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Persistent Hematuria and Kidney Disease Progression in IgA Nephropathy: A Cohort Study
Original Investigation
Persistent Hematuria and Kidney Disease Progression in IgA Nephropathy: A Cohort Study Gui-zhen Yu, Ling Guo, Jin-feng Dong, Su-fang Shi, Li-jun Liu, Jin-wei Wang, Gui-li Sui, Xu-jie Zhou, Ying Xing, Hai-xia Li, Ji-cheng Lv, and Hong Zhang Rationale & Objective: Hematuria is the most typical presentation of immunoglobulin A nephropathy (IgAN); however, its role in disease progression is still controversial. This study aimed to evaluate the association of hematuria and progression of IgAN. Study Design: Retrospective cohort study. Setting & Participants: A cohort of 1,333 patients with IgAN treated at a Chinese referral hospital with a median follow-up of 45 months. Predictors: Microhematuria was evaluated in fresh urine using a fully automated urine particle analyzer (automated method) and urine sediment examination by a skilled examiner (manual method). Hematuria was characterized as a timevarying attribute; namely, average hematuria level was calculated for every 6-month period for each patient during follow-up. Remission was defined as average red blood cell count ≤5/high-power field (manual method) or ≤28 red blood cells/μL (automated method) during the first 6 months of follow-up. Outcomes: Composite event of 50% decline in estimated glomerular filtration rate or development of kidney failure. Analytical Approach: Multivariable causespecific hazards models to analyze the relationship between hematuria and the composite kidney disease progression event. Results: Time-varying hematuria during follow-up was an independent risk factor for the composite kidney disease progression event (HR, 1.46;
I
95% CI, 1.13-1.87; P = 0.003). Hematuria remission during the 6 months after diagnosis was associated with a significantly lower rate of the composite kidney disease progression event (HR, 0.41; 95% CI, 0.28-0.61; P < 0.001). A significant interaction was detected between remission of proteinuria and remission of hematuria during the first 6 months (P < 0.001). The association between remission of hematuria and kidney disease progression was detectable (HR, 0.46; 95% CI, 0.32-0.68) within the subpopulation with persistent proteinuria (protein excretion > 1.0 g/d during the first 6 months), but not among patients whose proteinuria had remitted (HR, 0.64; 95% CI, 0.31-1.29; P = 0.2). The 2 techniques for hematuria evaluation were strongly and significantly linearly correlated (r = 0.948; P < 0.001), and results using these 2 methods were consistent.
Correspondence to J.-c. Lv ([email protected]) Am J Kidney Dis. XX(XX):110. Published online Month XX, XXXX. doi: 10.1053/ j.ajkd.2019.11.008 © 2020 by the National Kidney Foundation, Inc.
Limitations: A single-center retrospective study. Proportional hazards regression incorporating time-varying covariates may create time-varying confounding. The predictive value of reductions in hematuria was not directly evaluated. Conclusions: Level of hematuria was independently associated with kidney disease progression, whereas hematuria remission was associated with improved kidney outcomes in IgAN among patients with persistent proteinuria. Additionally, to monitor IgAN progression, automated methods to evaluate hematuria hold promise as a replacement for manual evaluation of urinary sediment.
mmunoglobulin A (IgA) nephropathy (IgAN) is the most common primary glomerular disease worldwide and a major cause of kidney failure requiring kidney replacement therapy (KRT).1-3 Clinical presentations in IgAN range from a benign incidental condition to rapidly progressive kidney failure, although most affected individuals develop chronic slowly progressive kidney injury and many patients will require KRT.4 Persistent proteinuria, hypertension, decreased glomerular filtration rate (GFR) at baseline, and the MEST-C scoring system are all established clinical and pathologic predictors of progression to kidney failure requiring KRT.5-12 Hematuria is the most typical presentation of IgAN. Approximately 70% to 100% of patients have microscopic hematuria, and macrohematuria often occurs after upper respiratory tract or intestinal infection.13 AJKD Vol XX | Iss XX | Month 2020
Complete author and article information provided before references.
However, the role of hematuria in IgAN progression is still controversial. In some studies, microhematuria has been shown to be a risk factor for kidney disease progression,14-21 whereas other studies demonstrated that it did not influence the risk for kidney failure.22-26 Reasons for the conflicting data include low quality of the studies, analysis of only 1 urinary sediment at the time of kidney biopsy, and lack of standardized measurements.13 Analysis of fresh urine sediment has interobserver variability, limiting the pooling of data from different laboratories, whereas 24-hour urine collection for red blood cell (RBC) count is also unsatisfactory given that it is cumbersome, expensive, and affected by collection errors.13 In addition, urine RBC counts vary over the short term much more than urine protein excretion. These problems have limited efforts to analyze the association of hematuria with long1
Original Investigation term outcomes. Recently, a small study indicated that time-averaged hematuria during follow-up, using a standard urine sediment analysis in one center, was strongly associated with poor renal prognosis.15,27 This finding needs further confirmation. In China, urine RBC measurement in fresh morning urine with a fully automated urine particle analyzer has been widely used. This method provides a standard measure without interobserver variability. In this study, we aimed to evaluate the association of microhematuria at baseline, hematuria during follow-up, and hematuria remission during the first 6 months with long-term kidney outcomes in a large cohort of patients with IgAN. We also compared automated analysis of hematuria with traditional urine sediment analysis. Methods Study Population A total of 1,333 patients with biopsy-proven IgAN diagnosed between 2003 and 2016 were selected from the prospective IgAN database at Peking University First Hospital, which is the first renal division and a renal reference center in China. Patients come from across China, especially from the north, and the center serves a population of 582 million people. All patients in the IgAN database were followed up regularly every 3 to 6 months, depending on the patients’ disease condition. Patients were diagnosed based on the dominant deposition of IgA in the glomerular mesangium by immunofluorescence, and patients with IgA vasculitis, systemic lupus, and other secondary IgAN were excluded. Written informed consent was obtained from all participants for use of the clinical and pathologic data in future studies when they entered the IgAN cohort. For this study, the protocol was reviewed and approved by the Ethics Committee of the institute but without getting additional informed consent. Data Collection and Pathologic Manifestations Examination of hematuria in urine was performed using 2 methods: measurement using a fully automated urine particle analyzer (UF-1000; Sysmex; automated method), and microscopic urine sediment examination performed by a skilled examiner (manual method). All urine samples were simultaneously analyzed using these 2 methods within 2 hours of collection at every visit and recorded as RBCs/μL in the automated method and RBCs/high-power field (HPF) in the manual method. Clinical data, including sex, age at the time of kidney biopsy, systolic/diastolic blood pressure, serum creatine level, hematuria, and 24-h urine protein excretion, were measured and recorded at the time of kidney biopsy and each visit. Pathologic lesions were evaluated according to the Oxford classification. Baseline (and the beginning of follow-up) was defined as the time of kidney biopsy. 2
Information was collected for medications used during follow-up, including renin-angiotensin-aldosterone system blockade, steroids, and other immunosuppressants. The use of immunosuppressive (IS) agents during the first 6 months after kidney biopsy was as follows: for patients with persistent proteinuria with protein excretion >1 g/ d and at least 3 to 6 months of optimal blood pressure control and receiving the maximum tolerated dose of renin-angiotensin-aldosterone system inhibitors, corticosteroid therapy was added. For patients with >25% crescents on biopsy and progressive GFR loss, we usually add steroids plus cyclophosphamide or mycophenolate mofetil. Definitions Hypertension was defined as systolic blood pressure ≥140 mm Hg, diastolic blood pressure ≥90 mm Hg, or taking antihypertension medication. Mean arterial pressure (MAP) was calculated as the sum of one-third of the pulse pressure and diastolic blood pressure. Because of the high variability of hematuria, a 6-month interval was used to calculate average hematuria. Average hematuria was determined for each patient for each 6-month block during follow-up that ended upon occurrence of a kidney disease progression event, death, or end of follow-up, whichever occurred first (baseline hematuria was included in the first block), and the mean of the average of hematuria calculated for every 6-month period was expressed using time-averaged hematuria. Time-averaged proteinuria and time-averaged MAP were calculated using the same method. Estimated GFR (eGFR) was calculated according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.28 Kidney failure was defined as eGFR persistently <15 mL/min/1.73 m2 or the need for KRT (including peritoneal dialysis, hemodialysis, or kidney transplantation). The composite kidney disease progression event was defined as 50% eGFR decline or kidney failure. Hematuria remission was defined as hematuria with ≤5 RBC/HPF using the manual method (consistent with a prior study27) or ≤28 RBC/μL using the automated method. Persistent no hematuria was defined as both baseline and time-varying hematuria ≤5 RBC/HPF using the manual method or ≤28 RBC/μL using the automated method. Persistent hematuria was defined as both baseline and time-varying hematuria > 5 RBC/HPF using the manual method or >28 RBC/μL using the automated method. Macroscopic hematuria was defined as urine that is visibly tea colored, cola colored, pink, or even red. Proteinuria remission was defined as proteinuria with protein excretion ≤1.0 g/d. Statistical Analyses Normally distributed variables are presented as mean ± standard deviation and compared using t test; nonnormally distributed data such as time-averaged hematuria and time-averaged proteinuria are expressed as median and interquartile range (IQR) and compared using AJKD Vol XX | Iss XX | Month 2020
Original Investigation Kruskal-Wallis test. All categorical data are summarized as frequency or percentage and tested using χ 2 test. Spearman correlation was used to analyze the correlation between the 2 techniques for hematuria measurement. Hematuria was statistically analyzed in 2 ways: as a continuous time-varying covariate and as a categorical variable characterizing its remission during the first 6 months following biopsy diagnosis. The relationships of time-varying hematuria and hematuria remission with the composite kidney disease progression outcome were estimated using cause-specific hazards models by treating death as competing events. Cumulative incidence function methods and Gray test were used to evaluate the association of kidney disease progression events and measures of hematuria remission. Statistical analysis was performed using IBM SPSS, version 24.0, and Stata software, version 14.0 (StataCorp). P < 0.05 (2 sided) was considered statistically significant. Results Clinical and Pathologic Data The cohort included 1,333 patients with IgAN, including 8 and 172 cases without baseline hematuria data using manual examination and automated analysis, respectively. During follow-up, participants had 429 automated urine examinations per 100 person-years and 457 per 100 person-years using the manual method. The Oxford classification was not done in 29 patients with fewer than 8 glomeruli on kidney biopsy. Clinical and pathologic characteristics are summarized in Table 1. At baseline there were 675 (50.64%) men and mean age was 35.07 ± 11.96 years. eGFR was 82.2 ± 30.6 mL/min/1.73 m2, median protein excretion was 1.31 (IQR, 0.68-2.54) g/d, and MAP was 93.73 ± 11.89 mm Hg, with 645 (48.39%) patients being hypertensive at baseline. Initial hematuria was 12.50 (IQR, 4.50-45.00) RBC/HPF using the manual method and 97.60 (IQR, 35.20-273.75) RBC/μL using the automated method. A total of 375 (28.13%) patients were recorded as having a history of macroscopic hematuria. The distributions of M1, E1, S1, T1-T2, and C1-C2 were 39.88%, 33.05%, 62.27%, 34.51%, and 58.67%, respectively. Median follow-up time was 45.00 (IQR, 24.00-80.00) months. Time-averaged hematuria was 11.31 (IQR, 4.1226.63) RBC/HPF using the manual method and 68.66 (IQR, 25.31-160.35) RBC/μL using the automated method. For time-averaged proteinuria, median protein excretion was 0.86 (IQR, 0.49-1.45) g/d. Overall, 207 (15.53%) patients reached the composite kidney disease progression event, including 123 kidney failure events. Characteristics of IgAN With Hematuria Remission As shown in Figure 1, the 2 techniques for hematuria measurement (manual and automated methods) displayed a strong linear correlation (r = 0.948; P < 0.001). The conversion coefficient between the 2 types of hematuria AJKD Vol XX | Iss XX | Month 2020
Table 1. Clinical and Pathologic Characteristics of Patients With IgAN Characteristic Baseline Male sex Age, y MAP, mm Hg Hypertension History of macroscopic hematuriaa Hematuria Manual method, RBC/HPFb Automated method, RBC/μLc Proteinuria, g/d eGFR, mL/min/1.73 m2 Oxford classificationd M1 E1 S1 T1-T2 C1-C2 Follow-up Follow-up duration, mo Time-averaged hematuria Manual method, RBC/HPF Automated method, RBC/μL Time-averaged proteinuria, g/d Time-averaged MAP, mm Hg Treatment with steroids and/or other IS agents during first 6 mo Outcome 50% decline in eGFR Kidney failure Composite outcome
Value 675 (50.64%) 35.07 ± 11.96 93.73 ± 11.89 645 (48.39%) 375 (28.13%) 12.50 [4.50-45.00] 97.60 [35.20-273.75] 1.31 [0.68-2.54] 82.2 ± 30.6 520 (39.88%) 431 (33.05%) 812 (62.27%) 450 (34.51%) 765 (58.67%) 45.00 [24.00-80.00] 11.31 [4.12-26.63] 68.66 [25.31-160.35] 0.86 [0.49-1.45] 90.21 ± 7.75 639 (47.94%) 190 (14.25%) 123 (9.23%) 207 (15.53%)
Note: Values for continuous variables given as mean ± standard deviation or median [interquartile range]; for categorical variables, as count (percent). The composite outcome was a composite kidney disease progression event defined as 50% eGFR decline or kidney failure. Abbreviations: C, presence of crescent; E1, endocapillary hypercellularity; eGFR, estimated glomerular filtration rate; HPF, high-powered field; IgAN, immunoglobulin A nephropathy; IS, immunosuppressive; M1, mesangial hypercellularity; MAP, mean arterial pressure; RBC, red blood cell; S, segmental glomerulosclerosis/adhesion; T, severity of tubular atrophy/interstitial fibrosis. a Six patients had macroscopic hematuria (urine visibly tea-colored, cola-colored, pink, or even red) at the time of kidney biopsy. b Eight cases were without baseline hematuria data using the manual examination method due to the loss of urine examination results; 359 (27.09%) patients had ≤5 RBC/HPF. c There were 172 cases without baseline hematuria data using the automated method because the automated method was not widely used at the time of their kidney biopsy; 241 (20.76%) patients had ≤28 RBC/μL. d Oxford classification was not performed in 29 patients because there were fewer than 8 glomeruli.
examination was 5.6, so hematuria remission was defined as ≤5 RBC/HPF using the manual method or ≤28 RBC/μL using the automated method. There were 253 (18.98%) patients with ≤28 RBC/μL during the first 6 months, as shown in Table 2, and they showed a higher prevalence of hypertension and lower prevalence of M1, E1, S1, C1, and C2 lesions. There was no significant difference in the prevalence of T lesions evaluated using the Oxford classification. As shown in Table S1, a total of 639 (47.94%) patients received steroids and/or other IS agents. In the IS therapy 3
Original Investigation
Figure 1. Correlation between the 2 techniques for hematuria measurement. Manual method: urine sediment examination with manual counting per high-power field (hpf) by a skilled professional nurse; automated method: fresh urine sample measurement using fully automated urine particle analyzer. The conversion coefficient between the 2 hematuria examinations is 5.6, which means that the value of hematuria in the manual examination method (RBC/hpf) multiplied by 5.6 equals the value of hematuria in automated measurement (RBC/μl). (A) Full results of time-averaged (TA)-hematuria. (B) Results shown in A, restricted to TA-hematuria values <500 RBC/hpf in the manual method and <4,000 RBC/μl in the automated method.
group, patients had more hematuria at baseline than the untreated group (median values of 117.55 [IQR, 42.20336.20] vs 81.20 [IQR, 30.90-226.95] RBC/μL; P < 0.001), but similar time-averaged hematuria during follow-up (67.32 [IQR, 24.83-159.90] vs 77.14 [IQR, 28.30-170.34] RBC/μL; P = 0.07). Risk Factors for Kidney Disease Progression in IgAN As shown in Table 3, in cause-specific hazards models, time-varying hematuria using the automated method was an independent risk factor for the composite kidney disease progression event in model 3 (hazard ratio [HR], 1.46; 95% confidence interval [CI], 1.13-1.87; P = 0.003) after adjustment for sex, age, time-varying proteinuria, time-varying MAP, baseline eGFR, use of steroids or other IS agents, and Oxford classification. Results were consistent when hematuria was measured using the manual method in model 3 (HR, 1.46; 95% CI, 1.15-1.86; P = 0.002). During the first 6 months, 31 (12.3%) patients in the hematuria remission group and 176 (16.3%) in the nonremission group reached the composite kidney disease progression event (P = 0.1; Table 2). Hematuria remission was significantly associated with reduced risk for the composite event (HR, 0.41; 95% CI, 0.28-0.61; P < 0.001) in model 3 (Table 4). Cumulative incidence functions analysis demonstrated that patients who had hematuria remission (evaluated using either method) had a lower incidence of the composite kidney disease progression event (automated method, P < 0.001; Fig 2A; manual method, P < 0.001; Fig 2B). Results were consistent when the end point was kidney failure (Table S2; Fig S1A and B). Because IS therapy might influence hematuria reduction (Table S1), a subgroup analysis was done between patients with and without IS 4
therapy. This showed no heterogeneity of the association of hematuria remission with kidney disease progression (HRs of 1.15 [95% CI, 0.93-1.42] and 1.34 [95% CI, 1.19-1.52] for the without and with IS therapy groups, respectively; P for interaction = 0.4). In the proportional hazard model that included baseline clinical and pathologic characteristics (Table S3), we found that baseline proteinuria, MAP, and S and T lesions were all independent risk factors. However, treatment with IS agents was not a protective factor in this model (HR, 1.70; 95% CI, 1.19-2.43; P = 0.004). When it was added in the model, time-varying hematuria was an independent risk factor (P < 0.001). Patients with persistent no hematuria had the lowest risk for kidney disease progression events, and the persistent hematuria group had the highest risk for kidney disease progression; the rest, defined as the intermittent hematuria group, had an intermediate level of risk (Fig 3). Association Between Hematuria Remission and Proteinuria Remission We further evaluated the roles of proteinuria and hematuria remission during the first 6 months on kidney disease progression. During the first 6 months, 138 (22.9%) patients in the proteinuria remission group (n = 602) and 191 (26.1%) in the non–proteinuria remission group (n = 731) had hematuria remission (P = 0.2). Hematuria remission during the first 6 months treated as a timevarying covariate significantly reduced the risk for the composite kidney disease progression event (HR, 0.46; 95% CI, 0.32-0.68; P < 0.001) in patients without proteinuria remission (protein excretion > 1.0 g/d) during the first 6 months. In patients with proteinuria remission during the first 6 months, hematuria remission during the first 6 months did not further improve kidney survival (HR, 0.64; 95% CI, 0.31-1.29; P = 0.2). AJKD Vol XX | Iss XX | Month 2020
Original Investigation Table 2. Characteristics by Hematuria Remission During the First 6 Months Hematuria Remission During First 6 mo Characteristics No. of patients Baseline Male sex Age, y MAP, mm Hg Hypertension Proteinuria, g/d History of macroscopic hematuriaa Hematuria Manual method, RBC/HPF Automated method, RBC/μL eGFR, mL/min/1.73 m2 Oxford classificationb M1 E1 S1 T1-T2 C1 C2 Follow-up Time-averaged hematuria Automated method, RBC/μL Manual method, RBC/HPF Time-averaged proteinuria, g/d Time-averaged MAP, mm Hg Treatment with steroids and/or other IS agents during first 6 mo Outcome 50% decline in eGFR Kidney failure Composite outcome
Yes (mean of ≤28 RBC/μL) 253
No (mean of >28 RBC/μL) 1,080
P
154 (60.91%) 35.16 ± 12.43 95.13 ± 11.60 137 (54.20%) 1.29 [0.70-2.72] 45 (17.8%)
521 (48.20%) 35.05 ± 11.85 93.41 ± 11.94 508 (47.00%) 1.24 [0.67-2.51] 330 (30.6%)
<0.001 0.9 0.04 0.04 0.08 <0.001
2.00 [0.50-4.00] 12.40 [7.70-23.10] 84.31 ± 30.44
20.00 [7.50-55.00] 130 [58.20-347.30] 81.65 ± 30.60
<0.001 <0.001 0.2
69 (28.5%) 61 (25.2%) 132 (54.5%) 71 (29.3%) 85 (35.1%) 4 (1.7%)
451 (42.5%) 370 (34.8%) 680 (64.0%) 379 (35.7%) 527 (49.6%) 149 (14.0%)
<0.001 0.004 0.006 0.06 <0.001 <0.001
11.98 [7.44-20.33] 1.95 [1.02-3.96] 0.86 [0.44-1.49] 91.71 ± 6.73 117 (46.2%)
93.87 [42.19-188.11] 14.78 [7.07-31.18] 0.86 [0.50-1.45] 89.84 ± 7.94 522 (48.3%)
<0.001 <0.001 0.7 <0.001 0.6
30 (11.9%) 15 (5.9%) 31 (12.3%)
160 (14.8%) 108 (10.0%) 176 (16.3%)
0.2 0.04 0.1
Note: Composite outcome defined as 50% eGFR decline or kidney failure. Abbreviations: C, presence of crescent; E1, endocapillary hypercellularity; eGFR, estimated glomerular filtration rate; HPF, high-powered field; IS, immunosuppressive; M1, mesangial hypercellularity; MAP, mean arterial pressure; RBC, red blood cell; S, segmental glomerulosclerosis/adhesion; T, severity of tubular atrophy/interstitial fibrosis. a Six patients presented macroscopic hematuria at the time of kidney biopsy. b Oxford classification was not performed in 29 patients because there were fewer than 8 glomeruli.
As shown in Figure 4, among patients with proteinuria (protein excretion > 1.0 g/d) during the first 6 months and based on time-varying hematuria, people in the hematuria remission group had a lower incidence of kidney disease progression events than those without hematuria remission (P < 0.001; Fig 4A). However, no significant differences were observed between the time-varying hematuria remission and nonremission groups in patients with proteinuria remission (P = 0.3; Fig 4B). An interaction analysis was performed, and statistically significant multiplicative interactions for kidney disease progression events were found between proteinuria remission and hematuria remission during the first 6 months (P for interaction < 0.001). Results were consistent when the end point was kidney failure (Fig S2). Risks for kidney failure in those treated with IS therapy among patients with hematuria or proteinuria remission are listed in Table S4; those without hematuria and proteinuria remission during AJKD Vol XX | Iss XX | Month 2020
the first 6 months had a higher proportion of kidney disease progression events. Discussion Although hematuria is a typical presentation of IgAN,4,29,30 its role in disease progression is still not well established. Persistent heavy proteinuria rather than hematuria is an indication for adding steroid therapy in our clinical practice, which is consistent with KDIGO (Kidney Disease: Improving Global Outcomes) guideline recommendations.31 However, in this cohort study with more than 1,000 participants and long-term follow-up, we found that the severity of persistent hematuria during follow-up was significantly associated with kidney disease progression. Hematuria remission was associated with reduced incidence of kidney disease progression events, especially in those without proteinuria remission, and hematuria remission 5
Original Investigation Table 3. Time-Varying Hematuria as a Risk Factor for the Composite Kidney Disease Progression Event in IgAN Hazard Ratio for Composite Outcomea (95% Confidence Interval); P Hematuria by automated method, per 1 unit greaterb Hematuria by manual method, per 1 unit greaterb
Unadjusted 1.50 (1.23-1.83); <0.001 1.51 (1.27-1.80); <0.001
Model 1 1.63 (1.33-2.01); <0.001 1.61 (1.34-1.93); <0.001
Model 2 1.44 (1.13-1.85); 0.004 1.45 (1.14-1.84); 0.002
Model 3 1.46 (1.13-1.87); 0.003 1.46 (1.15-1.86); 0.002
Note: Model 1 was adjusted for age and sex; sex was expressed as a dichotomous variable. Model 2 was adjusted for covariates in model 1 plus log-transformed proteinuria and mean arterial pressure, which were as time-varying covariates; eGFR; and Oxford classification (MEST-C scores). Model 3 was adjusted for covariates in model 2 plus steroids or other immunosuppressive agents. Use of treatment with steroids and/or other immunosuppressive agents was expressed as a dichotomous variable. Abbreviation: eGFR, estimated glomerular filtration rate; IgAN, immunoglobulin A nephropathy. a Composite outcome defined as 50% decline in eGFR or kidney failure.29 b Hematuria was measured at multiple visits, log-transformed value of the mean of every 6-month hematuria was treated as time-varying covariate.
was associated with greatly reduced risk for kidney failure. These results suggest the potential importance of therapy targeting hematuria remission in IgAN. Another key finding from this study was that hematuria evaluated using standard manual examination of urine sediment and automated analysis of a fresh urine sample were consistent, suggesting that a simple and rapid urine cytometry analysis could replace the traditional manual method for urine sediment examination in IgAN. Hematuria has arguably not been given sufficient attention in nephrology and is often considered benign in IgAN.13 In recent years, numerous cohort studies have evaluated risk factors for kidney failure in IgAN. Most studies found that proteinuria at baseline or during followup was the strongest risk factor for GFR decline or kidney failure, whereas hematuria was not a risk factor. In an early review, D’Amico32 evaluated risk factors for kidney failure in 23 cohorts with 5,507 patients with IgAN. Among them, 4 studies investigated baseline microhematuria and only 1 reported it as an independent risk factor for kidney failure.32 The conflicting results may relate to the analysis being based on only 1 urine sediment measurement at the time of kidney biopsy. The extent of erythrocyturia is often variable over the short term. For these reasons, a recent international risk prediction tool did not include hematuria as a key variable.33 However, time-averaged hematuria may be more valuable as a prognostic factor. In a large Chinese IgAN cohort, Le et al15 found that time-averaged hematuria was a strong independent risk factor for kidney failure. In a
recent European cohort of 112 patients and 14 years of follow-up, time-averaged hematuria was strongly associated with kidney failure, and hematuria remission had a significant favorable effect on IgAN outcomes.27 In another European cohort of 141 patients with IgAN and 108 months of follow-up, patients with hematuria and minimal or no proteinuria showed an excellent outcome with only 1 kidney disease progression event (doubling of serum creatinine level).34 Given the conflicting results, persistent hematuria was not included in the risk evaluation system in the KDIGO guideline. In this much larger cohort study with 1,333 participants, we confirmed that the extent of hematuria during follow-up was strongly associated with the development of kidney failure. Importantly, we found that the effect of microhematuria on kidney failure had an interaction with proteinuria, being much greater in patients with protein excretion > 1.0 g/d. In patients without proteinuria remission during follow-up, hematuria remission decreased the risk for kidney failure by 54%. For those with proteinuria remission (protein excretion < 1.0 g/d), persistent hematuria was not a risk factor for kidney disease progression. This result is consistent with prior studies finding that patients with IgAN with isolated hematuria or mild proteinuria had a benign course23,34,35 and highlights the importance of hematuria remission during the therapy of patients with IgAN, especially in those who do not achieve proteinuria remission. We noted that in our cohort 59% of patients had crescents on biopsy. Several studies have suggested that
Table 4. Association of Hematuria Remission and the Composite Kidney Disease Progression Event in IgAN Hazard Ratio for Composite Outcomea (95% Confidence Interval); P Hematuria remission by automated methodb Hematuria remission by manual methodb
Unadjusted 0.45 (0.32-0.64); <0.001 0.49 (0.35-0.68); <0.001
Model 1 0.42 (0.30-0.59); <0.001 0.45 (0.32-0.62); <0.001
Model 2 0.42 (0.28-0.62); <0.001 0.46 (0.31-0.67); <0.001
Model 3 0.41 (0.28- 0.61); <0.001 0.44 (0.30- 0.65); <0.001
Note: Model 1 was adjusted for age and sex; sex was expressed as a dichotomous variable. Model 2 was adjusted for covariates in model 1 plus log-transformed proteinuria and mean arterial pressure, which were as time-varying covariates; eGFR; and Oxford classification (MEST-C scores). Model 3 was adjusted for covariates in model 2 plus steroids or other immunosuppressive agents. Use of treatment with steroids and/or other immunosuppressive agents was expressed as a dichotomous variable. Abbreviations: eGFR, estimated glomerular filtration rate; IgAN, immunoglobulin A nephropathy; RBC, red blood cell. a Composite outcome defined as 50% decline in eGFR or kidney failure.29 b Hematuria remission was defined as the first 6-month average hematuria being ≤5 RBC/high-powered field (manual method) or ≤28 RBC/μL (automated method).
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Original Investigation
Figure 2. Cumulative incidence of the composite kidney disease progression outcome in patients with immunoglobulin A nephropathy according to magnitude of time-varying hematuria. (A) Hematuria examined using automated method. H-: time-varying hematuria ≤ 28 red blood cells (RBC)/μL; H+: time-varying hematuria > 28 RBC/μL. (B) Hematuria examined using manual method. H-: Time-varying hematuria ≤ 5 RBC/high-powered field (hpf); H+: time-varying hematuria > 5 RBC/hpf.
Asian patients more frequently have crescents than the European population. In a multicenter cohort with 1,026 Chinese participants, 48% of patients had crescents.36 In another study, Katafuchi et al37 reported that 63% of Japanese patients had crescents on biopsy. By contrast, in the VALIGA cohort with 1,147 patients from 13 European countries, only 11% of patients had crescents.38 The current study confirms the high prevalence of crescents in the Asian IgAN population.
Figure 3. Cumulative incidence of the composite kidney disease progression outcome in patients according to baseline hematuria and time-varying hematuria. Group 1 (persistent no hematuria), baseline hematuria ≤ 28 red blood cells (RBC)/μL, time-varying hematuria ≤ 28 RBC/μL. Group 2 (intermittent hematuria), baseline hematuria ≤28 RBC/μL, time-varying hematuria >28 RBC/μL; or baseline hematuria >28 RBC/μL, time-varying hematuria < 28 RBC/μL. Group 3 (persistent hematuria), baseline hematuria > 28 RBC/μL, time-varying hematuria > 28 RBC/μL.
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Until now, most interventional studies of IgAN have focused on proteinuria reduction and few have reported effects on hematuria. This study suggests that IS therapy may reduce hematuria. This finding is consistent with those from the TESTING trial, in which steroid therapy resulted in hematuria remission in 58% of patients.39 In our study, we found that patients with hematuria remission had a more severe disease profile at baseline. This observation may be influenced by the treatment regimen because this group of patients was more prone to receive steroid therapy. Other new therapies, including budesonide modified-released capsules and complement activation inhibition therapy, should also be evaluated for their effect on hematuria.40,41 The standard hematuria evaluation is urine sediment analysis, which is time-consuming and requires specific expertise. Unlike the consistency of proteinuria measurement when using a standard method, the interobserver variance among different laboratories for hematuria evaluation limits the study of hematuria on kidney disease progression, especially in multicenter studies. A key finding from this study was that the fresh urine sample automated method provides a simple and easy hematuria evaluation method in IgAN, which can avoid the interobserver bias inherent in urine sediment, whether within the same laboratory or between different laboratories. In this cohort study, we found that the extent of hematuria was a moderate risk factor for kidney disease progression. The strength of this study includes the large sample size and long-term follow-up, with a large number of kidney failure events observed. This provided good study power to detect the association of hematuria with hard end points. The association is independent of MESTC, time-varying proteinuria, and other established clinical predictors. 7
Original Investigation
Figure 4. Cumulative incidence of the composite kidney disease progression outcome according to the magnitude of time-varying hematuria in patients with and without proteinuria remission during the first 6 months. Patients (A) without and (B) with proteinuria remission during the first 6 months. H-: time-varying hematuria ≤ 28 red blood cells (RBC)/μL; H+: time-varying hematuria > 28 RBC/μL.
Limitations of this study were as follows: single-center design with a single ethnicity, using time-varying covariates in proportional hazards models may create timevarying confounding, and the predictive value of reductions in hematuria was not directly evaluated. Thus, the findings still need further confirmation. In conclusion, in this large cohort study, we confirmed that the extent of microhematuria during follow-up was independently associated with kidney disease progression in IgAN, suggesting it should be included in the risk scoring system for predicting renal outcomes of IgAN. Hematuria remission significantly reduced the incidence of kidney disease progression events. Importantly, we found this effect much greater in patients without proteinuria remission, for whom hematuria remission during therapy may reduce the risk for kidney failure. For those with no or mild proteinuria, persistent microhematuria did not significantly increase the risk for kidney failure. These findings may help inform clinical management decisions. Hematuria evaluated using the fresh urine automated method had similar associations with renal outcomes as those evaluated using the manual urine sediment method. When it is confirmed as a standard and rapid method, the fresh urine automated method should be considered for IgAN clinical practice in the future. Supplementary Material Supplementary File (PDF) Figure S1: Cumulative incidence of kidney failure in IgAN patients, by magnitude of time-varying hematuria. Figure S2: Cumulative incidence of kidney failure, by magnitude of time-varying hematuria in patients with and without proteinuria remission during the first 6 months. Table S1: Characteristics of groups with or without treatment of steroids and/or other immunosuppressive agents. 8
Table S2: Risk of kidney failure with hematuria as a time-varying variable and dichotomous grouping. Table S3: Factors associated with the composite kidney disease progression outcome in univariate and multivariate analyses. Table S4: Risk of kidney failure in those treated with immunosuppressive therapy, by hematuria or proteinuria remission.
Article Information Authors’ Full Names and Academic Degrees: Gui-zhen Yu, PhD, Ling Guo, MD, Jin-feng Dong, BS, Su-fang Shi, MD, Li-jun Liu, MD, Jin-wei Wang, PhD, Gui-li Sui, BS, Xu-jie Zhou, PhD, Ying Xing, MD, Hai-xia Li, PhD, Ji-cheng Lv, MD, PhD, and Hong Zhang, MD, PhD. Authors’ Affiliations: Renal Division, Peking University First Hospital (G-zY, LG, J-fD, S-fS, L-jL, J-wW, G-lS, X-jZ, J-cL, HZ); Peking University Institute of Nephrology (G-zY, LG, J-fD, S-fS, L-jL, J-wW, G-lS, X-jZ, J-cL, HZ); Key Laboratory of Renal Disease, Ministry of Health of China (G-zY, LG, J-fD, S-fS, L-jL, J-wW, G-lS, X-jZ, J-cL, HZ); Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Peking University, Ministry of Education, China (G-zY, LG, J-fD, S-fS, L-jL, J-wW, G-lS, X-jZ, J-cL, HZ); and Clinical Laboratory, Peking University First Hospital, Peking, China (YX, H-xL). Address for Correspondence: Ji-cheng Lv, MD, PhD, Renal Division, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China. E-mail: jichenglv75@ gmail.com Authors’ Contributions: Research idea and study design: J-cL; data acquisition: G-zY, J-fD, G-lS, LG; data analysis/interpretation: G-zY, J-wW; statistical analysis: G-zY, J-wW; supervision or mentorship: J-cL, S-fS, L-jL, X-jZ, YX, H-xL, HZ. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. Support: The work was supported by grants from the National Natural Science Foundation of China (81670649), the Capital Health Research and Special Development, China (201824073). the Natural Science Foundation for Innovation Research Groups of China (81621092), the National Key Research and Development Program of China (2018YFC1314004), and grants from the AJKD Vol XX | Iss XX | Month 2020
Original Investigation Science and Technology Project of Beijing, China (D18110700010000). The funders had no role in study design; collection, analysis, and interpretation of data; writing the report; and the decision to submit the report for publication.
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Financial Disclosure: The authors declare that they have no other relevant financial interests. Peer Review: Received April 25, 2019. Evaluated by 3 external peer reviewers, with direct editorial input from a Statistics/Methods Editor, an Associate Editor, and the Editor-in-Chief. Accepted in revised form November 21, 2019.
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survival in IgA nephropathy: a retrospective study. Am J Kidney Dis. 2007;49(6):763-775. Goto M, Wakai K, Kawamura T, Ando M, Endoh M, Tomino Y. A scoring system to predict renal outcome in IgA nephropathy: a nationwide 10-year prospective cohort study. Nephrol Dial Transplant. 2009;24(10):3068-3074. Feng CY, Xia YH, Wang WJ, et al. Persistent asymptomatic isolated hematuria in children: clinical and histopathological features and prognosis. World J Pediatr. 2013;9(2):163-168. Shen P, He L, Li Y, Wang Y, Chan M. Natural history and prognostic factors of IgA nephropathy presented with isolated microscopic hematuria in Chinese patients. Nephron Clin Pract. 2007;106(4):c157-c161. Yuste C, Rubio-Navarro A, Barraca D, et al. Haematuria increases progression of advanced proteinuric kidney disease. PLoS One. 2015;10(5):e0128575. Szeto CC, Lai FM, To KF, et al. The natural history of immunoglobulin A nephropathy among patients with hematuria and minimal proteinuria. Am J Med. 2001;110(6):434-437. Iwasaki C, Moriyama T, Tanaka K, Takei T, Nitta K. Effect of hematuria on the outcome of immunoglobulin A nephropathy with proteinuria. J Nephropathol. 2016;5(2):72-78. Tanaka K, Moriyama T, Iwasaki C, Takei T, Nitta K. Effect of hematuria on the outcome of IgA nephropathy with mild proteinuria. Clin Exp Nephrol. 2015;19(5):815-821. D'Amico G, Ferrario F, Colasanti G, Ragni A, Bestetti Bosisio M. IgA-mesangial nephropathy (Berger's disease) with rapid decline in renal function. Clin Nephrol. 1981;16(5):251257. Ibels LS, Gyory AZ. IgA nephropathy: analysis of the natural history, important factors in the progression of renal disease, and a review of the literature. Medicine (Baltimore). 1994;73(2):79-102. Coppo R, D'Amico G. Factors predicting progression of IgA nephropathies. J Nephrol. 2005;18(5):503-512. Sevillano AM, Gutierrez E, Yuste C, et al. Remission of hematuria improves renal survival in IgA nephropathy. J Am Soc Nephrol. 2017;28(10):3089-3099. Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009;150(9):604-612. D'Amico G. Natural history of idiopathic IgA nephropathy: role of clinical and histological prognostic factors. Am J Kidney Dis. 2000;36(2):227-237. Wyatt RJ, Julian BA. IgA nephropathy. N Engl J Med. 2013;368(25):2402-2414. KDIGO Working Group. KDIGO clinical practice guidelines for glomerulonephritis. Kidney Int Suppl. 2012;2(2):209-217. D'Amico G. Natural history of idiopathic IgA nephropathy and factors predictive of disease outcome. Semin Nephrol. 2004;24(3):179-196. Barbour SJ, Coppo R, Zhang H, et al. Evaluating a new international risk-prediction tool in IgA nephropathy. JAMA Intern Med. 2019;179(7):942-952. Gutierrez E, Zamora I, Ballarin JA, et al. Long-term outcomes of IgA nephropathy presenting with minimal or no proteinuria. J Am Soc Nephrol. 2012;23(10):1753-1760. Kim BS, Kim YK, Shin YS, et al. Natural history and renal pathology in patients with isolated microscopic hematuria. J Am Soc Nephrol. 2009;24(4):356-361. Zeng CH, Le W, Ni Z, et al. A multicenter application and evaluation of the oxford classification of IgA nephropathy in adult chinese patients. Am J Kidney Dis. 2012;60(5):812820.
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Original Investigation 37. Katafuchi R, Ninomiya T, Nagata M, et al. Validation study of Oxford classification of IgA nephropathy: the significance of extracapillary proliferation. Clin J Am Soc Nephrol. 2011;6(12): 2806-2813. 38. Coppo R, Troyanov S, Bellur S, et al. Validation of the Oxford classification of IgA nephropathy in cohorts with different presentations and treatments. Kidney Int. 2014;86(4):828-836. 39. Lv J, Zhang H, Wong MG, et al. Effect of oral methylprednisolone on clinical outcomes in patients with IgA nephropathy: the
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TESTING randomized clinical trial. JAMA. 2017;318(5):432442. 40. Rauen T, Eitner F, Fitzner C, et al. Intensive supportive care plus immunosuppression in IgA nephropathy. N Engl J Med. 2015;373(23):2225-2236. 41. Fellstrom BC, Barratt J, Cook H, et al. Targeted-release budesonide versus placebo in patients with IgA nephropathy (NEFIGAN): a double-blind, randomised, placebo-controlled phase 2b trial. Lancet. 2017;389(10084):2117-2127.
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Persistent Hematuria and Kidney Disease Progression in IgA Nephropathy: A Cohort Study Gui-zhen Yu, Ling Guo, Jin-feng Dong, Su-fang Shi, Li-jun Liu, Jin-wei Wang, Gui-li Sui, Xu-jie Zhou, Ying Xing, Hai-xia Li, Ji-cheng Lv, and Hong Zhang Rationale & Objective: Hematuria is the most typical presentation of immunoglobulin A nephropathy (IgAN); however, its role in disease progression is still controversial. This study aimed to evaluate the association of hematuria and progression of IgAN. Study Design: Retrospective cohort study. Setting & Participants: A cohort of 1,333 patients with IgAN treated at a Chinese referral hospital with a median follow-up of 45 months. Predictors: Microhematuria was evaluated in fresh urine using a fully automated urine particle analyzer (automated method) and urine sediment examination by a skilled examiner (manual method). Hematuria was characterized as a timevarying attribute; namely, average hematuria level was calculated for every 6-month period for each patient during follow-up. Remission was defined as average red blood cell count ≤5/high-power field (manual method) or ≤28 red blood cells/μL (automated method) during the first 6 months of follow-up. Outcomes: Composite event of 50% decline in estimated glomerular filtration rate or development of kidney failure. Analytical Approach: Multivariable causespecific hazards models to analyze the relationship between hematuria and the composite kidney disease progression event. Results: Time-varying hematuria during follow-up was an independent risk factor for the composite kidney disease progression event (HR, 1.46;
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95% CI, 1.13-1.87; P = 0.003). Hematuria remission during the 6 months after diagnosis was associated with a significantly lower rate of the composite kidney disease progression event (HR, 0.41; 95% CI, 0.28-0.61; P < 0.001). A significant interaction was detected between remission of proteinuria and remission of hematuria during the first 6 months (P < 0.001). The association between remission of hematuria and kidney disease progression was detectable (HR, 0.46; 95% CI, 0.32-0.68) within the subpopulation with persistent proteinuria (protein excretion > 1.0 g/d during the first 6 months), but not among patients whose proteinuria had remitted (HR, 0.64; 95% CI, 0.31-1.29; P = 0.2). The 2 techniques for hematuria evaluation were strongly and significantly linearly correlated (r = 0.948; P < 0.001), and results using these 2 methods were consistent.
Correspondence to J.-c. Lv ([email protected]) Am J Kidney Dis. XX(XX):110. Published online Month XX, XXXX. doi: 10.1053/ j.ajkd.2019.11.008 © 2020 by the National Kidney Foundation, Inc.
Limitations: A single-center retrospective study. Proportional hazards regression incorporating time-varying covariates may create time-varying confounding. The predictive value of reductions in hematuria was not directly evaluated. Conclusions: Level of hematuria was independently associated with kidney disease progression, whereas hematuria remission was associated with improved kidney outcomes in IgAN among patients with persistent proteinuria. Additionally, to monitor IgAN progression, automated methods to evaluate hematuria hold promise as a replacement for manual evaluation of urinary sediment.
mmunoglobulin A (IgA) nephropathy (IgAN) is the most common primary glomerular disease worldwide and a major cause of kidney failure requiring kidney replacement therapy (KRT).1-3 Clinical presentations in IgAN range from a benign incidental condition to rapidly progressive kidney failure, although most affected individuals develop chronic slowly progressive kidney injury and many patients will require KRT.4 Persistent proteinuria, hypertension, decreased glomerular filtration rate (GFR) at baseline, and the MEST-C scoring system are all established clinical and pathologic predictors of progression to kidney failure requiring KRT.5-12 Hematuria is the most typical presentation of IgAN. Approximately 70% to 100% of patients have microscopic hematuria, and macrohematuria often occurs after upper respiratory tract or intestinal infection.13 AJKD Vol XX | Iss XX | Month 2020
Complete author and article information provided before references.
However, the role of hematuria in IgAN progression is still controversial. In some studies, microhematuria has been shown to be a risk factor for kidney disease progression,14-21 whereas other studies demonstrated that it did not influence the risk for kidney failure.22-26 Reasons for the conflicting data include low quality of the studies, analysis of only 1 urinary sediment at the time of kidney biopsy, and lack of standardized measurements.13 Analysis of fresh urine sediment has interobserver variability, limiting the pooling of data from different laboratories, whereas 24-hour urine collection for red blood cell (RBC) count is also unsatisfactory given that it is cumbersome, expensive, and affected by collection errors.13 In addition, urine RBC counts vary over the short term much more than urine protein excretion. These problems have limited efforts to analyze the association of hematuria with long1
Original Investigation term outcomes. Recently, a small study indicated that time-averaged hematuria during follow-up, using a standard urine sediment analysis in one center, was strongly associated with poor renal prognosis.15,27 This finding needs further confirmation. In China, urine RBC measurement in fresh morning urine with a fully automated urine particle analyzer has been widely used. This method provides a standard measure without interobserver variability. In this study, we aimed to evaluate the association of microhematuria at baseline, hematuria during follow-up, and hematuria remission during the first 6 months with long-term kidney outcomes in a large cohort of patients with IgAN. We also compared automated analysis of hematuria with traditional urine sediment analysis. Methods Study Population A total of 1,333 patients with biopsy-proven IgAN diagnosed between 2003 and 2016 were selected from the prospective IgAN database at Peking University First Hospital, which is the first renal division and a renal reference center in China. Patients come from across China, especially from the north, and the center serves a population of 582 million people. All patients in the IgAN database were followed up regularly every 3 to 6 months, depending on the patients’ disease condition. Patients were diagnosed based on the dominant deposition of IgA in the glomerular mesangium by immunofluorescence, and patients with IgA vasculitis, systemic lupus, and other secondary IgAN were excluded. Written informed consent was obtained from all participants for use of the clinical and pathologic data in future studies when they entered the IgAN cohort. For this study, the protocol was reviewed and approved by the Ethics Committee of the institute but without getting additional informed consent. Data Collection and Pathologic Manifestations Examination of hematuria in urine was performed using 2 methods: measurement using a fully automated urine particle analyzer (UF-1000; Sysmex; automated method), and microscopic urine sediment examination performed by a skilled examiner (manual method). All urine samples were simultaneously analyzed using these 2 methods within 2 hours of collection at every visit and recorded as RBCs/μL in the automated method and RBCs/high-power field (HPF) in the manual method. Clinical data, including sex, age at the time of kidney biopsy, systolic/diastolic blood pressure, serum creatine level, hematuria, and 24-h urine protein excretion, were measured and recorded at the time of kidney biopsy and each visit. Pathologic lesions were evaluated according to the Oxford classification. Baseline (and the beginning of follow-up) was defined as the time of kidney biopsy. 2
Information was collected for medications used during follow-up, including renin-angiotensin-aldosterone system blockade, steroids, and other immunosuppressants. The use of immunosuppressive (IS) agents during the first 6 months after kidney biopsy was as follows: for patients with persistent proteinuria with protein excretion >1 g/ d and at least 3 to 6 months of optimal blood pressure control and receiving the maximum tolerated dose of renin-angiotensin-aldosterone system inhibitors, corticosteroid therapy was added. For patients with >25% crescents on biopsy and progressive GFR loss, we usually add steroids plus cyclophosphamide or mycophenolate mofetil. Definitions Hypertension was defined as systolic blood pressure ≥140 mm Hg, diastolic blood pressure ≥90 mm Hg, or taking antihypertension medication. Mean arterial pressure (MAP) was calculated as the sum of one-third of the pulse pressure and diastolic blood pressure. Because of the high variability of hematuria, a 6-month interval was used to calculate average hematuria. Average hematuria was determined for each patient for each 6-month block during follow-up that ended upon occurrence of a kidney disease progression event, death, or end of follow-up, whichever occurred first (baseline hematuria was included in the first block), and the mean of the average of hematuria calculated for every 6-month period was expressed using time-averaged hematuria. Time-averaged proteinuria and time-averaged MAP were calculated using the same method. Estimated GFR (eGFR) was calculated according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.28 Kidney failure was defined as eGFR persistently <15 mL/min/1.73 m2 or the need for KRT (including peritoneal dialysis, hemodialysis, or kidney transplantation). The composite kidney disease progression event was defined as 50% eGFR decline or kidney failure. Hematuria remission was defined as hematuria with ≤5 RBC/HPF using the manual method (consistent with a prior study27) or ≤28 RBC/μL using the automated method. Persistent no hematuria was defined as both baseline and time-varying hematuria ≤5 RBC/HPF using the manual method or ≤28 RBC/μL using the automated method. Persistent hematuria was defined as both baseline and time-varying hematuria > 5 RBC/HPF using the manual method or >28 RBC/μL using the automated method. Macroscopic hematuria was defined as urine that is visibly tea colored, cola colored, pink, or even red. Proteinuria remission was defined as proteinuria with protein excretion ≤1.0 g/d. Statistical Analyses Normally distributed variables are presented as mean ± standard deviation and compared using t test; nonnormally distributed data such as time-averaged hematuria and time-averaged proteinuria are expressed as median and interquartile range (IQR) and compared using AJKD Vol XX | Iss XX | Month 2020
Original Investigation Kruskal-Wallis test. All categorical data are summarized as frequency or percentage and tested using χ 2 test. Spearman correlation was used to analyze the correlation between the 2 techniques for hematuria measurement. Hematuria was statistically analyzed in 2 ways: as a continuous time-varying covariate and as a categorical variable characterizing its remission during the first 6 months following biopsy diagnosis. The relationships of time-varying hematuria and hematuria remission with the composite kidney disease progression outcome were estimated using cause-specific hazards models by treating death as competing events. Cumulative incidence function methods and Gray test were used to evaluate the association of kidney disease progression events and measures of hematuria remission. Statistical analysis was performed using IBM SPSS, version 24.0, and Stata software, version 14.0 (StataCorp). P < 0.05 (2 sided) was considered statistically significant. Results Clinical and Pathologic Data The cohort included 1,333 patients with IgAN, including 8 and 172 cases without baseline hematuria data using manual examination and automated analysis, respectively. During follow-up, participants had 429 automated urine examinations per 100 person-years and 457 per 100 person-years using the manual method. The Oxford classification was not done in 29 patients with fewer than 8 glomeruli on kidney biopsy. Clinical and pathologic characteristics are summarized in Table 1. At baseline there were 675 (50.64%) men and mean age was 35.07 ± 11.96 years. eGFR was 82.2 ± 30.6 mL/min/1.73 m2, median protein excretion was 1.31 (IQR, 0.68-2.54) g/d, and MAP was 93.73 ± 11.89 mm Hg, with 645 (48.39%) patients being hypertensive at baseline. Initial hematuria was 12.50 (IQR, 4.50-45.00) RBC/HPF using the manual method and 97.60 (IQR, 35.20-273.75) RBC/μL using the automated method. A total of 375 (28.13%) patients were recorded as having a history of macroscopic hematuria. The distributions of M1, E1, S1, T1-T2, and C1-C2 were 39.88%, 33.05%, 62.27%, 34.51%, and 58.67%, respectively. Median follow-up time was 45.00 (IQR, 24.00-80.00) months. Time-averaged hematuria was 11.31 (IQR, 4.1226.63) RBC/HPF using the manual method and 68.66 (IQR, 25.31-160.35) RBC/μL using the automated method. For time-averaged proteinuria, median protein excretion was 0.86 (IQR, 0.49-1.45) g/d. Overall, 207 (15.53%) patients reached the composite kidney disease progression event, including 123 kidney failure events. Characteristics of IgAN With Hematuria Remission As shown in Figure 1, the 2 techniques for hematuria measurement (manual and automated methods) displayed a strong linear correlation (r = 0.948; P < 0.001). The conversion coefficient between the 2 types of hematuria AJKD Vol XX | Iss XX | Month 2020
Table 1. Clinical and Pathologic Characteristics of Patients With IgAN Characteristic Baseline Male sex Age, y MAP, mm Hg Hypertension History of macroscopic hematuriaa Hematuria Manual method, RBC/HPFb Automated method, RBC/μLc Proteinuria, g/d eGFR, mL/min/1.73 m2 Oxford classificationd M1 E1 S1 T1-T2 C1-C2 Follow-up Follow-up duration, mo Time-averaged hematuria Manual method, RBC/HPF Automated method, RBC/μL Time-averaged proteinuria, g/d Time-averaged MAP, mm Hg Treatment with steroids and/or other IS agents during first 6 mo Outcome 50% decline in eGFR Kidney failure Composite outcome
Value 675 (50.64%) 35.07 ± 11.96 93.73 ± 11.89 645 (48.39%) 375 (28.13%) 12.50 [4.50-45.00] 97.60 [35.20-273.75] 1.31 [0.68-2.54] 82.2 ± 30.6 520 (39.88%) 431 (33.05%) 812 (62.27%) 450 (34.51%) 765 (58.67%) 45.00 [24.00-80.00] 11.31 [4.12-26.63] 68.66 [25.31-160.35] 0.86 [0.49-1.45] 90.21 ± 7.75 639 (47.94%) 190 (14.25%) 123 (9.23%) 207 (15.53%)
Note: Values for continuous variables given as mean ± standard deviation or median [interquartile range]; for categorical variables, as count (percent). The composite outcome was a composite kidney disease progression event defined as 50% eGFR decline or kidney failure. Abbreviations: C, presence of crescent; E1, endocapillary hypercellularity; eGFR, estimated glomerular filtration rate; HPF, high-powered field; IgAN, immunoglobulin A nephropathy; IS, immunosuppressive; M1, mesangial hypercellularity; MAP, mean arterial pressure; RBC, red blood cell; S, segmental glomerulosclerosis/adhesion; T, severity of tubular atrophy/interstitial fibrosis. a Six patients had macroscopic hematuria (urine visibly tea-colored, cola-colored, pink, or even red) at the time of kidney biopsy. b Eight cases were without baseline hematuria data using the manual examination method due to the loss of urine examination results; 359 (27.09%) patients had ≤5 RBC/HPF. c There were 172 cases without baseline hematuria data using the automated method because the automated method was not widely used at the time of their kidney biopsy; 241 (20.76%) patients had ≤28 RBC/μL. d Oxford classification was not performed in 29 patients because there were fewer than 8 glomeruli.
examination was 5.6, so hematuria remission was defined as ≤5 RBC/HPF using the manual method or ≤28 RBC/μL using the automated method. There were 253 (18.98%) patients with ≤28 RBC/μL during the first 6 months, as shown in Table 2, and they showed a higher prevalence of hypertension and lower prevalence of M1, E1, S1, C1, and C2 lesions. There was no significant difference in the prevalence of T lesions evaluated using the Oxford classification. As shown in Table S1, a total of 639 (47.94%) patients received steroids and/or other IS agents. In the IS therapy 3
Original Investigation
Figure 1. Correlation between the 2 techniques for hematuria measurement. Manual method: urine sediment examination with manual counting per high-power field (hpf) by a skilled professional nurse; automated method: fresh urine sample measurement using fully automated urine particle analyzer. The conversion coefficient between the 2 hematuria examinations is 5.6, which means that the value of hematuria in the manual examination method (RBC/hpf) multiplied by 5.6 equals the value of hematuria in automated measurement (RBC/μl). (A) Full results of time-averaged (TA)-hematuria. (B) Results shown in A, restricted to TA-hematuria values <500 RBC/hpf in the manual method and <4,000 RBC/μl in the automated method.
group, patients had more hematuria at baseline than the untreated group (median values of 117.55 [IQR, 42.20336.20] vs 81.20 [IQR, 30.90-226.95] RBC/μL; P < 0.001), but similar time-averaged hematuria during follow-up (67.32 [IQR, 24.83-159.90] vs 77.14 [IQR, 28.30-170.34] RBC/μL; P = 0.07). Risk Factors for Kidney Disease Progression in IgAN As shown in Table 3, in cause-specific hazards models, time-varying hematuria using the automated method was an independent risk factor for the composite kidney disease progression event in model 3 (hazard ratio [HR], 1.46; 95% confidence interval [CI], 1.13-1.87; P = 0.003) after adjustment for sex, age, time-varying proteinuria, time-varying MAP, baseline eGFR, use of steroids or other IS agents, and Oxford classification. Results were consistent when hematuria was measured using the manual method in model 3 (HR, 1.46; 95% CI, 1.15-1.86; P = 0.002). During the first 6 months, 31 (12.3%) patients in the hematuria remission group and 176 (16.3%) in the nonremission group reached the composite kidney disease progression event (P = 0.1; Table 2). Hematuria remission was significantly associated with reduced risk for the composite event (HR, 0.41; 95% CI, 0.28-0.61; P < 0.001) in model 3 (Table 4). Cumulative incidence functions analysis demonstrated that patients who had hematuria remission (evaluated using either method) had a lower incidence of the composite kidney disease progression event (automated method, P < 0.001; Fig 2A; manual method, P < 0.001; Fig 2B). Results were consistent when the end point was kidney failure (Table S2; Fig S1A and B). Because IS therapy might influence hematuria reduction (Table S1), a subgroup analysis was done between patients with and without IS 4
therapy. This showed no heterogeneity of the association of hematuria remission with kidney disease progression (HRs of 1.15 [95% CI, 0.93-1.42] and 1.34 [95% CI, 1.19-1.52] for the without and with IS therapy groups, respectively; P for interaction = 0.4). In the proportional hazard model that included baseline clinical and pathologic characteristics (Table S3), we found that baseline proteinuria, MAP, and S and T lesions were all independent risk factors. However, treatment with IS agents was not a protective factor in this model (HR, 1.70; 95% CI, 1.19-2.43; P = 0.004). When it was added in the model, time-varying hematuria was an independent risk factor (P < 0.001). Patients with persistent no hematuria had the lowest risk for kidney disease progression events, and the persistent hematuria group had the highest risk for kidney disease progression; the rest, defined as the intermittent hematuria group, had an intermediate level of risk (Fig 3). Association Between Hematuria Remission and Proteinuria Remission We further evaluated the roles of proteinuria and hematuria remission during the first 6 months on kidney disease progression. During the first 6 months, 138 (22.9%) patients in the proteinuria remission group (n = 602) and 191 (26.1%) in the non–proteinuria remission group (n = 731) had hematuria remission (P = 0.2). Hematuria remission during the first 6 months treated as a timevarying covariate significantly reduced the risk for the composite kidney disease progression event (HR, 0.46; 95% CI, 0.32-0.68; P < 0.001) in patients without proteinuria remission (protein excretion > 1.0 g/d) during the first 6 months. In patients with proteinuria remission during the first 6 months, hematuria remission during the first 6 months did not further improve kidney survival (HR, 0.64; 95% CI, 0.31-1.29; P = 0.2). AJKD Vol XX | Iss XX | Month 2020
Original Investigation Table 2. Characteristics by Hematuria Remission During the First 6 Months Hematuria Remission During First 6 mo Characteristics No. of patients Baseline Male sex Age, y MAP, mm Hg Hypertension Proteinuria, g/d History of macroscopic hematuriaa Hematuria Manual method, RBC/HPF Automated method, RBC/μL eGFR, mL/min/1.73 m2 Oxford classificationb M1 E1 S1 T1-T2 C1 C2 Follow-up Time-averaged hematuria Automated method, RBC/μL Manual method, RBC/HPF Time-averaged proteinuria, g/d Time-averaged MAP, mm Hg Treatment with steroids and/or other IS agents during first 6 mo Outcome 50% decline in eGFR Kidney failure Composite outcome
Yes (mean of ≤28 RBC/μL) 253
No (mean of >28 RBC/μL) 1,080
P
154 (60.91%) 35.16 ± 12.43 95.13 ± 11.60 137 (54.20%) 1.29 [0.70-2.72] 45 (17.8%)
521 (48.20%) 35.05 ± 11.85 93.41 ± 11.94 508 (47.00%) 1.24 [0.67-2.51] 330 (30.6%)
<0.001 0.9 0.04 0.04 0.08 <0.001
2.00 [0.50-4.00] 12.40 [7.70-23.10] 84.31 ± 30.44
20.00 [7.50-55.00] 130 [58.20-347.30] 81.65 ± 30.60
<0.001 <0.001 0.2
69 (28.5%) 61 (25.2%) 132 (54.5%) 71 (29.3%) 85 (35.1%) 4 (1.7%)
451 (42.5%) 370 (34.8%) 680 (64.0%) 379 (35.7%) 527 (49.6%) 149 (14.0%)
<0.001 0.004 0.006 0.06 <0.001 <0.001
11.98 [7.44-20.33] 1.95 [1.02-3.96] 0.86 [0.44-1.49] 91.71 ± 6.73 117 (46.2%)
93.87 [42.19-188.11] 14.78 [7.07-31.18] 0.86 [0.50-1.45] 89.84 ± 7.94 522 (48.3%)
<0.001 <0.001 0.7 <0.001 0.6
30 (11.9%) 15 (5.9%) 31 (12.3%)
160 (14.8%) 108 (10.0%) 176 (16.3%)
0.2 0.04 0.1
Note: Composite outcome defined as 50% eGFR decline or kidney failure. Abbreviations: C, presence of crescent; E1, endocapillary hypercellularity; eGFR, estimated glomerular filtration rate; HPF, high-powered field; IS, immunosuppressive; M1, mesangial hypercellularity; MAP, mean arterial pressure; RBC, red blood cell; S, segmental glomerulosclerosis/adhesion; T, severity of tubular atrophy/interstitial fibrosis. a Six patients presented macroscopic hematuria at the time of kidney biopsy. b Oxford classification was not performed in 29 patients because there were fewer than 8 glomeruli.
As shown in Figure 4, among patients with proteinuria (protein excretion > 1.0 g/d) during the first 6 months and based on time-varying hematuria, people in the hematuria remission group had a lower incidence of kidney disease progression events than those without hematuria remission (P < 0.001; Fig 4A). However, no significant differences were observed between the time-varying hematuria remission and nonremission groups in patients with proteinuria remission (P = 0.3; Fig 4B). An interaction analysis was performed, and statistically significant multiplicative interactions for kidney disease progression events were found between proteinuria remission and hematuria remission during the first 6 months (P for interaction < 0.001). Results were consistent when the end point was kidney failure (Fig S2). Risks for kidney failure in those treated with IS therapy among patients with hematuria or proteinuria remission are listed in Table S4; those without hematuria and proteinuria remission during AJKD Vol XX | Iss XX | Month 2020
the first 6 months had a higher proportion of kidney disease progression events. Discussion Although hematuria is a typical presentation of IgAN,4,29,30 its role in disease progression is still not well established. Persistent heavy proteinuria rather than hematuria is an indication for adding steroid therapy in our clinical practice, which is consistent with KDIGO (Kidney Disease: Improving Global Outcomes) guideline recommendations.31 However, in this cohort study with more than 1,000 participants and long-term follow-up, we found that the severity of persistent hematuria during follow-up was significantly associated with kidney disease progression. Hematuria remission was associated with reduced incidence of kidney disease progression events, especially in those without proteinuria remission, and hematuria remission 5
Original Investigation Table 3. Time-Varying Hematuria as a Risk Factor for the Composite Kidney Disease Progression Event in IgAN Hazard Ratio for Composite Outcomea (95% Confidence Interval); P Hematuria by automated method, per 1 unit greaterb Hematuria by manual method, per 1 unit greaterb
Unadjusted 1.50 (1.23-1.83); <0.001 1.51 (1.27-1.80); <0.001
Model 1 1.63 (1.33-2.01); <0.001 1.61 (1.34-1.93); <0.001
Model 2 1.44 (1.13-1.85); 0.004 1.45 (1.14-1.84); 0.002
Model 3 1.46 (1.13-1.87); 0.003 1.46 (1.15-1.86); 0.002
Note: Model 1 was adjusted for age and sex; sex was expressed as a dichotomous variable. Model 2 was adjusted for covariates in model 1 plus log-transformed proteinuria and mean arterial pressure, which were as time-varying covariates; eGFR; and Oxford classification (MEST-C scores). Model 3 was adjusted for covariates in model 2 plus steroids or other immunosuppressive agents. Use of treatment with steroids and/or other immunosuppressive agents was expressed as a dichotomous variable. Abbreviation: eGFR, estimated glomerular filtration rate; IgAN, immunoglobulin A nephropathy. a Composite outcome defined as 50% decline in eGFR or kidney failure.29 b Hematuria was measured at multiple visits, log-transformed value of the mean of every 6-month hematuria was treated as time-varying covariate.
was associated with greatly reduced risk for kidney failure. These results suggest the potential importance of therapy targeting hematuria remission in IgAN. Another key finding from this study was that hematuria evaluated using standard manual examination of urine sediment and automated analysis of a fresh urine sample were consistent, suggesting that a simple and rapid urine cytometry analysis could replace the traditional manual method for urine sediment examination in IgAN. Hematuria has arguably not been given sufficient attention in nephrology and is often considered benign in IgAN.13 In recent years, numerous cohort studies have evaluated risk factors for kidney failure in IgAN. Most studies found that proteinuria at baseline or during followup was the strongest risk factor for GFR decline or kidney failure, whereas hematuria was not a risk factor. In an early review, D’Amico32 evaluated risk factors for kidney failure in 23 cohorts with 5,507 patients with IgAN. Among them, 4 studies investigated baseline microhematuria and only 1 reported it as an independent risk factor for kidney failure.32 The conflicting results may relate to the analysis being based on only 1 urine sediment measurement at the time of kidney biopsy. The extent of erythrocyturia is often variable over the short term. For these reasons, a recent international risk prediction tool did not include hematuria as a key variable.33 However, time-averaged hematuria may be more valuable as a prognostic factor. In a large Chinese IgAN cohort, Le et al15 found that time-averaged hematuria was a strong independent risk factor for kidney failure. In a
recent European cohort of 112 patients and 14 years of follow-up, time-averaged hematuria was strongly associated with kidney failure, and hematuria remission had a significant favorable effect on IgAN outcomes.27 In another European cohort of 141 patients with IgAN and 108 months of follow-up, patients with hematuria and minimal or no proteinuria showed an excellent outcome with only 1 kidney disease progression event (doubling of serum creatinine level).34 Given the conflicting results, persistent hematuria was not included in the risk evaluation system in the KDIGO guideline. In this much larger cohort study with 1,333 participants, we confirmed that the extent of hematuria during follow-up was strongly associated with the development of kidney failure. Importantly, we found that the effect of microhematuria on kidney failure had an interaction with proteinuria, being much greater in patients with protein excretion > 1.0 g/d. In patients without proteinuria remission during follow-up, hematuria remission decreased the risk for kidney failure by 54%. For those with proteinuria remission (protein excretion < 1.0 g/d), persistent hematuria was not a risk factor for kidney disease progression. This result is consistent with prior studies finding that patients with IgAN with isolated hematuria or mild proteinuria had a benign course23,34,35 and highlights the importance of hematuria remission during the therapy of patients with IgAN, especially in those who do not achieve proteinuria remission. We noted that in our cohort 59% of patients had crescents on biopsy. Several studies have suggested that
Table 4. Association of Hematuria Remission and the Composite Kidney Disease Progression Event in IgAN Hazard Ratio for Composite Outcomea (95% Confidence Interval); P Hematuria remission by automated methodb Hematuria remission by manual methodb
Unadjusted 0.45 (0.32-0.64); <0.001 0.49 (0.35-0.68); <0.001
Model 1 0.42 (0.30-0.59); <0.001 0.45 (0.32-0.62); <0.001
Model 2 0.42 (0.28-0.62); <0.001 0.46 (0.31-0.67); <0.001
Model 3 0.41 (0.28- 0.61); <0.001 0.44 (0.30- 0.65); <0.001
Note: Model 1 was adjusted for age and sex; sex was expressed as a dichotomous variable. Model 2 was adjusted for covariates in model 1 plus log-transformed proteinuria and mean arterial pressure, which were as time-varying covariates; eGFR; and Oxford classification (MEST-C scores). Model 3 was adjusted for covariates in model 2 plus steroids or other immunosuppressive agents. Use of treatment with steroids and/or other immunosuppressive agents was expressed as a dichotomous variable. Abbreviations: eGFR, estimated glomerular filtration rate; IgAN, immunoglobulin A nephropathy; RBC, red blood cell. a Composite outcome defined as 50% decline in eGFR or kidney failure.29 b Hematuria remission was defined as the first 6-month average hematuria being ≤5 RBC/high-powered field (manual method) or ≤28 RBC/μL (automated method).
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Original Investigation
Figure 2. Cumulative incidence of the composite kidney disease progression outcome in patients with immunoglobulin A nephropathy according to magnitude of time-varying hematuria. (A) Hematuria examined using automated method. H-: time-varying hematuria ≤ 28 red blood cells (RBC)/μL; H+: time-varying hematuria > 28 RBC/μL. (B) Hematuria examined using manual method. H-: Time-varying hematuria ≤ 5 RBC/high-powered field (hpf); H+: time-varying hematuria > 5 RBC/hpf.
Asian patients more frequently have crescents than the European population. In a multicenter cohort with 1,026 Chinese participants, 48% of patients had crescents.36 In another study, Katafuchi et al37 reported that 63% of Japanese patients had crescents on biopsy. By contrast, in the VALIGA cohort with 1,147 patients from 13 European countries, only 11% of patients had crescents.38 The current study confirms the high prevalence of crescents in the Asian IgAN population.
Figure 3. Cumulative incidence of the composite kidney disease progression outcome in patients according to baseline hematuria and time-varying hematuria. Group 1 (persistent no hematuria), baseline hematuria ≤ 28 red blood cells (RBC)/μL, time-varying hematuria ≤ 28 RBC/μL. Group 2 (intermittent hematuria), baseline hematuria ≤28 RBC/μL, time-varying hematuria >28 RBC/μL; or baseline hematuria >28 RBC/μL, time-varying hematuria < 28 RBC/μL. Group 3 (persistent hematuria), baseline hematuria > 28 RBC/μL, time-varying hematuria > 28 RBC/μL.
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Until now, most interventional studies of IgAN have focused on proteinuria reduction and few have reported effects on hematuria. This study suggests that IS therapy may reduce hematuria. This finding is consistent with those from the TESTING trial, in which steroid therapy resulted in hematuria remission in 58% of patients.39 In our study, we found that patients with hematuria remission had a more severe disease profile at baseline. This observation may be influenced by the treatment regimen because this group of patients was more prone to receive steroid therapy. Other new therapies, including budesonide modified-released capsules and complement activation inhibition therapy, should also be evaluated for their effect on hematuria.40,41 The standard hematuria evaluation is urine sediment analysis, which is time-consuming and requires specific expertise. Unlike the consistency of proteinuria measurement when using a standard method, the interobserver variance among different laboratories for hematuria evaluation limits the study of hematuria on kidney disease progression, especially in multicenter studies. A key finding from this study was that the fresh urine sample automated method provides a simple and easy hematuria evaluation method in IgAN, which can avoid the interobserver bias inherent in urine sediment, whether within the same laboratory or between different laboratories. In this cohort study, we found that the extent of hematuria was a moderate risk factor for kidney disease progression. The strength of this study includes the large sample size and long-term follow-up, with a large number of kidney failure events observed. This provided good study power to detect the association of hematuria with hard end points. The association is independent of MESTC, time-varying proteinuria, and other established clinical predictors. 7
Original Investigation
Figure 4. Cumulative incidence of the composite kidney disease progression outcome according to the magnitude of time-varying hematuria in patients with and without proteinuria remission during the first 6 months. Patients (A) without and (B) with proteinuria remission during the first 6 months. H-: time-varying hematuria ≤ 28 red blood cells (RBC)/μL; H+: time-varying hematuria > 28 RBC/μL.
Limitations of this study were as follows: single-center design with a single ethnicity, using time-varying covariates in proportional hazards models may create timevarying confounding, and the predictive value of reductions in hematuria was not directly evaluated. Thus, the findings still need further confirmation. In conclusion, in this large cohort study, we confirmed that the extent of microhematuria during follow-up was independently associated with kidney disease progression in IgAN, suggesting it should be included in the risk scoring system for predicting renal outcomes of IgAN. Hematuria remission significantly reduced the incidence of kidney disease progression events. Importantly, we found this effect much greater in patients without proteinuria remission, for whom hematuria remission during therapy may reduce the risk for kidney failure. For those with no or mild proteinuria, persistent microhematuria did not significantly increase the risk for kidney failure. These findings may help inform clinical management decisions. Hematuria evaluated using the fresh urine automated method had similar associations with renal outcomes as those evaluated using the manual urine sediment method. When it is confirmed as a standard and rapid method, the fresh urine automated method should be considered for IgAN clinical practice in the future. Supplementary Material Supplementary File (PDF) Figure S1: Cumulative incidence of kidney failure in IgAN patients, by magnitude of time-varying hematuria. Figure S2: Cumulative incidence of kidney failure, by magnitude of time-varying hematuria in patients with and without proteinuria remission during the first 6 months. Table S1: Characteristics of groups with or without treatment of steroids and/or other immunosuppressive agents. 8
Table S2: Risk of kidney failure with hematuria as a time-varying variable and dichotomous grouping. Table S3: Factors associated with the composite kidney disease progression outcome in univariate and multivariate analyses. Table S4: Risk of kidney failure in those treated with immunosuppressive therapy, by hematuria or proteinuria remission.
Article Information Authors’ Full Names and Academic Degrees: Gui-zhen Yu, PhD, Ling Guo, MD, Jin-feng Dong, BS, Su-fang Shi, MD, Li-jun Liu, MD, Jin-wei Wang, PhD, Gui-li Sui, BS, Xu-jie Zhou, PhD, Ying Xing, MD, Hai-xia Li, PhD, Ji-cheng Lv, MD, PhD, and Hong Zhang, MD, PhD. Authors’ Affiliations: Renal Division, Peking University First Hospital (G-zY, LG, J-fD, S-fS, L-jL, J-wW, G-lS, X-jZ, J-cL, HZ); Peking University Institute of Nephrology (G-zY, LG, J-fD, S-fS, L-jL, J-wW, G-lS, X-jZ, J-cL, HZ); Key Laboratory of Renal Disease, Ministry of Health of China (G-zY, LG, J-fD, S-fS, L-jL, J-wW, G-lS, X-jZ, J-cL, HZ); Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Peking University, Ministry of Education, China (G-zY, LG, J-fD, S-fS, L-jL, J-wW, G-lS, X-jZ, J-cL, HZ); and Clinical Laboratory, Peking University First Hospital, Peking, China (YX, H-xL). Address for Correspondence: Ji-cheng Lv, MD, PhD, Renal Division, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China. E-mail: jichenglv75@ gmail.com Authors’ Contributions: Research idea and study design: J-cL; data acquisition: G-zY, J-fD, G-lS, LG; data analysis/interpretation: G-zY, J-wW; statistical analysis: G-zY, J-wW; supervision or mentorship: J-cL, S-fS, L-jL, X-jZ, YX, H-xL, HZ. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. Support: The work was supported by grants from the National Natural Science Foundation of China (81670649), the Capital Health Research and Special Development, China (201824073). the Natural Science Foundation for Innovation Research Groups of China (81621092), the National Key Research and Development Program of China (2018YFC1314004), and grants from the AJKD Vol XX | Iss XX | Month 2020
Original Investigation Science and Technology Project of Beijing, China (D18110700010000). The funders had no role in study design; collection, analysis, and interpretation of data; writing the report; and the decision to submit the report for publication.
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Financial Disclosure: The authors declare that they have no other relevant financial interests. Peer Review: Received April 25, 2019. Evaluated by 3 external peer reviewers, with direct editorial input from a Statistics/Methods Editor, an Associate Editor, and the Editor-in-Chief. Accepted in revised form November 21, 2019.
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