Crescents formations are independently associated with higher mortality in biopsy-confirmed immunoglobulin A nephropathy.

BACKGROUND AND OBJECTIVES: Immunoglobulin A Nephropathy (IgAN) is the most common type of glomerulonephritis with variable renal outcome. The association between IgAN and patient survival is limited. The effect of crescents on patient survival was never studied. MATERIALS: We conducted a retrospective cohort study between January 2003 and December 2013. All patients with the biopsy-proved IgAN was enrolled for the analysis of patient survival and renal survival. Cox regression model was used analyze the associated factors for patient survival.
RESULTS: All 388 participants with IgAN were enrolled, in which 45 patients with crescents. The mean percentage of glomeruli involvement was 23±18.9%. After long-term follow-up, crescents group had both worse renal (p = 0.034) and patient survivals (p = 0.016). In univariate Cox regression model, the age (HR = 1.08, 95% CI = 1.05-1.12, p<0.001), crescents (HR = 3.93, 95% CI = 1.18-13.07, p = 0.025), serum albumin (HR = 0.023, 95%CI = 0.11-0.50, p<0.001), blood total protein (HR = 0.46, 95%CI = 0.28-0.75, p = 0.002), HDL (HR = 0.95, 95%CI = 0.91-0.99, p = 0.009), daily urine protein (HR = 1.14, 95%CI = 1.01-1.29, p = 0.038), urine PCR (HR = 1.07, 95%CI = 1.02-1.12, p = 0.003), serum IgM (HR = 0.98, 95%CI = 0.96-1.00, p = 0.036), BUN (HR = 1.02, 95%CI = 1.01-1.02, p = 0.005), and eGFR (HR = 0.097, 95%CI = 0.94-0.99, p = 0.0011) were associated with patient survival. After multivariate Cox regression analysis, age (HR = 1.08, 95%CI = 1.01-1.13, p = 0.013), crescents (HR = 5.57, 95%CI = 1.14-29.05, p = 0.034), and HDL (HR = 0.94, 95%CI = 0.90-0.99, p = 0.026) were associated with patient survival. Crescents IgAN is with the highest risk (up to 5.75 of HR) for patient mortality.
CONCLUSIONS: The major strengths of the present study is that crescents IgAN had worse patient survival compared to non-crescents IgAN. Clinicians should be more careful to care patients with crescents IgAN.

Introduction

Immunoglobulin A Nephropathy (IgAN) is the most common type of glomerulonephritis throughout the world [1, 2] with a wide range of histologic patterns and complex clinical manifestations. Even with standard treatment, IgAN still remains an important burden of end-stage renal disease (ESRD) [3]. Currently, certain clinical situations and histopathological features, including the degrees of glomerular and interstitial fibrosis, tubular atrophy, and glomerular hypercellularity are considered independent risk factors for the progression of IgAN to ERSD. The poor histological conditions can predict renal outcome. The Oxford Classification of IgAN does not include glomerular crescents before. Recently, there was a major update in the Oxford classification system [4]: crescents to the graded parameters, consisting of the MEST-C scores. This update was based on a large scale (3096 patients) performed by Haas et al in 2017 [4]. In that study [4], crescents in at least one sixth or one fourth of glomeruli associated with a significantly higher hazard ratio (HR) for a combined event of 1.63 or 2.29, respectively. Even the patients with crescents in less than 1/10 of biopsied glomeruli still showed worse composite renal outcome [adjusted HR 1.38 (95% CI 1.13–1.69), p = 0.002] than those without crescents [5]. The abovementioned findings all support that crescents is with clear association with renal outcome in patients with IgAN no matter the treatment. However, the association of crescents formation in IgAN and patients outcome is still unknown.

The association between IgAN and ESRD was much stronger than between IgAN and patient death [6, 7]. The patient survival in patients with IgAN had been studied more frequently recently. In a study in Norwegian in 2013, an age- and sex-adjusted mortality rate in Norwegian patients with IgAN is approximately twice that of the general Norwegian population [6]. However, previous studies were with limitations, including single center only [8], some IgAN subgroups [9, 10], and limited to IgAN already with ESRD [11]. Recently, a nationwide population-based cohort study was conducted in Sweden and the authors compared 3622 patients with IgAN with 18041 matched general population controls [12].They found a 53% relative increase in mortality and a modest increase in absolute death rate. On average, patients with IgAN died 6 years earlier than people without the disease. However, the association between detailed pathological findings of IgAN and patients mortality is still limited. Furthermore, the predictive value for patient mortality of crescents formation is also rare. Until now, there was only one study revealing that increasing crescents proportion was identified as an independent predictor for unfavorable clinical outcomes in IgAN [13]. However, the increased mortality in crescents IgAN was only based on indirect evidence and that patient outcome is only secondary outcome [4]. In IgAN with crescents, aggressive immunosuppressant may rescue renal function but the effect on patient outcome is still unknown. On the contrary, over-suppression of immune system could be harmful for patient survival. The outcome of crescents IgAN should be clarified first before our aggressive treatment. In our previous study, we found that a lower serum IgG (≤907 mg/dL) and serum C3 (≤79.7 mg/dL) were both risk factors for poor renal outcome in our database of IgAN [14]. Here, in this study, we aimed to study the association between crescents formation and the patient mortality in IgAN in the same patients group [14].

Materials and methods

Study population

This retrospective cohort study was performed between January 2003 and December 2013. Participants of the age should be more than 20 years old and the diagnosis of IgAN was based on their first renal (native kidney only) biopsy in our medical center (Taichung Veterans General Hospital, TCVGH) in Taiwan. Our institute has possessed the largest patient population undergoing renal biopsies. Until now, the accumulative case numbers of renal biopsy was more than 8000. This study was approved by Ethics Committee of TCVGH, IRB number:CE15125B. All methods were performed in accordance with relevant guidelines and regulations and informed consent was obtained from all subjects.

Data collection and outcome assessment

All data was obtained from this cohort via the reviewing of medical records. Baseline data while diagnosis was collected at the time of each patient’s renal biopsy, including gender, age, body height (cm), body weight (kg), and systolic or diastolic blood pressure (mmHg) (SBP and DBP). Data from blood sample was also collected for blood urea nitrogen (BUN) (mg/dl), serum creatinine (mg/dl), estimated glomerular filtration rate (eGFR from Modification of Diet in Renal Disease equation) (ml/min/1.732m2) [15], white blood cell (WBC) (/cumm), red blood cell (RBC) (/cumm), hemoglobin (g/dl), neutrophil percentage (%), platelet count (/cumm), uric acid (mg/dl), sodium (meq/L), potassium (meq/L), calcium (mg/dl), phosphate (mg/dl), magnesium (mg/dl), albumin (g/dl), total protein (g/dl), glutamate oxaloacetate transaminase (GOT) (U/L), glutamate-pyruvate transaminase (GPT) (U/L), total cholesterol (mg/dl), triglyceride (mg/dl), low-density lipoprotein (LDL) (mg/dl), high-density lipoprotein (HDL) (mg/dl), fasting and postprandial blood sugar (mg/dl), glycated hemoglobin (%). Chronic infection or inflammatory markers were included as follows; hepatitis B status, hepatitis C status, Anti-Nuclear Ab (ANA), anti-double stranded DNA (anti-dsDNA), Anti-neutrophil Cytoplasmic Antibodies (ANCAs), Proteinase 3 (PR3) and Myeloperoxidase (MPO). Urine samples were tested for daily urine proteinuria (g/day), urinary protein/creatinine ratio (PCR) (g/g) and urinary albumin/creatinine ratio (ACR) (mg/g).

All pathological samples were analyzed by an experienced pathologist while all enrolled participants had their diagnosis of IgAN based upon the criteria in the World Health Organization monograph of kidney disease [16]. The diagnosis of crescents IgAN was confirmed once with the report of crescents whatever the percentage of crescents formation. The proportion of crescents in biopsied tissue was also recorded as percentage of all glomeruli. The detailed pathological findings of IgAN were based on Oxford classification [17]. The study major outcome is patient death and secondary outcome is ESRD, whose who needed the initiation of dialysis, or those receiving transplantation according to local guidelines.

Statistical methods

Data was expressed as the mean ± SD in continuous variables and as numbers (percentages) in categorical data. A Mann–Whitney U test was used for continuous variables and the Chi-square test was used for categorical variables. A Kaplan-Meier curve was implemented for measuring both patient survival and renal survival. A Cox proportional hazard regression (shown as HR, 95% confidence interval (CI)) was used to analyze the possible factors for patient survival (both the univariate and multivariate Cox models). Initially in the univariate analysis, all possible factors were analyzed. Then we chose each possible associated factors from each associated categories (such as baseline physical condition (age, and gender), pathological data for IgAN (crescents formation), blood laboratory data for IgAN (secondary hyperlipidemia, low albumin, low total protein, low LDL and low HDL), urinary laboratory data for IgAN (daily urine protein, and urinary PCR) and data for renal function evaluation (serum creatinine, BUN, and eGFR)), which were significantly associated factors in univariate analysis, to be analyzed in multivariate Cox regression model.

A value of p<0.05 was considered statistically significant. All statistical procedures were performed using the SPSS statistical software package, version 17.0 (Chicago, IL).

Results

All 388 participants (age > 20 years old) were enrolled in this study because of the diagnosis of IgAN between January 2003 and December 2013. Forty-five IgAN-patients were with crescents whatever the percentage of glomeruli involvement. The whole duration of follow-up was 11 years (January 2003 to December 2013). The mean duration of follow-up was 7.2 ± 3.1 years. In Table 1, patients with crescents IgAN were more stages of E, S, and T scores of Oxford classification, hypocalcemia (p = 0.001), hypoalbuminemia (p<0.001), higher LDL (p = 0.002), lower HDL (p = 0.047), more daily urine protein (p = 0.005), higher urinary PCR (p<0.001), higher blood IgG (p = 0.034), lower IgM (p = 0.035), and more positive ANCA (p<0.001). As for the crescents IgAN (S1 Table), the mean percentage of glomeruli involvement was 23±18.9% (medium percentage of crescents IgAN is 18%). Only 4 (8.9%) patient had more than 50% crescents formation of IgAN (S2 Table and S1 Fig).

Table 1

Basic characteristics of IgA nephropathy based on crescents or non-crescents.

	Total (n = 388)	Non Crescents(n = 343)	Crescents (n = 45)	p value
Sex: female	175 (45.1%)	157 (45.8%)	18 (40.0%)	0.567
Age (years old)	40.91±15.39	40.85±15.14	41.40±17.42	0.970
Oxford classification (n, %)
M0	247	223 (90.3%)	24 (53.3%)	0.078
M1	138	118 (34.4%)	20 (44.4%)	0.087
M2	3	2 (0.6%)	1 (2.2%)	0.227
E0	227	296 (86.3%)	31 (68.9%)	0.04
E1	62	48 (14.0%)	14 (31.1%)	0.002
S0	341	322 (93.9%)	19 (45.2%)	<0.001
S1	47	21 (6.1%)	26 (57.8%)	<0.001
T0	332	298 (86.9%)	34 (75.6%)	0.073
T1	38	29 (8.5%)	9 (20%)	0.028
T2	18	16 (4.7%)	2 (4.4%)	0.788
C0	388	343 (100%)	0 (0%)	<0.001
C1	35	0	35 (77.8%)	<0.001
C2	10	0	10 (22.2%)	<0.001
SBP (mmHg)	133.43±19.11	133.88±18.81	130.00±21.17	0.250
DBP (mmHg)	84.20±14.03	84.45±13.61	82.33±16.90	0.616
Body height (cm)	102.61±48.92	102.36±49.25	104.51±46.78	0.330
Body weight (kg)	101.09±49.19	101.71±49.57	96.29±46.39	0.944
Blood data
Blood urea nitrogen (mg/dl)	31.14±26.61	30.43±26.43	36.51±27.61	0.150
Creatinine (mg/dl)	2.43±3.15	2.37±3.18	2.94±2.93	0.112
eGFR (ml/min/1.732m2)	57.15±40.00	58.05±40.20	50.10±38.15	0.190
Blood WBC (/μL)	8016.25±2898.53	7928.69±2911.87	8681.78±2734.85	0.076
Hemoglobin (g/dl)	12.16±2.23	12.20±2.20	11.90±2.50	0.715
Neutrophil (%)	65.34±10.78	65.09±10.56	67.28±12.32	0.247
Platelet (x103/μL)	248.05±90.16	244.01±73.29	278.78±169.07	0.336
Uric acid (mg/dl)	7.45±2.19	7.39±2.14	7.86±2.48	0.285
Sodium (meq/L)	139.76±3.49	139.81±3.54	139.38±3.12	0.195
Potassium (meq/L)	4.28±0.55	4.27±0.56	4.37±0.51	0.249
Calcium (mg/dl)	8.51±1.07	8.57±1.02	8.08±1.33	0.001**
Phosphate (mg/dl)	4.00±1.29	3.97±1.25	4.26±1.54	0.223
Magnesium (mg/dl)	2.28±0.46	2.26±0.46	2.37±0.44	0.490
Albumin (g/dl)	3.69±0.65	3.73±0.64	3.37±0.62	<0.001**
Total protein (g/dl)	6.54±0.94	6.59±0.92	6.19±0.97	0.010*
GOT (U/L)	23.62±19.46	23.84±20.25	22.00±12.25	0.783
GPT (U/L)	23.94±22.17	23.87±22.47	24.49±20.05	0.693
Total cholesterol (mg/dl)	193.50±54.59	192.17±54.25	204.26±56.87	0.062
Triglyceride (mg/dl)	153.31±136.16	149.52±132.02	184.24±165.00	0.140
Low-density lipoprotein(mg/dl)	117.21±43.30	114.21±39.49	141.54±62.12	0.002**
High-density lipoprotein	55.02±19.52	55.60±19.31	49.64±21.03	0.047*
Fasting glucose (mg/dl)	95.70±21.20	96.16±22.08	92.19±12.37	0.552
Postprandial glucose (mg/dl)	143.40±66.99	143.68±67.36	140.00±76.22	1.000
Glycated hemoglobin (%)	5.81±1.15	5.83±1.20	5.66±0.63	0.567
IgG (mg/dl)	1093.09±342.02	1103.93±326.09	1011.76±440.59	0.034*
IgA (mg/dl)	353.51±151.70	352.20±151.35	363.34±155.82	0.588
IgM (mg/dl)	111.20±53.12	113.09±53.16	97.08±51.30	0.035*
IgE (mg/dl)	256.94±563.22	252.09±576.56	290.90±476.11	0.765
C3 (mg/dl)	109.40±24.66	108.93±24.29	112.61±27.12	0.368
C4 (mg/dl)	29.56±10.14	29.52±10.10	29.83±10.53	0.867
HBsAg positive	10 (18.5%)	8 (20.5%)	2 (13.3%)	0.398
AntiHBs positive	17 (40.5%)	11 (36.7%)	6 (50.0%)	0.735
AntiHCV positive	11 (3.2%)	11 (3.6%)	0 (0.0%)	0.384
ANA positive	31 (8.7%)	25 (8.0%)	6 (13.3%)	0.500
dsDNA positive	19.98±17.88	19.76±17.09	21.15±22.02	0.732
ANCA positive	6 (2.8%)	1 (0.5%)	5 (15.6%)	<0.001**
Myeloperoxidase	10.16±27.08	2.30±1.96	28.21±45.58	0.070
Proteinase-3	3.47±3.14	2.88±2.54	5.11±4.14	0.150
Rapid plasma reagin	1 (0.8%)	1 (0.9%)	0 (0.0%)	1.000
Daily urine protein (g/day)	2.40±2.78	2.28±2.74	3.36±2.89	0.005**
Urinary protein/creatinine ratio (g/g)	2.45±4.26	2.28±4.24	3.74±4.18	<0.001**
Urinary albumin creatinine ration (mg/g)	778.37±1159.31	728.21±1146.22	1254.96±1253.97	0.101

Chi-square test. Mann-Whitney U test.

*p<0.05,

**p<0.01.

The 1-year, 3-year and 5-year renal survival were 98.4% vs. 92.8%, 93.4% vs. 89.8%, and 86.7% vs. 77.3% in non-crescents IgAN and crescents IgAN (Fig 1). After long-term follow-up, the renal survival was better in patients with IgAN without crescents formation (p = 0.034). As for patient survival (Fig 2), the 1-year, 3-year and 5-year renal survival were 98.8 vs. 95.2%, 97.8 vs. 95.2%, and 97.1 vs. 86.5% in non-crescents IgAN and crescents IgAN. After long-term follow-up, the patient survival was better in patients with IgAN without crescents formation (p = 0.016). The infection related cause of death was 62.5% and 75% in non-cresent IgAN and cresent IgAN (S3 Table).

Fig 1

Real survivals based on crescent or non-crescent.

Fig 2

Patient survival based on crescent or non-crescent.

The possible variables associated with patient survival were list in Table 2. In univariate Cox regression model, the age (HR = 1.08 (95% CI = 1.05–1.12), p<0.001), crescents formation (HR = 3.93 (95% CI = 1.18–13.07), p = 0.025), blood albumin (HR = 0.023 (95% CI = 0.11–0.50), p<0.001), blood total protein (HR = 0.46 (95% CI = 0.28–0.75), p = 0.002), HDL (HR = 0.95 (95% CI = 0.91–0.99), p = 0.009), daily urine protein (HR = 1.14 (95% CI = 1.01–1.29), p = 0.038), urine PCR (HR = 1.07 (95% CI = 1.02–1.12), p = 0.003), serum IgM (HR = 0.98 (95% CI = 0.96–1.00), p = 0.036), BUN (HR = 1.02 (95% CI = 1.01–1.03), p = 0.005), and eGFR (HR = 0.097 (95% CI = 0.94–0.99), p = 0.0011) were associated with patient survival. After multivariate Cox regression analysis, older age (HR = 1.08 (95% CI = 1.01–1.13), p = 0.013), crescents (HR = 5.57 (95% CI = 1.14–29.05), p = 0.034), and HDL (HR = 0.094 (95% CI = 0.90–0.99), p = 0.026) were associated with patient survival. Crescents IgAN is with the highest risk (up to 5.75 of HR) for patient mortality.

Table 2

Univariate and multivariate Cox regression analyses on patient survivals.

	Univariate			Multivariate
	Hazard ratio	95%CI	p value	Hazard ratio	95%CI	p value
Sex
Female	Reference	Reference
Male	4.44	(0.97–20.25)	0.055
Age	1.08	(1.05–1.12)	<0.001**	1.07	(1.01–1.13)	0.013*
Crescents
0	Reference	Reference		Reference	Reference
1	3.93	(1.18–13.07)	0.025*	5.75	(1.14–29.05)	0.034*
Albumin	0.23	(0.11–0.50)	<0.001**	0.38	(0.05–3.09)	0.367
Total protein	0.46	(0.28–0.75)	0.002**	0.85	(0.22–3.22)	0.809
Low-density lipoprotein	0.99	(0.97–1.00)	0.154
High-density lipoprotein	0.95	(0.91–0.99)	0.009**	0.94	(0.90–0.99)	0.026*
Daily urine protein	1.14	(1.01–1.29)	0.038*	0.97	(0.78–1.20)	0.771
Urinary protein/creatinine ratio	1.07	(1.02–1.12)	0.003**
IgG	1.00	(1.00–1.00)	0.398
IgM	0.98	(0.96–1.00)	0.036*	0.99	(0.97–1.02)	0.453
IgA	1.00	(1.00–1.00)	0.075
C3	0.98	(0.95–1.00)	0.094
C4	1.02	(0.97–1.07)	0.447
Blood urea nitrogen	1.02	(1.01–1.03)	0.005**
Creatinine	1.06	(0.96–1.17)	0.277
eGFR	0.97	(0.94–0.99)	0.011*	1.00	(0.96–1.03)	0.915
ANA
Negative	Reference	Reference
Positive	1.56	(0.19–12.84)	0.678
ANCA
Negative	Reference	Reference
Positive	7.59	(0.88–65.49)	0.065

Cox proportional hazard regression.

*p<0.05,

**p<0.01.

Discussion

In this study, focusing on the effect of crescents of IgAN on patient survival revealed that mortality was increased independently from other traditional risk factors for patient mortality. According to previous studies [11, 12], IgAN is associated with more hypertension and vascular disease after long-term follow-up. Investigators also found a 59% increased risk for death from cardiovascular disease (CVD) [12]. Another study also showed that 45% of all death in IgAN was due to CVD [6]. Both decreased eGFR and increased proteinuria increase the risk of CVD from many studies [18-21]. Once the IgAN cannot be cured, all patients will go into chronic kidney disease (CKD). CKD was also considered as a coronary heart disease risk equivalent based on several studies [22-24]. Besides, as CKD progression, the CV outcome also got worse, including death from CV causes, re-infarction, congestive heart failure, stroke, resuscitation and composite end points [25]. In our study, the baseline eGFRs between non-crescents and crescents group were similar (58.05 vs. 50.10 ml/min/1.732m2, p = 0.190). However, after long-term follow-up of renal function, renal function was significantly worse in crescents group than non-crescents group (p = 0.034). Therefore, crescents group went into more advanced stage of CKD and would experience more CVD. Moreover, worse controlled IgAN will have more severe secondary hyperlipidemia, which further predisposed more CVD after long-term follow-up.

In addition to worse renal function related CV complication in crescents IgAN, increased mortality in patients treated with steroids or immunosuppressive agents was also reported in IgAN from other study [12]. In a prematurely terminated trial to evaluate the efficacy and safety of steroids in patients with IgAN with more than 1g/day proteinuria, oral methylprednisolone was associated with an increased risk of serious infections [26]. Rauen et al conducted a multicenter, open-label, randomized, controlled study that more adverse effects were observed among the patients who received immunosuppressive therapy, with no change in the rate of decrease in the eGFR [27]. Another randomized controlled trial showed that after a median of 2.1 years, serious infection occurred in 14.7% percent of patients who received methylprednisolone compared with 3.2 percent of those who received placebo [26]. Crescents IgAN tend to receive more immunosuppressant then non-crescents group. Reasonably, patients with crescents IgAN would experience more infection and infection related death. That was consistent with our data (75% vs. 62.5%).

Apart from the above reasons, more crescents IgAN went into ESRD and received hemodialysis or peritoneal dialysis. They were prone to experience dialysis related complications, including CVD and infection. From a nation-wide stud, a strongly increased mortality (HR = 4.9) is observed in IgAN undergoing dialysis [6]. Our data also showed similar result that the patient survival between no crescents and crescents differ more significantly after longer follow-up (3.6%, 4.6%, and 10.6% for 1-year, 3-year and 5-year, respectively).

In 2012, a Japanese prognostic model was validated in a Norwegian cohort [28]. In that scoring system, only one variable was histological score (5 scores if grade 3 or 4). However, no detailed analysis of crescents was performed. A single-center study following 30 years for the mortality of IgAN showed that more severe in both glomerular and tubulointerstitial areas in ESRD patients and in those who died [8]. However, the effect of crescents on mortality was not studied. Another study conducted by Knoop et al in 2013 showed increased mortality if baseline eGFR< 60 ml/min/1.732m2 or DUP≧3g [6]. Our study also supports this risk prediction to mortality. As for eGFR, both crescents (50.10 ml/min/1.732m2) and non-crescents (58.05 ml/min/1.732m2) group are moderate risk group according to that study [8]. As for proteinuria, crescents group is high risk (3.36 g/day) group but non-crescents group is moderate risk group (2.28 g/day). Our data further support their results [8] based on new pathological evidence.

Our study is similar to a previous study [13], conducted by Zhang et al in 2017. In that study, authors found that less than 50% crescents involvement was associated with worse renal outcome without statistical significance (p = 0.077). The composite outcome (renal and patient survival) showed significant association (p = 0.003). Multivariate Cox regression analyses adjusting for eGFR, hypertension, proteinuria, and the Oxford-MEST classification demonstrated the predictive significance of an increasing crescents proportion with composite outcome (HR = 1.51). However, there was no direct analysis (Kaplan–Meier curves and Multivariate Cox regression analysis) for patient survival in that study [13]. They just showed renal outcome and incorporated (renal and patient survival) outcome. It is worth mentioning that our study is the first one focusing on patient survival of IgAN with crescents. In summary, crescents formation in IgAN is associated with higher mortality in our study with direct evidence and in previous study with indirect evidence [13]. As for renal survival, the 5-your renal outcome was worse in our cohort compared to that study (77.3 vs. 86.9%) [13]. That reason was as follows. Comparing to that cohort, our patients with crescents were with older age (41.4 vs. 32 years old), more severe baseline renal function (50.1 vs. 84 ml/min/1.732m2 of eGFR, and 3.36 vs. 0.8 g/day of DUP) and more severe crescents involvement (6.7, 17.8, 37.8 and 37.8% vs. 28.0, 31.2, 32.7, and 8.0%, for <5%, 5–9%, 10–24%, and ≥ 25% crescents, respectively).

There are some limitations for this study. First, the case number of this study is relatively low. Second, we did not include the treatment for IgAN (especially the dose of corticosteroid). However, because this study was conducted in single institute, the treatment protocol is almost the same. For IgAN without crescents, steroid based therapy was applied. As for IgAN with crescents, we always applied induction therapy with therapeutic plasmapheresis for five times and methylprednisolone 500mg for three days. All patients with IgAN should receive renin–angiotensin–aldosterone system blockader as possible. Third, we did not evaluate the association between detailed MEST-C scores and patient outcome. However, the main object of this study focused on crescents and all other histological parameters for patient outcome had been already evaluated in previous studies. Fourth, we did not exclude 6 patients with ANCA+ and IgAN. That was because they did have systemic manifestation of vasculitis and they were not diagnosed as pauci-immune glomerulonephritis according to immunofluorescence microscopy. Finally, we did not have information of blood pressure and other disease after long-term follow-up.

Conclusion

The major strengths of the present study is that crescents IgAN had worse patient survival compared to non-crescents IgAN. Clinicians should be more careful to care patients with crescents IgAN.

All patients with crescents IgAN.

(DOCX)

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Distribution of the proportions of crescents.

(DOCX)

Click here for additional data file.

Cause of death.

(DOCX)

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Distribution of the proportions of glomeruli with crescents.

(DOCX)

Click here for additional data file.

12 Jun 2020

PONE-D-20-15033

Crescent formations are independently associated with higher mortality in biopsy-confirmed immunoglobulin A nephropathy

PLOS ONE

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Reviewer #2: Yes

**********

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Reviewer #2: Yes

**********

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Reviewer #1: Chen et al have studied the effect of glomerular crescents in 388 biopsy-proven IgA nephropathy (IgAN) patients included in a retrospective study with a mean follow-up of 7.3±3.1 years. Crescents were present in 12% of cases. Results demonstrated that age, crescents and HDL were associated with patient survival at multivariate Cox regression analysis. Moreover, crescents expressed the highest risk for patient mortality.

The paper has many scientific drawbacks:

1. Kidney biopsies were not analyzed according to the Oxford Classification (MEST-C).

The number of crescents and their type (proliferative, fibrous) are not reported. This referee suggests to follow the paper of M. Haas et al on the crescents in IgAN kidney biopsies.

The percentage of florid crescents is not reported.

The percentage of tubulo-interstitial lesions is not reported. These lesions are responsible for the prognosis and outcome of the disease.

2. The Haas classification is out of date because the international Oxford classification is used by all pathologists.

3. ANCA positivity was present in 15.6% of IgAN cases with crescents. This means that these cases are not idiopathic IgAN. Therefore, the authors should exclude these patients.

4. Fig 1 and 2 are not correct. The number of cases observed at each time for the two subgroups is not reported.

5. The therapy should be described in details in material and methods. How many patients received plasmapheresis?

6. Dosage of corticosteroids is not reported.

7. Conclusions on aggressive immunosuppressive therapy are not supported by clinical data in the study that shows many lacking points.

8. The authors should evaluate the number of crescents; therefore, the term crescent is not appropriate and should be replaced by the term crescents.

Minor points

1. Reference No.1 is out of date because there are recent published papers on the frequency of IgAN in the world

2. P9 line 13 controlled trial

3. P10 eGFR>60 ml/min/1.732m2; should be eGFR<60 ml/min/1.732m2

Reviewer #2: I have no competing interests.

Interesting study with some limitations.

Abstract: why authors in the conclusion spoke about "Aggressive immunosuppressants should not

be given for all crescent IgAN and should be individualized". This does not seem to have relation with reported results

Abstract. CI should be added for all dependent variables

Methods: normality should be checked for

Methods: levels of significance for values inserted in multvariate analysis should be added

Methods/results: ratio of n of events/n of variavbles seems to low (see PMID: 8970487)

**********

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Reviewer #1: No

Reviewer #2: Yes: Fabrizio D'Ascenzo

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13 Jun 2020

Editor comments

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

Ok.

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type of consent you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed).

All methods were carried out in accordance with relevant guidelines and regulations and informed consent was obtained from all subjects.

3. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://www.mdpi.com/2077-0383/8/6/848/html

The text that needs to be addressed involves the Introduction.

We addressed that in the part of introduction.

“In our previous study, we found that a lower serum IgG (≤907 mg/dL) and serum C3 (≤79.7 mg/dL) were both risk factors for poor renal outcome in our database of IgAN[J Clin Med

. 2019 Jun 14;8(6):848] ”

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

We rephrased that. Thanks for your comments.

“This We conducted a retrospective cohort study was performed between January 2003 and December 2013. Participants of the age should be more than > 20 years old and the diagnosis of IgAN was based on who had undergone their first renal (native kidney only) biopsy with the diagnosis of IgAN were enrolled in our medical center (Taichung Veterans General Hospital, TCVGH) in Taiwan. Graft renal biopsies were excluded. Our institute has This medical center possessesd the largest patient population undergoing renal biopsiesfor those who have undergone renal biopsies (m. Until now, the accumulative case numbers of renal biopsy was more than ore than 8000 within the last 30 years). This study was approved by Ethics Committee of TCVGHTaichung Veterans General Hospital, IRB number：CE15125B. All methods were carried outperformed in accordance with relevant guidelines and regulations and informed consent was obtained from all subjects. ”

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement.

We removed that.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

We cite all supplementary data in the text. Also, we added the information of supplementary data at the end of our manuscript (as follows).

Supplementary data

Table 1. All patients with crescent IgAN.

Table 2. Distribution of the proportions of crescents

Table 3. Cause of death

Figure 1 Distribution of the proportions of glomeruli with crescents

Reviewer #1

1. Kidney biopsies were not analyzed according to the Oxford Classification (MEST-C).

The number of crescents and their type (proliferative, fibrous) are not reported. This referee suggests to follow the paper of M. Haas et al on the crescents in IgAN kidney biopsies. The percentage of florid crescents is not reported. The percentage of tubulo-interstitial lesions is not reported. These lesions are responsible for the prognosis and outcome of the disease.

Thanks for this comment. The focus of this article is impact of the presence of crescent formation on patients’ survival. And our result showed that the significant association between crescent formation and mortality. We did not aim to elucidate the type of crescent formation (proliferative, fibrous), which maybe more associated with renal outcome, rather than patient outcome. Similarly, our goal is this study is not to research the tubular or interstitial involvement of IgAN. Besides, the typical finding of IgAN is on glomerular change, rather than tubular or interstitial change. Thus, we did not show the detailed pathological finding in our study.

2. The Haas classification is out of date because the international Oxford classification is used by all pathologists.

Thanks for this comment. We also agreed that Oxford classification is used by most pathologists. There have been 22 validation studies of the Oxford classification [Curr Opin Nephrol Hypertens 2017, 26, 165-171]. Recently, Park el al suggested that the Haas and the Oxford classifications are comparable in predicting the progression of IgAN [Human pathology 2014, 45, 236-243]. In our previous study [J Clin Med . 2019 Jun 14;8(6):848.], we proved that Haas classification is also useful for establishing predictive values in Asian groups. Therefore, the validation of Haas classification in our IgAN cohort had been done in our previous study. In addition, our aim of this study is crescent formation or not in IgAN. Not only Oxford but also Haas classification can show crescent formation. So, types of classification of IgAN do not affect the study design or outcome.

3. ANCA positivity was present in 15.6% of IgAN cases with crescents. This means that these cases are not idiopathic IgAN. Therefore, the authors should exclude these patients.

Totally, there are only 6 patients (2.8%) with ANCA+ in this IgAN cohort. It is debatable whether IgG-ANCA plays a pathogenic role in IgAN. We did not exclude this ANCA+ IgAN patents because of following reasons. First, in this study, we enrolled patients with IgAN (including idiopathic or not, primary or secondary type). Therefore, we did not mention in the text that our patients are with idiopathic IgAN. Second (more important), all 6 patients were not diagnosed as ANCA related crescentic GN because of not pauci-immune finding in immunofluorescence microscopy. Third, these 5 patients with ANCA+ IgAN did not have systemic manifestation of vasculitis. Based on the above reasons, we did not exclude these 6 patients in this study. We mentioned this in the part of limitation, page 11.

4. Fig 1 and 2 are not correct. The number of cases observed at each time for the two subgroups is not reported.

Thanks for this comment. We only showed the rate (%). As your suggestion, we added the information of “Number at risk (n) and survival rate (%)”

5. The therapy should be described in details in material and methods. How many patients received plasmapheresis?

Thanks for this comment. That is because we did not have detailed information of therapy of crescent IgAN. We mentioned this limitation in the part of limitation, page 11. However, because this study was conducted in single institute, the treatment protocol is almost the same. For IgAN without crescent, steroid based therapy was applied. As for IgAN with crescent, we always applied induction therapy with therapeutic plasmapheresis for five times (all patients with crescent IgAN) and methylprednisolone 500mg for three days. All patients with IgAN should receive renin–angiotensin–aldosterone system blockader as possible. We acknowledged this limitation.

6. Dosage of corticosteroids is not reported.

Thanks for this comment. That is because we did not have detailed information of therapy of crescent IgAN. We mentioned this limitation in the part of limitation, page 11. However, because this study was conducted in single institute, the treatment protocol is almost the same. For IgAN without crescent, steroid based therapy was applied. As for IgAN with crescent, we always applied induction therapy with therapeutic plasmapheresis for five times (all patients with crescent IgAN) and methylprednisolone 500mg for three days. All patients with IgAN should receive renin–angiotensin–aldosterone system blockader as possible. We acknowledged this limitation. The information regarding doses of steroid is lacking. We emphasize this in the part of limitation, page 11.

“Second, we did not include the treatment for IgAN (especially the dose of corticosteroid)”

7. Conclusions on aggressive immunosuppressive therapy are not supported by clinical data in the study that shows many lacking points.

Thanks for this comment. We revised the part of conclusion as follows. “The major strengths of the present study is that crescent IgAN had worse patient survival compared to non-crescent IgAN. Clinicians should be more careful to care patients with crescent IgAN.

”

8. The authors should evaluate the number of crescents; therefore, the term crescent is not appropriate and should be replaced by the term crescents.

We replaced crescent with crescents. Thanks for your suggestion.

Minor points

1. Reference No.1 is out of date because there are recent published papers on the frequency of IgAN in the world

Thanks for this comment. We cited a new study regarding the prevalence/frequency of IgAN in this world [CJASN April 2017, 12 (4) 677-686].

2. P9 line 13 controlled trial

Sorry about this typo.

3. P10 eGFR>60 ml/min/1.732m2; should be eGFR<60 ml/min/1.732m2

Sorry about this typo.

Reviewer #2:

� I have no competing interests.

Interesting study with some limitations.

Thanks for this comment.

� Abstract: why authors in the conclusion spoke about "Aggressive immunosuppressants should not

be given for all crescent IgAN and should be individualized". This does not seem to have relation with reported results

That’s only based on indirect evidence. We revised that as “Clinicians should be more careful to care of patients with crescent IgAN. Thanks for this comment.

� Abstract. CI should be added for all dependent variables

We added all 95% CI. Thanks for this comment.

� Methods: normality should be checked for

OK.

� Methods: levels of significance for values inserted in multvariate analysis should be added

A Cox proportional hazard regression (shown as HR, 95% confidence interval (CI)) was used to analyze the possible factors for patient survival (both the univariate and multivariate Cox models).

� Methods/results: ratio of n of events/n of variavbles seems to low (see PMID: 8970487)

Thanks for this comment. For EPV (number of events per variable) values of 10 or greater, no major problems occurred (J Clin Epidemiol . 1996 Dec;49(12):1373-9). EPV is 12/8, which is less than 10. However, in this study, the mean duration of follow-up was 7.2 ± 3.1 years. As for study regarding patient mortality, the possible bias maybe inevitable. After meticulous statistical analysis (Cox regression model, univariate and multivariate), the hazard ratio is significant. The result is consistent to previous studies. Therefore, we believed that the result of this study is true. Thanks for this comment.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

6 Jul 2020

PONE-D-20-15033R1

Crescents formations are independently associated with higher mortality in biopsy-confirmed immunoglobulin A nephropathy

PLOS ONE

Dear Dr. Tsai,

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.

Please revise Kidney biopsies according to the Oxford classification and try to address all other issues raised by Referee 1.

Please submit your revised manuscript by Aug 20 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Fabio Sallustio

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

**********

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: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This referee invite the authors to revise the manuscript according to the suggestions listed in the first

comments, otherwise the paper will be rejected by this referee.

Kidney biopsies must be revised according to the Oxford classification described by Haas et al in JASN 28:691-701, 2017.

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

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7 Jul 2020

We reviewed our data regarding renal biopsy, and we re-scored it by Oxford classification with MEST-C score. We replaced Haas classification with Oxford classification in table 1. We also revised some parts in text. Thanks for your suggestions.

Total (n=388) Non Crescents(n=343) Crescents (n=45) p value

Oxford classification (n, %)

M0 247 223 (90.3%) 24 (53.3%) 0.078

M1 138 118 (34.4%) 20 (44.4%) 0.087

M2 3 2 (0.6%) 1 (2.2%) 0.227

E0 227 296 (86.3%) 31 (68.9%) 0.04

E1 62 48 (14.0%) 14 (31.1%) 0.002

S0 341 322 (93.9%) 19 (45.2%) <0.001

S1 47 21 (6.1%) 26 (57.8%) <0.001

T0 332 298 (86.9%) 34 (75.6%) 0.073

T1 38 29 (8.5%) 9 (20%) 0.028

T2 18 16 (4.7%) 2 (4.4%) 0.788

C0 388 343 (100%) 0 (0%) <0.001

C1 35 0 35 (77.8%) <0.001

C2 10 0 10 (22.2%) <0.001

Submitted filename: response to reviewer comment.docx

Click here for additional data file.

21 Jul 2020

Crescents formations are independently associated with higher mortality in biopsy-confirmed immunoglobulin A nephropathy

PONE-D-20-15033R2

Dear Dr. Tsai,

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.

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Fabio Sallustio

Academic Editor

PLOS ONE

23 Jul 2020

PONE-D-20-15033R2

Crescents formations are independently associated with higher mortality in biopsy-confirmed immunoglobulin A nephropathy

Dear Dr. Tsai:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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