapy.

aAs determined by the Chronic Kidney Disease–Epidemiology Collaboration equation.

Immunohistochemistry for CD68 (Fig 1) was performed to look into macrophage-mediated tissue inflammation in 76 patients, nine from the group of 21 patients with TMA (43%) and 67 without TMA (69.1%). Fig 1 shows immunohistochemistry images derived from renal biopsy specimens. No statistically significant difference was found when patients with and without TMA were compared for number of CD68 positive glomerular (4.3 vs. 2.25, p = 0.12) or interstitial (25.4 vs. 18.3, p = 0.25) cells. The comparison between patients progressing or not to CKD-KRT did not yield significant differences for glomerular cells labeled positive for CD68 (3.35 vs. 2.24, p = 0.38), but significantly more CD68 interstitial cells were seen in individuals progressing to CKD-KRT (32.5 vs. 15.6, p<0.0001) (Table 4). In total, 72 kidney biopsies were available to stain for C4d, including 9 tissue sections from patients with TMA and 63 tissue sections from those without TMA. Glomerular deposits of C4d were more frequent in patients with TMA as compared to those without TMA (n/M = 7/9 and n/N = 26/63; P = 0.04).

Fig 1

CD68 immunohistochemistry of kidney biopsies of IgAN patients labeled positive in the A) glomerulus (x400) and B) tubulointerstitium (x200). C) C4d immunohistochemistry revealing positive glomerular staining (x400).

Logistic regression performed to assess risk factors independently related to the primary end point in all the 118 patients found that female sex was protective against the condition (Hazard Ratio—HR 0.24; 95% CI 0.07–0.54, p = 0.03), while T1/T2 scores (HR: 8.17; 95% CI 2.17–30.89, p = 0.001) and histologic signs of TMA (HR: 8.34; 95% CI 1.66; 41.96, p<0.01) were associated with progression to CKD-KRT (Table 6). Fig 2 shows the differences in renal survival for patients with and without evidences of TMA in kidney biopsy.

Table 6

Logistic regression analysis for the primary outcome adjusted for sex, hypertension, creatinine, Oxford classification parameters (M,E,S,T,C) and TMA.

		95% Confidence Interval for HR
Variable	HR	Lower	Upper	p
Female	0.24	0.07	0.54	0.03
Hypertension	1.02	0.19	5.36	0.98
Creatinine >1.2mg/dl	3.12	0.51	19.03	0.21
M1	1.53	0.28	8.52	0.62
E1	0.56	0.14	2.27	0.41
S1	10.2	0.88	118.1	0.06
T1/T2	8.17	2.17	30.89	0.002
C1/C2	3.62	0.87	14.94	0.07
TMA	8.34	1.66	41.96	0.01

HR hazard ratio. e-GFR estimated glomerular filtration rate M1: Mesangial hypercellularity. E1: Endocapillary hypercellularity. S1: Segmental glomerulosclerosis. T1/T2: Tubular atrophy or interstitial fibrosis. C1/C2 cellular crescent. CI confidence interval. T1/T2 mild to severe tubular atrophy or interstitial fibrosis. TMA: Thrombotic Microangiopathy.

aCKD requiring RRT: Chronic Kidney Disease requiring Renal Replacement Therapy.

Fig 2

Survival free of dialysis of patients with IgAN with and without evidence of TMA in kidney biopsy specimens.

Discussion

Some clinical findings associated with worse outcomes in individuals with IgAN–male sex, older age, persistent microscopic hematuria, hypertension, proteinuria, and creatinine levels on kidney biopsy [21-24]–have been described as a factor in the progression to CKD-KRT within five years seen in approximately 30% of the individuals with the condition [4,22,25]. In light of this fact and the variety of histologic findings to consider, the need to standardize the analysis of kidney biopsy of IgAN patients and the search for histology-related prognostic factors led to the creation of histologic classifications [6-8,26], with the Oxford Classification standing as the most widely accepted among pathologists and nephrologists. However, the OC does not consider TMA despite its role in disease progression and although it has been described to affect 2.2% to 53% of the individuals with IgAN [11,26]. Discrepancies in the frequency of involvement by TMA may be ascribed to differences among populations, although the adoption of diverse criteria to diagnose the condition–some including electron microscopy examination and immunohistochemistry staining protocols–is a relevant matter to consider.

El Karoui et al [11] studied a multi-center population of patients with IgAN and TMA. The author found that 68 (53.15%) of 128 patients had histologic evidence of TMA, a proportion higher than the reported in other studies. Diagnosis of TMA was based on light microscopy and immunohistochemistry staining for CD61. Patients with TMA progressed more frequently to CKD-KRT. A multicenter study carried out in Spain [27] included patients with IgAN and malignant hypertension. In the study, 13/186 patients (7%) were diagnosed with malignant hypertension. Patients with the condition were predominantly men in the fourth decade of life. Ten patients were on KRT at the end of the follow-up period. Three of the six individuals submitted to kidney transplantation returned to dialysis due to chronic allograft nephropathy associated with recurrent IgAN, although they did not have signs of malignant hypertension. Cai et al [10] studied 944 patients with IgAN from a single center in China, in which 194 patients with histologic evidence of TMA were found based on light and electron microscopy examination. Patients with TMA were older than their counterparts. Most were males with worse renal function, more proteinuria, and, interestingly enough, no differences in serum TMA markers. As seen in previous literature reports, patients with TMA reached the target endpoint more frequently and quickly. Haas et al [26] looked into 2290 patients with IgAN and found 49 (2.2%) with TMA based on light and electron microscopy examination. The patients in this group were younger and had worse renal function and proteinuria on admission. They were not analyzed for progression to CKD-KRT. Other studies support these findings [10,28-31]. Our study found histologic evidence of TMA in 17.8% of the included patients, confirming some of the clinical and workup findings described previously in the literature (more individuals with hypertension, worse renal function, and worse outcomes) [9-11]. As reported in other studies [9,10], patients often do not present with clinical or workup findings indicative of TMA. Therefore, in our study only anemia and increased schistocyte counts stood out in the comparison of individuals with and without histologic evidence of TMA, since the groups were not different in terms of platelet counts, serum LDH, or haptoglobin levels.

Among the etiologies associated with TMA we must consider malignant hypertension, thrombotic thrombocytopenic purpura, atypical hemolytic uremic syndrome (aHUS), shiga toxin-producing Escherichia coli (STEC-HUS), scleroderma renal crisis, hypertensive pregnancy disorders, antiphospholipide syndrome, infections, drug toxicity and metabolic diseases [12,32]. All of those conditions described above can manifest themselves as microangiopathic hemolytic anemia, with presentation in the form of hypertensive urgency/emergency and the differential diagnosis becomes important since the treatment varies according the base disease [33,34]. Timermans et al [35] analyzed a series of 9 patients with diagnostic hypertension-associated TMA and compatible findings at funduscopic examination. An interesting and high relevant finding of this study is that 67% of the patients had mutations in genes associated with aHUS, despite the neither absence of a family history of TMA or compatible hematological markers of the syndrome in most patients. Facing the high frequency of complement disorders in these cases, the authors highlight the importance to perform genetic tests in patients with malignant hypertension without evidences of secondary causes, since the progression to CKD-KRT is frequent and specific treatment can prevent recurrence of the disease after transplant. Such findings are supported by the studies of El Karoui et al [36] and Cavero et al [37] which showed that cases of TMA that present with hypertensive emergency are more often associated to complement disorders than other secondary etiologies.

Cases of IgAN associated with complement factor mutation have been described [31,38-41], some of which effectively treated with Eculizumab [32]. What is not known is whether patients with IgAN and TMA actually present with a combination of the two diseases or if immunoglobulin deposition associated with the onset of the disease might produce local manifestations of TMA. The early pathogenesis of TMA has been linked to endothelial lesion [41,42]. Studies have shown that in addition to tropism by immunoglobulin degradation and complement factors in the mesangium, patients with IgAN also produce anti-endothelial cell antibodies [43,44], thereby causing endothelial lesion and dissociation of the endothelium and the glomerular basement membrane, activation of the immune system (cytokines, inflammatory cells, complement system) and the coagulation cascade, producing local glomerular thrombosis [41,45,46]. On the other hand, the complement system may be activated via the lectin pathway as hypoglycosylated IgA1 –linked to the pathogenesis of IgAN–binds to endothelial cells [47-50]. This finding may be demonstrated by the overwhelming positivity for C4d in patients with IgAN and TMA without co-deposition of C1q [10,41]. Both theories are plausible, since it is not rare to find patients with only histologic and no clinical signs of TMA. We did not observe differences in the profile or intensity of immunoglobulin deposition (IgG, IgM) or complement proteins (C3, C1q) in our series between patients with and without TMA or individuals that progressed versus patients that did not progress to CKD-KRT. However, none of the patients with TMA had deposition of IgG or C1q, while 10.3% of the patients without TMA had deposition of IgG and 7.3% of C1q. Important contributions to IgAN pathological understanding of complement disturbances came from histochemical studies of kidney tissue C4d staining. On clinical settings C4d deposition has been associated with aggressive histology and worse clinical course [51,52]. Drachenberg et al [53] showed positive mesangial staining in 13 out of 34 IgAN patients and additional positivity in capillary walls in 9. Mesangial staining did not correlate with any of the Oxford scores while capillary wall staining correlated with E1 (endocapillary hypercelularity). Considering TMA patients, predominantly with aHUS diagnosis, a well-known complement disturbance condition, in the same study of Drachenberg et al [53], glomerular and/or arteriolar TMA lesions stained positive for C4d in all cases (100%) Chua et al [9] employed markers not used in routine clinical practice to assess the complement system and analyze depositions of C4d, C5b-9, mannose-binding lectin and Factor B in the renal tissue of patients with IgAN with and without TMA. The authors found an association between C4d and TMA and the presence of the two conditions with worse renal survival, thus stressing the role of the complement system in the pathogenesis of TMA in patients with IgAN. Similar findings were observed in our patients, where the group with TMA expressed more frequently glomerular staining for C4d than those without TMA. Since only 2 of 10 patients with TMA had negative immunostaining for C4d, we opted for not analyze renal survival curve for the three groups (no-TMA vs TMA/C4d+ vs TMA/C4d-).

By approaching the complement system from a systemic standpoint, we found, in our series, more patients with low C3 levels in the TMA group when compared with individuals without TMA and with the group of patients that progressed to CKD requiring KRT. This finding is not reflected in renal tissue, in which C3 deposition did not occur differently in patients with or without TMA (85.7 vs. 77.3, p = 0.57) or in individuals progressing or not to CKD-KRT (80.5 vs. 78.0, p = 0.95). In regard to MEST-C and TMA parameters, we found associations only with parameter E, which identifies active glomerular endothelial lesions in association with findings consistent with TMA. Since parameter E is modifiable via treatment, it is important to notice that no significant difference was seen in the treatment prescribed to the two groups of patients.

Recent studies [2,49] pointed to the relevance of glomerular tissue expression of CD68 as an additional marker that allows pathologists to more accurately define parameter E for endocapillary hypercellularity, as described by Soares et al [49]. In our series, this marker did not elicit differences in the number of cells labeled for CD68 in the glomeruli or tubulointerstitium of patients with and without TMA. However, expression was increased in the tubulointerstitium of patients progressing to CKD requiring KRT on account of the histologic evidence of chronic disease described previously by Soares et al [49].

The limitations of this study derive from the fact that is was carried out in a single center and that diagnostic examination for TMA was based only on light microscopy, without the aid of immunohistochemistry staining or electron microscopy. However, studies performed in a single center are known for more consistent compliance with procedure, follow-up, and treatment processes. We are starting in our institution a study of genetic analysis of patients with TMA associated to primary and secondary glomerulopathies. At the moment, patients were not tested but we plan to perform analysis by whole exome sequencing and results will be performed as soon as those are available.

In conclusion, our study found that patients with IgAN and histologic evidences of TMA had clinically worse kidney function, more hypertension and hematuria, greater proportions of low serum C3 at kidney biopsy, with a larger amount of individuals with endocapillary hypercellularity (E1). Histologic evidence of TMA were not concurrent with clinical/laboratory markers of the condition. Patients with histologic evidence of TMA progressed more frequently and quickly to CKD-KRT when compared to individuals without TMA. Having signs of TMA in kidney biopsy specimens was an independent marker for progression to CKD-KRT when compared to other histologic predictors in the Oxford Classification. Additional studies are required to investigate the role of the complement system in TMA and IgAN and support the development of new therapeutic targets.

10 Jun 2020

PONE-D-20-12431

Evidences of histologic Thrombotic Microangiopathy and the impact in renal outcomes of patients with IgA nephropathy

PLOS ONE

Dear Dr. Neves,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript focuses on a topic of potential interest. However, the study has several shortcomings that should be addressed. To mention few of them, i) need to provide an explanation on the rational behind using CD68 IHC staining; ii) unclear whether there is any difference in the prevalence of TMA among races; iii) unclear whether there is any difference among races in the progression of kidney failure in those with TMA, iv) unclear whether genetic studies in complement genes were performed to evaluate possible genetic causes of TMA; v) unclear whether patients classified as M0 have other morphologic features indicated in the Oxford Classification; vi) need to provide the clinical course of these M0 patients; vii) need to provide a more detailed description of the morphologic feature of TMA in the Results section; viii) provide detailed information regarding extra-renal target organ damage related to hypertension; ix) need to elaborate on complement dysregulation in patients presenting with hypertensive emergency; x) explore the possibility to stain of C4d and/or MBI to dissect the complement cascade; xi) unclear what means p25 and p75; xii) need to clarify the statement on statistical significance attributed to p-values <0,05; xiii) unclear for the logistic regression what covariates have been used and what the sample size was to train the model.

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Reviewer #1: The authors describe the prevalence and outcomes of TMA in patients with IgAN in a retrospective single center cohort.

Please consider the following comments and suggestions:

- Please include in Methods the timeline of the data analyzed in this cohort (i.e. This study was conducted in patients from X year through X year)

- Please provide small explanation on the rational behind using CD68 IHC staining for the readers to understand why it was used in these cases.

- Authors mention 73% of subjects were whites. Please mention the different races in the remaining 27% of the cohort population, and add this information to your baseline characteristics table

- Is there any difference in the prevalence of TMA among races?

- Is there any difference among races in the progression of kidney failure in those with TMA?

- Was the cause of TMA investigated in any those 21 patients included for analysis? If so, this should be mentioned in the manuscript

- Did any genetic studies in complement genes were performed to further evaulate possible genetic causes of TMA in these subjects?

Reviewer #2: Variables with non-

146 parametric distributions were expressed as median values (p25 and p75)

What is p25 and p75? Is it the first and third quartile? Please clarify this in detail.

Statistical significance was attributed to p-values <0.05.

I guess the authors mean the significance level was chosen to be alpha=0.05?

https://www.mdpi.com/2504-4990/1/3/54

If so please correct the statement or clarify.

In the results section, the authors present a survival analysis but in the methods section this is not discussed. Please revise this section correspondingly.

See

https://www.mdpi.com/2504-4990/1/3/58

how results should be presented.

For the Logistic regression it is unclear what covariates have been used. Also it is unclear what the sample size was to train the model.

The authors mention that 'These findings indicate that vascular compartment may also be a prognostic marker in IgAN patients.' however it is unclear what is the connection of this to the statistical analysis conducted. Please explain if results from the statistical analysis point to this finding and elaborate on the robustness of this finding.

Reviewer #3: Neves et al. analyzed the prognostic role of TMA on kidney biopsy in 118 patients with IgA nephropathy, a prevalent form of GN with variable prognosis, and state that TMA lesions affect the prognosis and, in particular, the risk of ESKD. However, I do have some points that should be discussed.

- Mesangial IgA deposits on immunofluorescence microscopy are common in the general population and are not always related to disease. This is particular the case in patients with no mesangial proliferation. Neves et al. showed that 76.3% patients had significant mesangial proliferation, that is, M1, indicating that mesangial abnormalities were lacking in about 25% patients. Do these patients classified as M0 have other morphologic features indicated in the Oxford classification, mesangial C3c deposits, and/or electron dense deposits on electron microscopy? Also, the clinical course of these “M0 patients” would be interesting to know if none of the aforementioned morphologic features are present on kidney biopsy to exclude patients with “normal” IgA deposits on immunofluorescence microscopy as such patients may bias the results.

- The authors should provide a more detailed description of the morphologic features of TMA in the Results section. How many patients present with acute and/or chronic lesions? In my experience, patients with chronic features of TMA often have persistent phospholipid serum reactivity, either fulfilling clinical criteria for the antiphospholipid syndrome or not, drug-induced TMA (e.g., anti-VEGF treatment, chemotherapy), or genetic variants linked to low CD46 activity (this has also been found in patients carrying C3 p.R161W), etcetera. It is important to note that patients with a MPGN pattern on light microscopy should not have electron dense deposits along the glomerular capillary wall on electron microscopy.

- Do patients with TMA on kidney biopsy often have complement deposits co-localized with thrombi and/or other lesions related to the TMA?

- Patients with TMA on kidney biopsy invariably presented with hypertension. The TMA was often localized to the kidneys without profound hematologic abnormalities on peripheral blood. This has been observed in patients with TMA presenting with hypertensive emergency and thus, extrarenal target organ damage, e.g., hypertensive retinopathy, left ventricular hypertrophy. Many of such patients may present with complement-mediated TMA. I would suggest to add detailed information regarding extrarenal target organ damage related to hypertension. The authors should elaborate on complement dysregulation in patients presenting with hypertensive emergency. (For example, genotyping if feasible, recurrent disease prior to and after kidney transplantation.)

- Previous studies suggested that activation of the lectin pathway of complement may affect the prognosis. C4d can be found in about 25% patients with IgA nephropathy and appear as prevalent as morphologic features of TMA. MASP2, downstream of MBL, has been implicated in thrombosis. Is it possible to stain of C4d and/or MBL to better dissect the complement cascade?

- Table 1 should be updated. ACE inhibitor or ARB alone indicates no concomitant medication, although >50% patients had been treated with immunosuppressive drugs.

- Table 5 should also include the HRs of hypertension, serum creatinine, eGFR, and the complete Oxford classification (unless the authors only corrected for IF/TA). I would suggest to pick either serum creatinine or eGFR as a confounder instead of both.

**********

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Reviewer #1: Yes: MARIA L. GONZALEZ SUAREZ

Reviewer #2: No

Reviewer #3: Yes: Sjoerd A.M.E.G. Timmermans

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17 Jul 2020

July 16th, 2020

To Dr. Joerg Heber

Editor-in-Chief of PLos One

Dear Editor,

Please find attached the revised version of the manuscript “The impact of histologic evidence of thrombotic microangiopathy in the renal outcomes of patients with IgA nephropathy”, by Neves et al, which is being submitted for the “Original Research Article” section.

We thank you for the follow-up and the reviewers for the helpful comments and suggestions, which certainly improved our manuscript quality.

We believe that we have appropriately addressed all points raised by the reviewers and that the manuscript is now suitable for publication. The specific responses to the editors and reviewers are outlined below.

REVIEWER 1 REPORT:

The authors describe the prevalence and outcomes of TMA in patients with IgAN in a retrospective single center cohort. Please consider the following comments and suggestions:

1. Please include in Methods the timeline of the data analyzed in this cohort (i.e. This study was conducted in patients from X year through X year)

Response: Thank you for your point. We have added the information as requested (Page 4, line 91).

2. Please provide small explanation on the rational behind using CD68 IHC staining for the readers to understand why it was used in these cases.

Response: Thank you for your relevant comment. Recent studies (Soares et al, Histopathology. 2019;74(4):629-637; and Trimarchi H et al Kidney Int. 2019;95(4):750-756) have highlighted the relevance of glomerular staining of CD68 as an additional tool to define endocapillary hypercellularity (E) more accurately. (Page 13, lines 333-339).

3. Authors mention 73% of subjects were whites. Please mention the different races in the remaining 27% of the cohort population, and add this information to your baseline characteristics table.

Response: Thank you for your comment. We have described the detailed races in the text (Page 7, line 166-167) and in the Table 1.

4. Is there any difference in the prevalence of TMA among races?

Response: Thank you for your comment. There was no statistical significant difference in the prevalence of TMA among races, as described in the text (Page 8, line 187) and in the Table 4.

5. Is there any difference among races in the progression of kidney failure in those with TMA?

Response: Thank you for your comment. In patients with histological evidences of TMA, 15 (71.4%) undergone Chronic Kidney Disease requiring dialysis. When we describe dialysis patients according race, 1/7 (14.2%) was east-asian, 4/25 (16%) were black and 10/86 (11.6%) were white. Comparing the proportions in the total population, there were no difference among races in the progression of TMA (p=0.792). (Page 8, line 187)

6. Was the cause of TMA investigated in any those 21 patients included for analysis? If so, this should be mentioned in the manuscript.

Response: Thank you for comment. Among our patients there are no solid organs transplanted patients, bone marrow transplant, pregnant women or patients diagnosed with cancer. We have no description of possible medications associated with TMA. Serologies were performed for Hepatitis B, C and HIV as well as research for autoimmune diseases (ANA, ANCA, Rheumatoid Factor, lupus anticoagulant). No genetic tests have been performed. We have described all these information in the manuscript (Page 4, Lines 95-99).

7. Did any genetic studies in complement genes were performed to further evaluate possible genetic causes of TMA in these subjects?

Response: Thank you for your comment. We are starting in our institution a study of genetic analysis of patients with TMA associated to primary and secondary glomerulopathies. At the moment, patients were not tested but we plan to perform analysis by whole exome sequencing. (Page 14, lines 344-347).

REVIEWER 2 REPORT:

1. Variables with non- parametric distributions were expressed as median values (p25 and p75). What is p25 and p75? Is it the first and third quartile? Please clarify this in detail.

Response: Thank you for your comment. The terms p25 and p75 refers to first and third quartile. We have corrected it in the Material and Methods section as suggested (Page 6, line 156).

2. Statistical significance was attributed to p-values <0.05. I guess the authors mean the significance level was chosen to be alpha=0.05? https://www.mdpi.com==/2504-4990/1/3/54. If so please correct the statement or clarify.

Response: Thank you for your comment. We have made the correction in the Material and Methods section as requested (Page 7, line 160-161).

3. In the results section, the authors present a survival analysis but in the methods section this is not discussed. Please revise this section correspondingly. See: https://www.mdpi.com/2504-4990/1/3/58

how results should be presented.

Response: Thank you for your raised point. We have described the analysis in the Material and Methods section as suggested (Page 7, lines 161-162).

4. For the Logistic regression it is unclear what covariates have been used. Also it is unclear what the sample size was to train the model.

Response: Thank you for your valuable comment. For Logistic regression we have analyzed data from all 118 patients. We have detailed the covariants in in the Material and Methods section (Page 7, lines 157-160) and in the Table 6 as suggested.

5. The authors mention that 'These findings indicate that vascular compartment may also be a prognostic marker in IgAN patients.' however it is unclear what is the connection of this to the statistical analysis conducted. Please explain if results from the statistical analysis point to this finding and elaborate on the robustness of this finding.

Response: Thank you for your high relevant comment. In our manuscript we demonstrated that evidences of TMA in renal biopsy were an independent risk factor for reaching CKD-KRT. This finding corroborates previous published data; however, our sample is of 118 patients. In this context, we opted to replace the sentence “These findings indicate that vascular compartment may also be a prognostic marker in IgAN patients” to “Further studies are necessary to analyze the impact of vascular lesions on IgAN prognosis.” (Page 2, Line 49) and (Page 14, lines 356-357).

REVIEWER 3 REPORT:

Neves et al. analyzed the prognostic role of TMA on kidney biopsy in 118 patients with IgA nephropathy, a prevalent form of GN with variable prognosis, and state that TMA lesions affect the prognosis and, in particular, the risk of ESKD. However, I do have some points that should be discussed.

1. Mesangial IgA deposits on immunofluorescence microscopy are common in the general population and are not always related to disease. This is particular the case in patients with no mesangial proliferation. Neves et al. showed that 76.3% patients had significant mesangial proliferation, that is, M1, indicating that mesangial abnormalities were lacking in about 25% patients. Do these patients classified as M0 have other morphologic features indicated in the Oxford classification, mesangial C3c deposits, and/or electron dense deposits on electron microscopy? Also, the clinical course of these “M0 patients” would be interesting to know if none of the aforementioned morphologic features are present on kidney biopsy to exclude patients with “normal” IgA deposits on immunofluorescence microscopy as such patients may bias the results.

Response: Thank you for your interesting comment. In our sample we have 24 patients (20.3%) that does not fulfill M1 criteria of Oxford Classification. We have made a table to make the analysis of clinical and histological M0 patients easier. The table with clinical, laboratory tests, renal biopsy findings and outcomes of patients with M0 is following:

Table: Clinical profile, laboratory tests, renal biopsy findings and outcomes of patients with M0

Clinical and Laboratory data at renal biopsy Renal Biopsy Findings End of follow-up

Gender SCr (mg/dL) CKD-EPI

(ml/min/1,73m2) Proteinuria

(g/dia) HMT HTN M E S T C TMA IgG IgA C3 IgM C1q SCr

(mg/dL) CKD-EPI

(ml/min/1,73m2) Dialysis

Male 1,46 52,95 0,81 Yes Yes 0 1 1 0 1 No neg ++ ++ neg neg 0,8 111 No

Male 3,5 17 2,4 Yes Yes 0 0 1 1 1 No + ++ neg neg neg 1,2 58 No

Female 0,8 111 1,5 Yes No 0 0 0 0 0 No neg + neg neg neg 0,65 135 No

Male 0,92 100 0,82 Yes No 0 0 0 0 0 No neg + + + neg 0,72 99,93 No

Female 1,89 26 2,98 Yes Yes 0 0 1 0 1 No neg +++ ++ neg neg 1,07 49,34 No

Female 3,05 18 1,36 Yes Yes 0 0 1 1 0 No neg +++ neg neg neg 3,91 14,27 Yes

Male 1 117 1,27 No No 0 0 0 0 0 No neg + neg + neg 1,17 81,53 No

Female 0,7 92 0,51 Yes No 0 0 0 0 0 No neg + + neg neg 0,62 102 No

Male 1,6 62 9,7 Yes Yes 0 0 1 1 1 No neg ++ + neg neg 5,7 13 Yes

Male 0,75 113,6 1,39 No Yes 0 0 0 0 0 No neg + + neg neg 0,92 94,71 No

Male 1 94 2,35 Yes No 0 0 1 0 0 No neg ++ + neg neg 0,73 120,59 No

Female 0,8 91 0,33 Yes No 0 0 0 0 0 No neg ++ + neg neg 0,6 95 No

Female 0,74 120 2,68 Yes Yes 0 1 0 0 1 No + +++ + neg + 0,81 102,7 No

Male 1,23 81,24 0,09 Yes No 0 0 1 0 0 No neg ++ + neg neg 0,84 105,24 No

Female 2,13 33 0,71 Yes Yes 0 0 0 1 0 Yes neg ++ + neg neg 2,24 26,05 No

Female 1,44 39,73 1,2 Yes Yes 0 0 0 1 0 No neg +++ neg neg neg 1,24 46,78 No

Female 0,99 59,56 2,55 Yes Yes 0 0 1 0 0 No neg ++ neg neg neg 0,7 82 No

Female 0,9 74 8,1 No No 0 0 0 0 0 No neg ++ neg neg neg 0,71 97,06 No

Female 1,2 52 2,5 Yes No 0 0 0 0 0 No neg + + + neg 1,1 55 No

Male 2,6 36 3,3 No Yes 0 0 1 0 0 No neg +++ ++ neg neg 6,96 9 Yes

Male 1,24 71,62 4,7 Yes Yes 0 0 1 1 0 No neg ++ ++ neg neg 0,82 122,88 No

Male 1,2 83 0,5 Yes Yes 0 0 1 0 0 No neg +++ + + neg 0,99 100 No

Male 1,06 86 2,03 Yes No 0 0 1 0 1 No neg ++ + neg neg 12,7 4,91 Yes

Male 1,2 83 0,5 Yes Yes 0 0 1 0 0 No neg +++ ++ + neg 0,99 100 No

HMT: hematúria; HTN: hypertension; Neg: negative; SCr: sérum creatinine; TMA: Thrombotic Microangiopathy

The table shows that all patients with M0 has clinical, laboratory and/or histological evidences of current IgAN disease, what exclude the possibility of normal IgA deposits on renal biopsy.

Normal IgA deposits on immunofluorescence microscopy of patients are scarcely mentioned in literature. Waldherr et al (Nephrol. Dial Transpl. 1989;4(11):943-946), studying 250 consecutive autopsy cases with no renal diagnosed disease showed mesangial deposition in 12 (4.8). From those ones, 6 patients had cirrhosis (a well-known cause of secondary IgAN) while 6 suffered from various other con-ditions including endocarditis, bronchial asthma, cardio-vascular disease, and neoplasia. Two of these patients had completely negative urine analysis on repeated investigations, whereas three patients exhibited microscopic haematuria and/or mild proteinuria. C3c deposits in glomeruli were detected in one kidney.

2. The authors should provide a more detailed description of the morphologic features of TMA in the Results section. How many patients present with acute and/or chronic lesions? In my experience, patients with chronic features of TMA often have persistent phospholipid serum reactivity, either fulfilling clinical criteria for the antiphospholipid syndrome or not, drug-induced TMA (e.g., anti-VEGF treatment, chemotherapy), or genetic variants linked to low CD46 activity (this has also been found in patients carrying C3 p.R161W), etcetera. It is important to note that patients with a MPGN pattern on light microscopy should not have electron dense deposits along the glomerular capillary wall on electron microscopy.

Response: Thank you for your highly relevant comment. We decided to create a new table (Table 2) where we describe the frequencies of histological findings of TMA in renal biopsies of patients with IgAN. We also describe those finding in the manuscript (Page 7, lines 173-180).

3. Do patients with TMA on kidney biopsy often have complement deposits co-localized with thrombi and/or other lesions related to the TMA?

Response: Thank you for your comment. Our pathologist as a routine analysis describes complement deposits only in glomerular compartment, so, there is no information about co-localization of protein complement in TMA lesion. However, in 21 patients described with acute and/or chronic glomerular lesions of TMA, glomerular C3 deposition was evidenced in 18 patients. Most renal biopsy has no available fragment, hence, we will not able to reassess this information.

4. Patients with TMA on kidney biopsy invariably presented with hypertension. The TMA was often localized to the kidneys without profound hematologic abnormalities on peripheral blood. This has been observed in patients with TMA presenting with hypertensive emergency and thus, extrarenal target organ damage, e.g., hypertensive retinopathy, left ventricular hypertrophy. Many of such patients may present with complement-mediated TMA. I would suggest to add detailed information regarding extrarenal target organ damage related to hypertension. The authors should elaborate on complement dysregulation in patients presenting with hypertensive emergency. (For example, genotyping if feasible, recurrent disease prior to and after kidney transplantation.)

Response: Thank you for your comment. Unfortunately we have no data about extrarenal organ damage of most patients. We added to the discussion a paragraph describing about complement dysregulation in patients presenting with hypertensive emergency (Page 11, lines 268-285).

5. Previous studies suggested that activation of the lectin pathway of complement may affect the prognosis. C4d can be found in about 25% patients with IgA nephropathy and appear as prevalent as morphologic features of TMA. MASP2, downstream of MBL, has been implicated in thrombosis. Is it possible to stain of C4d and/or MBL to better dissect the complement cascade?

Response: Thank you for your valuable comment. From 118 patients, 72 had renal biopsy fragments that allowed immunohistochemistry for C4d, being 9/21 with TMA and 63/97 without TMA. The results of Immunohistochemistry for C4d is described in the text (Page 9, lines 216-220), and Table 4.

6. Table 1 should be updated. ACE inhibitor or ARB alone indicates no concomitant medication, although >50% patients had been treated with immunosuppressive drugs.

Response: Thank you for your comment. We have corrected it on Table 4.

7. Table 5 should also include the HRs of hypertension, serum creatinine, eGFR, and the complete Oxford classification (unless the authors only corrected for IF/TA). I would suggest to pick either serum creatinine or eGFR as a confounder instead of both.

Response: Thank you for your comment. We have detailed the covariants analyzed in in the Material and Methods section (Page 7, lines 157-160) and in Table 6 as suggested.

Sincerely,

Precil Diego Miranda de Menezes Neves, MD

Division of Nephrology

University of São Paulo School of Medicine

São Paulo - Brazil

Submitted filename: Neves et al_LettertoReviewers_2020.docx

Click here for additional data file.

27 Aug 2020

PONE-D-20-12431R1

Evidences of histologic Thrombotic Microangiopathy and the impact in renal outcomes of patients with IgA nephropathy

PLOS ONE

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Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The revised version of the manuscript is improved. However, few additional issues remain to be addressed. In particular, i) recommendation to exclude patients with no mesangial hypercellularity (M0) and no other morphologic features included in the MEST-C classification; ii) change the results section (page 9, line 218) as indicated by Reviewer 3.

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Reviewer #1: I Don't Know

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Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: Thank you for addressing reviewers comments. It is an interesting topic, and I think this revised version has improved manuscript content.

Reviewer #3: Almost all comments have been addressed appropriately. However, I would recommend to exclude patients with no mesangial hypercellularity (M0) and no other morphologic features included in the MEST-C classification. This is particular the case if electron dense deposits were not found/electron microscopy was not performed.

Also, I would sugges to change the results section page 9 line 218 into: In total, 72 kidney biopsies were available to stain for C4d, including 9 tissue sections from patients with TMA and 63 tissue sections from those without TMA. Glomerular deposits of C4d were more frequent in patients with TMA as compared to those without TMA (n/M=7/9 and n/N=26/63; P=0.04).

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Reviewer #1: Yes: Maria L. Gonzalez Suarez

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18 Sep 2020

September 16th, 2020

To Dr. Joerg Heber

Editor-in-Chief of PLos One

Dear Editor,

Please find attached the revised version of the manuscript “The impact of histologic evidence of thrombotic microangiopathy in the renal outcomes of patients with IgA nephropathy”, by Neves et al, which is being submitted for the “Original Research Article” section.

We thank you for the follow-up and the reviewers for the helpful comments and suggestions, which certainly improved our manuscript quality.

We believe that we have appropriately addressed all points raised by the reviewers and that the manuscript is now suitable for publication. The specific responses to the editors and reviewers are outlined below.

REVIEWER 1 REPORT:

Thank you for addressing reviewers comments. It is an interesting topic, and I think this revised version has improved manuscript content.

Response: Thank you for your kind comment. Improve the quality of our manuscript was just possible once you have been contributed with your valuable suggestions.

REVIEWER 3 REPORT:

1. Almost all comments have been addressed appropriately. However, I would recommend to exclude patients with no mesangial hypercellularity (M0) and no other morphologic features included in the MEST-C classification. This is particular the case if electron dense deposits were not found/electron microscopy was not performed.

Response: Thank you for your relevant comment We do understand your concern including patients with MEST-C score zero (M0/E0/S0/T0/C0) in our protocol particularly lacking electronic microscopy analysis. Those patients could be misdiagnosed as IgAN instead of C3 Glomerulopathy (C3GP) . It is well defined that light microscopy (LM) and immunofluorescence analysis (IF) are sufficient for IgAN diagnosis while MEST-C score adds morphological parameters to clinical prognosis and not to diagnosis.

MEST-C score shows high variation among studies. In Valiga study (N=647) and North American Validation study (N=87) M0 is described in 73% to 10% of the patients, respectively, E0 from 88% to 69%, S0 from 25% to 35%, T0 from 78% to 80%. C parameter was added to MEST score recently and C0 is described in 80 to 90 % of the patients.

In our 118 IgAN patients, MEST-C parameters distribution M0 20.4% , E0 64.4% , S0 29.7% , T0 61.7% , C0 71.2 % was not different from world’s report while 8 patients showed MEST-C score zero in each parameter concurrently. Three out of those 8 patients did not show any C3 deposition making impossible the hypothetical diagnosis of C3GP that is defined as “C3 dominant and at least two orders of intensity stronger than any combination of IgG, IgM, IgA and C1q “ ( Jean Hou et al).

So far, 5 patients with C3 deposition and MEST-C score concurrently zero could be on hazard of a misdiagnosis of IgAN instead of C3GP (see table below describing each patient)

Going deeply on G3GP clinical features described on literature we can emphasize some characteristics :

• C3GP is an uncommon glomerulopathy when compared to IgAN.

• C3GP more frequent light microscopy pattern is Membranoproliferative (MPGN ) from 73% to 78 % (Caravaca-Fontan et al, and Avasare et al) while IgAN is mostly Mesangioproliferative, and in some patients depicts a FSGS pattern. No IgAN patient from our protocol discloses MPGN pattern.

• Considering IF findings in C3GP Avasare et al showed exclusive C3 deposition in 33%, C3+IgM in 17% and do not mention important IgA deposition while in IgAN that deposition is mandatory as disclosed in all our patients.

• Clinical evolution and prognosis are very different between IgAN and C3GP. Our questioned 5 patients showed on last follow-up visit , around 4 years, almost normal kidney function as expected in many non-progressive IgAN patients much different from C3GP patients reaching aroud 50% of kidney failure on the same time

REFERENCES

1. Jean Hou et al. Toward a working definition of C3 glomerulopathy by immunofluorescence. KI 85: 450-456, 2014

2. Caravaca-Fontan et al Mycophenolate Mofetil in C3 glomerulopathy and pathogenic drivers of the disease CJASN 15: 1287-1298, 2020

3. Avasare et al Mycophenolate Mofetil in combination with steroids for treatment ok C3 glomerulopathy : a case series CJASN 13: 406-413, 2018

We hope having cleared your question. Nevertheless we would be glad processing EM on those specific 5 patients. Unfortunately we have no biopsy material left.

We are Very gratefull for your comments that highly enriched our paper.

Table: Clinical profile, laboratory tests, renal biopsy findings and outcomes of patients with M0, E0, S0, T0, C0 and C3+

Clinical and Laboratory data at renal biopsy Renal Biopsy Findings End of follow-up

N Gender SCr (mg/dL) CKD-EPI

(ml/min/1,73m2) Proteinuria

(g/dia) HMT HTN M E S T C TMA IgG IgA C3 IgM C1q SCr

(mg/dL) CKD-EPI

(ml/min/1,73m2) Dialysis

4 Male 0,92 100 0,82 Yes No 0 0 0 0 0 No neg + + + neg 0,72 99,93 No

8 Female 0,7 92 0,51 Yes No 0 0 0 0 0 No neg + + neg neg 0,62 102 No

10 Male 0,75 113,6 1,39 No Yes 0 0 0 0 0 No neg + + neg neg 0,92 94,71 No

12 Female 0,8 91 0,33 Yes No 0 0 0 0 0 No neg ++ + neg neg 0,6 95 No

19 Female 1,2 52 2,5 Yes No 0 0 0 0 0 No neg + + + neg 1,1 55 No

HMT: hematúria; HTN: hypertension; Neg: negative; SCr: sérum creatinine; TMA: Thrombotic Microangiopathy

2. Also, I would suggest to change the results section page 9 line 218 into: In total, 72 kidney biopsies were available to stain for C4d, including 9 tissue sections from patients with TMA and 63 tissue sections from those without TMA. Glomerular deposits of C4d were more frequent in patients with TMA as compared to those without TMA (n/M=7/9 and n/N=26/63; P=0.04).

Response: Thank you for your suggestion. We have replaced the sentence as suggested (Page 9, lines 216-220).

Sincerely,

Precil Diego Miranda de Menezes Neves, MD

Division of Nephrology

University of São Paulo School of Medicine

São Paulo - Brazil

Submitted filename: Neves et al_LettertoReviewers_2020.docx

Click here for additional data file.

1 Oct 2020

Evidences of histologic Thrombotic Microangiopathy and the impact in renal outcomes of patients with IgA nephropathy

PONE-D-20-12431R2

Dear Dr. Neves,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

The re-revised manuscript is definitely improved. The authors have adequately addressed all the remaining comments

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Kind regards,

Giuseppe Remuzzi

Academic Editor

PLOS ONE

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Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #1: I Don't Know

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: Thank you for submitting this manuscript after addressing comments. All my comments and suggestions have been addressed.

Reviewer #3: (No Response)

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Reviewer #1: Yes: MARIA L. GONZALEZ SUAREZ

Reviewer #3: Yes: Dr. Sjoerd A.M.E.G. Timmermans

21 Oct 2020

PONE-D-20-12431R2

Evidences of histologic Thrombotic Microangiopathy and the impact in renal outcomes of patients with IgA nephropathy

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