Article

Soluble Urokinase-Type Plasminogen Activator Receptor in Black Americans with CKD

Shengyuan Luo ,1,2 Josef Coresh,1,2,3 Adrienne Tin,1,2 Casey M. Rebholz,1,2 Teresa K. Chen,4 Salim S. Hayek ,5 Melissa Tracy,6 Michael S. Lipkowitz,7 Lawrence J. Appel,1,2,3 Andrew S. Levey,8 Lesley A. Inker,8 Jochen Reiser,9 and Morgan Erika Grams1,2,4

Abstract 1 Background and objectives Black Americans with and without APOL1 kidney disease risk variants face high risk Department of Epidemiology, Johns of ESKD. Soluble urokinase-type plasminogen activator receptor (suPAR), a circulating signaling protein and Hopkins Bloomberg marker of immune activation, constitutes a promising biomarker of CKD-associated risks. We aimed to quantify School of Public the associations between serum suPAR concentration and adverse outcomes in Black Americans with and without Health, Baltimore, 2 APOL1 kidney disease risk variants, over and above iodine-125 iothalamate measured GFR and proteinuria. Maryland; Welch Center for Prevention, Epidemiology, and Design, setting, participants, & measurements Using data from the African-American Study of Kidney Disease Clinical Research, and Hypertension, a multicenter clinical trial followed by a cohort phase with a median total follow-up of 9.7 years 3Division of General (interquartile range, 6.5–10.9 years), we examined the associations of suPAR with CKD progression (defined Internal Medicine, as doubling of serum creatinine or ESKD), ESKD, worsening proteinuria (defined as pre-ESKD doubling of Department of $ Medicine, and 24-hour urine protein-to-creatinine ratio to 220 mg/g), and all-cause death. 4Division of Nephrology, Results At baseline, the median suPAR was 4462 pg/ml, mean measured GFR was 46 ml/min per 1.73 m2,and Department of median 24-hour urine protein-to-creatinine ratio was 80 mg/g. After controlling for baseline demographics, Medicine, Johns Hopkins University, randomization arm, GFR, proteinuria, APOL1 risk status, and clinical risk factors, there was a 1.26-times higher Baltimore, Maryland; risk for CKD progression per SD higher baseline log-transformed suPAR (hazard ratio [HR], 1.26; 95% confidence 5Division of interval [95% CI], 1.11 to 1.43; P,0.001). Higher suPAR was also independently associated with risk of ESKD (HR, Cardiology, Emory , University, Atlanta, 1.36; 95% CI, 1.17 to 1.58; P 0.001) and death (HR, 1.25; 95% CI, 1.08 to 1.45; P=0.003). suPAR was only associated 6 with worsening proteinuria in patients with two APOLI risk alleles (HR, 1.46; 95% CI, 1.08 to 1.99; P=0.02). Georgia; Division of Cardiology and 9Department of Conclusions Higher suPAR was associated with various adverse outcomes in Black Americans with CKD, with Medicine, Rush and without APOL1 kidney disease risk variants, independently of proteinuria and GFR. University Medical Center, Chicago, Clin J Am Soc Nephrol 13: 1013–1021, 2018. doi: https://doi.org/10.2215/CJN.13631217 Illinois; 7Division of Nephrology, Georgetown University, Introduction Observational studies in primarily European popula- Washington, DC; More than 4% of deaths worldwide are attributable to tions suggest that suPAR may be associated with and 8Division of CKD (1). Despite the extensive resources devoted to incident CKD (7–10). There is less evidence of asso- Nephrology, Tufts CKD treatment, the incidence of ESKD remains high, ciations between suPAR and adverse outcomes in Medical Center, Boston, Massachusetts particularly among Black Americans. Higher ESKD Black Americans with CKD. We recently found faster risk in Black Americans is explained in part by the suPAR-associated eGFR decline in Black Americans Correspondence: Dr. prevalence of a high-risk genotype (two APOL1 risk with two APOL1 risk alleles compared with zero or Morgan E. Grams, alleles), present in 13% of Black Americans; however, one risk allele in two populations (11). However, Welch Center for the majority of ESKD cases occur in people with the evidence of associations of suPAR with eGFR decline Prevention, low-risk genotype (2,3). Novel biomarkers and treat- in Black Americans with the low-risk genotype was Epidemiology, and Clinical Research, ment targets are needed in order to ameliorate adverse mixed, and the associations between suPAR and Room 2-638, 2024 outcomes in CKD among individuals with and with- clinical outcomes, independent of measured GFR East Monument Street, out APOL1 risk alleles. and proteinuria, were not explored. Baltimore, MD 21205. Soluble urokinase-type plasminogen activator re- Using a baseline measurement of suPAR in the Email: mgrams2@ ceptor (suPAR), a cell membrane glycosylphosphati- African-American Study of Kidney Disease and Hy- jhmi.edu dylinositol-anchored protein expressed in many cell pertension (AASK), a clinical trial of Black Americans types (e.g., podocytes and immune, endothelial, and with hypertension-attributed kidney disease, mea- bone marrow cells), is released into the circulation sured GFR, per-protocol proteinuria assessments, during immune activation. Circulating suPAR par- and APOL1 genotyping, we characterized the associ- ticipates in cell adhesion, migration, and survival, ations between suPAR and CKD progression, ESKD, and may mediate podocyte injury in FSGS (4–6). worsening proteinuria, and all-cause death. www.cjasn.org Vol 13 July, 2018 Copyright © 2018 by the American Society of Nephrology 1013 1014 Clinical Journal of the American Society of Nephrology

Materials and Methods analyses. By suPAR quartile, continuous variables were Study Design summarized using means with SD (for symmetrically In brief, AASK was a three3two factorial design, distributed variables), or medians and interquartile ranges randomized, controlled trial evaluating the effects of three (for variables with skewed distributions). Categorical antihypertensives (ramipril, metoprolol, and amlodipine) variables were summarized using proportions. Baseline and two BP control goals (mean arterial pressure #92 mm characteristics were compared using tests for linear trends. Hg versus 102–107 mm Hg) in slowing CKD progression Cumulative incidences of CKD progression and ESKD (12,13). Participants were self-identified Black Americans were plotted using competing risks regression accounting (18–70 years of age) with hypertension-attributed CKD for death as the competing event, and for worsening (measured GFR between 20 and 65 ml/min per 1.73 m2). proteinuria, accounting for ESKD and death as competing After completion of the trial (in April of 2002), 691 events. Kaplan–Meier functions were used to plot cumu- participants without ESKD entered the cohort phase (14). lative mortality. Cox proportional hazards regression was Participants were censored at loss to follow-up or the end used to assess the associations of baseline suPAR (log- of study (in June of 2007). Informed consent was obtained transformed and scaled to 1 SD) with outcomes. Multivari- from all participants. The study protocol was approved by able models included baseline demographics (age and sex), the institutional review boards at Johns Hopkins Univer- trial arms (BP control goal and trial medication), baseline sity and all other participating institutions. kidney measures (measured GFR and proteinuria), CRP, APOL1 risk status (modeled as a three-category variable: zero or one risk variant, two risk variants, and not Measurement of Exposure Blood samples were taken at enrollment and stored at genotyped), history of heart disease, and history of smok- 280°C. Serum suPAR concentrations were measured in 955 ing. The assumption of a log-linear relationship between participants with available samples, using an ELISA suPAR and outcomes was tested by modeling suPAR using fi (suPARnostic kit; ViroGates, Copenhagen, Denmark), in cubic splines, and comparing model t with the original 2017. In a blind duplicate study (n=21), the mean coefficient model using Akaike information criterion. Interactions of variation was 3.9% (median, 2.3%), and the correlation between kidney function and suPAR on outcomes were (r) was 0.97. assessed by including an interaction term of baseline GFR and suPAR in the regression models, with Wald test for statistical significance. To assess consistency by APOL1 risk Outcomes, Kidney Function Measures, and Risk Factors status, analyses were repeated with an interaction term by fi The primary outcome was CKD progression, de ned APOL1 risk status (zero or one risk allele versus two risk as doubling of serum creatinine from baseline or ESKD alleles versus not genotyped). The ability of suPAR to (requiring dialysis or kidney transplantation). Secondary improve risk discrimination when added to prediction outcomes included incident ESKD, worsening proteinuria models was assessed using Harrell C-statistic. To assess the (pre-ESKD doubling of 24-hour urine protein-to-creatinine robustness of results, the associations of baseline suPAR $ ratio [UPCR] to 220 mg/g), and all-cause mortality. with CKD progression, worsening proteinuria, and ESKD Baseline GFR was measured by urinary clearance of iodine- were tested using multivariable competing risks regression 125 iothalamate. Serum creatinine was collected every 6 as sensitivity analyses. A two-sided a value of 0.05 was months and measured with an autoanalyzer (AASK Cen- chosen as the cut-off for statistical significance. All statis- tral Biochemistry Laboratory, Cleveland Clinic, Cleveland, tical analyses were performed using Stata 14.0 (StataCorp, OH), 24-hour urine creatinine and protein were measured College Station, TX). yearly using the modified Jaffe reaction and the pyrogallol red technique, respectively, and used to calculate UPCR (15,16). Serum C-reactive protein (CRP) was measured by Results nephelometry using a high-sensitivity assay (Dade Behring Baseline Characteristics BN II System). Details about APOL1 genotyping among Among 955 AASK participants with available serum AASK participants have been described in detail previ- samples for suPAR quantification, the median suPAR con- ously (17,18). Briefly, the APOL1 risk variants (G1 centration was 4462 pg/ml (interquartile range, 3425–5923 [rs73885319 or rs60910145] and G2[rs71785313]) were de- pg/ml). The mean baseline age was 55 years, the mean termined using an ABI TaqMan assay (Applied Biosystems, measured GFR was 46 ml/min per 1.73 m2, and the median Foster City, CA) in 607 participants who gave consent to 24-hour UPCR was 80 mg/g (Table 1). When stratified by genotyping. Participants with zero or one APOL1 risk baseline suPAR quartile, participants with higher suPAR alleles were classified as low risk, and those with two risk were more often women, had higher prevalence of current alleles were classified as high risk. Other variables collected smoking, longer history of hypertension, lower diastolic at baseline included age, sex, duration of hypertension, BP, higher CRP, higher proteinuria, and lower GFR (all , family history of ESKD, body mass index, history of Ptrend 0.05). The presence of two APOL1 risk alleles was smoking, systolic and diastolic BP, and history of heart not statistically significantly different by suPAR quartile. disease. Baseline suPAR and CKD Progression Statistical Analyses During a median follow-up time of 7.4 years, 363 (38.0%) Continuous variables with right-skewed distributions participants developed CKD progression. The incidence of (baseline suPAR, CRP, and UPCR) were logarithmically CKD progression was 2, 4, 7, and 10 per 100 person-years, transformed on a natural log scale in continuous statistical for the first, second, third, and fourth quartile of baseline Clin J Am Soc Nephrol 13: 1013–1021, July, 2018 suPAR in Black Americans with CKD, Luo et al. 1015

Table 1. Baseline characteristics of participants in the African-American Study of Kidney Disease and Hypertension, overall and by serum suPAR concentration

Baseline Serum suPAR Concentration, pg/ml Entire Cohort Variables (n=955) Below 3425 3425–4462 4462–5923 Above 5923 (n=239) (n=239) (n=239) (n=238)

Age, yr, mean (SD) 55 (11) 56 (10) 54 (11) 54 (11) 54 (10) Womena, N (%) 373 (39) 76 (32) 76 (32) 102 (43) 119 (50) Goal of BP control in AASK MAP#92 mm Hg, N (%) 472 (49) 108 (45) 129 (54) 121 (51) 114 (48) MAP=102–107 mm Hg, 483 (51) 131 (55) 110 (46) 118 (49) 124 (52) N (%) Trial medication in AASK Ramipril, N (%) 373 (39) 85 (36) 96 (40) 100 (42) 92 (39) Metoprolol, N (%) 394 (41) 106 (44) 93 (39) 95 (40) 100 (42) Amlodipine, N (%) 188 (20) 48 (20) 50 (21) 44 (18) 46 (19) Years with hypertensiona, 14 (10) 14 (9) 13 (10) 14 (10) 16 (11) mean (SD) Familial diagnosis of ESKD, 124 (13) 32 (13) 27 (11) 32 (13) 33 (14) N (%) Body mass index, kg/m2, 30.7 (6.6) 29.7 (5.3) 31.0 (6.4) 31.0 (6.7) 31.0 (7.8) mean (SD) History of smokinga Never smoker, N (%) 398 (42) 115 (48) 102 (43) 102 (43) 79 (33) Past smoker, N (%) 279 (29) 76 (32) 70 (29) 71 (30) 62 (26) Current smoker, N (%) 278 (29) 48 (20) 67 (28) 66 (28) 97 (41) Systolic BP, mm Hg, 150 (24) 151 (24) 150 (25) 151 (24) 149 (24) mean (SD) Diastolic BP, mm Hga, 95 (15) 97 (14) 95 (15) 96 (16) 94 (13) mean (SD) History of heart disease, 491 (51) 124 (52) 132 (55) 112 (47) 123 (52) N (%) C-reactive protein, mg/La, 4.3 (1.9, 9.1) 3.1 (1.4, 7.0) 3.9 (1.8, 8.9) 5.2 (2.3, 9.7) 5.8 (2.7, 10.8) median (IQR) APOL1 risk statusb Low risk: zero or one risk 464 (76) 127 (83) 119 (76) 115 (72) 103 (75) allele, N (%) High risk: two risk alleles, 143 (24) 26 (17) 38 (24) 45 (28) 34 (25) N (%) 24-h UPCR, mg/ga,median 80 (28, 359) 38 (21, 101) 57 (26, 230) 118 (40, 504) 201 (56, 735) (IQR) Measured GFR, ml/min per 46 (13) 53 (11) 50 (11) 43 (13) 38 (12) 1.73 m2a,mean(SD) suPARa, pg/ml, median (IQR) 4462 (3425, 5923) 2831 (2265, 3157) 3977 (3703, 4260) 5141 (4817, 5463) 7472 (6505, 8669)

suPAR, soluble urokinase-type plasminogen activator receptor; AASK, the African American Study of Kidney Disease and Hypertension; MAP, mean arterial pressure; UPCR, 24-hour urine protein-to-creatinine ratio; IQR, 25th and 75th quartile. aP for trend ,0.05. bA total of 348 participants did not have genotyping data.

suPAR, respectively. The unadjusted risk for CKD pro- status (Pinteraction=0.73; Table 3). When suPAR was added to a gression was significantly higher with higher quartiles of prediction model including age, sex, baseline GFR, pro- suPAR (P,0.001; Figure 1A). The continuous association teinuria, and history of heart disease, the risk discrimination remained statistically significant after adjustment for age, for CKD progression was not significantly improved (Harrell sex, and trial arm (hazard ratio [HR], 1.71 per SD higher; C-statistic, 0.800 versus 0.797; difference, 0.003; 95% CI, 20.01 95% confidence interval [95% CI], 1.54 to 1.90; P,0.001; to 0.01; P=0.16). In fully adjusted multivariable competing Table 2), and adjustment for baseline GFR and proteinuria risks regression accounting for death as the competing event, (HR, 1.29; 95% CI, 1.14 to 1.46; P,0.001). Further adjust- baseline suPAR remained associated with CKD progression ment for baseline clinical risk factors (history of heart (sub-HR, 1.18; 95% CI, 1.03 to 1.34; P=0.01). disease, history of smoking, CRP, and APOL1 risk status) did not alter the association (HR, 1.26; 95% CI, 1.11 to 1.43; Baseline suPAR and ESKD P,0.001). There was no significant interaction between During a median follow-up time of 8.9 years, 271 (28.4%) baseline GFR and suPAR (Pinteraction=0.52). The fully ad- participants developed ESKD. The incidence of ESKD was justed association between suPAR and CKD progression 1, 3, 5, and 8 per 100 person-years for the first, second, was log-linear (Figure 2A), and was consistent by APOL1 risk third, and fourth quartile of baseline suPAR, respectively. 1016 Clinical Journal of the American Society of Nephrology

Figure 1. | The unadjusted cumulative incidences of CKD progression, ESKD, worsening proteinuria, and mortality were higher with higher suPAR quartiles in the African-American Study of Kidney Disease and Hypertension. (A) Levels of baseline serum suPAR concentration and cumulative incidence of CKD progression. (B) Levels of baseline serum suPAR concentration and cumulative incidence of ESKD. (C) Levels of baseline serum suPAR concentration and cumulative incidence of worsening proteinuria. (D) Levels of baseline serum suPAR concentration and cumulative incidence of all-cause mortality. CKD progression was defined as doubling of serum creatinine from baseline or ESKD (requiring dialysis or kidney transplantation). Worsening proteinuria was defined as pre-ESKD doubling of 24-hour UPCR to $220 mg/g.

The unadjusted risk for incident ESKD was significantly with follow-up measures of proteinuria. The incidence of higher with higher suPAR quartiles (P,0.001; Figure 1B). worsening proteinuria was 6, 7, 11, and 12 per 100 person- In continuous analysis, the risk for ESKD was higher per years for the first, second, third, and fourth quartile of SD higher log-transformed baseline suPAR both before baseline suPAR, respectively. The unadjusted risk for (HR, 1.91; 95% CI, 1.69 to 2.16; P,0.001) and after worsening proteinuria was higher with higher suPAR adjustment for kidney measures and clinical risk factors quartiles (P=0.007; Figure 1C). In continuous analysis, , (HR, 1.36; 95% CI, 1.17 to 1.58; P 0.001). There was no there was 1.21-times higher risk for worsening protein- fi signi cant interaction between baseline GFR and suPAR uria per SD higher log-transformed baseline suPAR, (Pinteraction=0.65). The fully adjusted association between adjusting for age, sex, and trial arm (HR, 1.21; 95% CI, suPAR and ESKD was log-linear (Figure 2B), and was 1.10 to 1.34; P,0.001), but no association was found consistent by APOL1 risk status (Pinteraction=0.60). When after adjustment for kidney measures and clinical risk suPAR was added to a prediction model including age, sex, factors (HR, 1.07; 95% CI, 0.96 to 1.19; P=0.23). Similarly, baseline GFR, proteinuria, and history of heart disease, the fully adjusted competing risks regression accounting risk discrimination for ESKD was statistically significantly for ESKD and death as competing events showed no improved (Harrell C-statistic, 0.855 versus 0.851; differ- association (sub-HR, 1.01; 95% CI, 0.90 to 1.13; P=0.88), ence, 0.004; 95% CI, 0.00 to 0.01; P=0.05). Fully adjusted and there was no significant interaction between baseline competing risks regression accounting for death as the com- GFR and suPAR (P =0.83). However, the associa- peting event showed similar association between suPAR interaction tion was stronger among those with two APOL1 risk and ESKD (sub-HR, 1.27; 95% CI, 1.09 to 1.49; P=0.003). alleles (HR, 1.46; 95% CI, 1.08 to 1.99; P=0.02) compared with those with zero or one APOL1 risk alleles (HR, 1.12; Baseline suPAR and Worsening Proteinuria 95% CI, 0.97 to 1.31; P=0.13) and those who were not During a median follow-up of 4.7 years, worsening genotyped (HR, 0.81; 95% CI, 0.60 to 1.08; P=0.15) proteinuria occurred in 448 (41.7%) of the 951 participants (Pinteraction=0.008). Clin J Am Soc Nephrol 13: 1013–1021, July, 2018 suPAR in Black Americans with CKD, Luo et al. 1017

Table 2. Results of Cox regression analyses assessing the associations between baseline serum suPAR concentration and CKD progression, ESKD, worsening proteinuria, all-cause mortality in the African-American Study of Kidney Disease and Hypertension

Model 1: Adjusted for Model 2: Model 1 Model 3: Model 2Additionally Demographics and AASK Additionally Adjusted for Adjusted for Clinical Risk No. of Outcome Trial Arm Kidney Measures Factors events HRa (95% CI) P Value HRa (95% CI) P Value HRa (95% CI) P Value

CKD progression 363 1.71 (1.54 to 1.90) ,0.001 1.29 (1.14 to 1.46) ,0.001 1.26 (1.11 to 1.43) ,0.001 ESKD 271 1.91 (1.69 to 2.16) ,0.001 1.34 (1.15 to 1.55) ,0.001 1.36 (1.17 to 1.58) ,0.001 Doubling of UPCR 448 1.21 (1.10 to 1.34) ,0.001 1.06 (0.96 to 1.18) 0.26 1.07 (0.96 to 1.19) 0.23 to $220 mg/g All-cause mortality 218 1.50 (1.32 to 1.70) ,0.001 1.32 (1.14 to 1.53) ,0.001 1.25 (1.08 to 1.45) 0.003

Adjusting variables include demographics (age and sex), AASK trial arm (BP control goal and trial medication), kidney measures (UPCR and measured GFR), and clinical risk factors (history of heart disease, history of smoking, C-reactive protein, and APOL1 risk status) at baseline. CKD progression was defined as doubling of serum creatinine from baseline or ESKD (requiring dialysis or kidney transplantation). Worsening proteinuria was defined as pre-ESKD doubling of 24-hour UPCR to $220 mg/g. suPAR, soluble urokinase-type plasminogen activator receptor; AASK, the African American Study of Kidney Disease and Hypertension; HR, hazard ratio; 95% CI, 95% confidence interval; UPCR, 24-hour urine protein-to-creatinine ratio. aHR per SD higher log-transformed baseline serum suPAR concentration.

Baseline suPAR and All-Cause Mortality measurement into clinical practice and determine if suPAR During a median follow-up of 9.7 years, 218 (22.8%) plays a causal role in disease progression in humans. participants died, of whom 153 died without having ESKD. Originally proposed as the circulating “permeability The incidence of death was 2, 2, 2, and 4 per 100 person- factor” in FSGS (4), suPAR has attracted attention in years for the first, second, third, and fourth quartile of increasingly broad populations. Several studies have dem- baseline suPAR, respectively. The highest mortality risk onstrated cross-sectional associations of suPAR with pro- was observed in the highest suPAR quartile (P,0.001; teinuria or reduced GFR in the settings of minimal change Figure 1D). In continuous analysis, per SD higher log- disease, membranous nephropathy, IgA nephropathy, and transformed baseline suPAR, the risk for mortality adjusted diabetic nephropathy (19–21). A few studies also investi- for age, sex, and trial arm was 1.5-times higher (HR, 1.50; gated the association between suPAR and long-term 95% CI, 1.32 to 1.70; P,0.001). The effect size was not kidney outcomes in populations with glomerular disease, substantially altered after full adjustment for other clinical cardiovascular disease, or in the general population (7–10). risk factors (HR, 1.25; 95% CI, 1.08 to 1.45; P=0.003). There Results from these studies, however, have been somewhat was no significant interaction between baseline GFR and inconsistent. Zhao et al. (9) found no association between suPAR (Pinteraction=0.32). The association between suPAR plasma suPAR and progression of IgA nephropathy among and mortality was log-linear (Figure 2D) and not signifi- 569 Chinese patients followed for a median of 52 months. cantly different by APOL1 risk status (Pinteraction=0.11). Spinale et al. (8) found higher proteinuria and greater eGFR When suPAR was added to a prediction model including decline with higher suPAR, but no association between age, sex, baseline GFR, proteinuria, and history of heart suPAR and a composite outcome of 50% decline in eGFR or disease, the risk discrimination for all-cause mortality was ESKD among 241 patients with glomerular disease fol- not significantly improved (Harrell C-statistic, 0.681 versus lowed for a median of 17 months. Compared with AASK, 0.674; difference, 0.007; 95% CI, 20.01 to 0.02; P=0.34). both studies included younger participants (mean baseline age ,40 years) with higher GFR (mean baseline eGFR .80 ml/min per 1.73 m2) and relatively short follow-up Discussion (median follow-up ,5 years). Higher suPAR was associ- In this prospective study of Black Americans with CKD, ated with incident CKD in the Emory Cardiovascular we demonstrated strong associations of higher baseline Biobank (EmCAB) cohort (n=3683, mean age 63 years, suPAR with risks for CKD progression, ESKD, and all- mean eGFR of 73 ml/min per 1.73 m2) (7) and a general cause mortality, which were independent of baseline European population-based cohort (n=5381, mean age 58 measured GFR and proteinuria. Higher suPAR was also years, mean eGFR of 76 ml/min per 1.73 m2) (10). Our work associated with worsening proteinuria, but only among expands on these studies by investigating, in a study with participants with two APOL1 risk alleles. These results long-term follow-up, the association of suPAR with mul- suggest that suPAR is an independent marker of risk in tiple adverse outcomes in Black Americans with existing CKD, regardless of APOL1 status, and suggest the possi- CKD, with and without adjustment for measured GFR and bility of a synergistic action between suPAR and APOL1 proteinuria, and with and without APOL1 kidney disease risk alleles to promote proteinuria. Because suPAR assays risk variants. are commercially available and relatively inexpensive, our Basic science studies investigating suPAR and kidney findings may have clinical implications. Future research disease have focused on the ability of suPAR to induce should evaluate the utility of incorporating suPAR proteinuria and histologic changes recapitulating kidney 1018 Clinical Journal of the American Society of Nephrology

Figure 2. | The adjusted HRs for CKD progression, ESKD, and mortality increased log-linearly with higher levels of baseline suPAR in the African-American Study of Kidney Disease and Hypertension. Solid lines indicate adjusted HRs. Shaded areas indicate 95% CIs. Dashed lines indicateupperandlower limitsof95% CIs. (A) AdjustedHRs forCKD progression plottedagainstbaselinesuPARconcentrationonalog-scale.(B) Adjusted HRs for ESKD plotted against baseline suPAR concentration on a log-scale. (C) Adjusted HRs for worsening proteinuria plotted against baseline suPAR concentration on a log-scale. (D) Adjusted HRs for mortality plotted against baseline suPAR concentration on a log-scale. HRs were adjusted for demographics (age and sex), AASK trial arm (BP control goal and trial medication), kidney measures (UPCR and measured GFR), and clinical risk factors (history of heart disease, history of smoking, CRP,and APOL1 risk status) at baseline. CKD progression was defined as doubling of serum creatinine from baseline or ESKD (requiring dialysis or kidney transplantation). Worsening proteinuria was defined as pre-ESKD doubling of 24-hour UPCR to $220 mg/g. disease. In mouse models, suPAR promotes kidney disease suPAR was associated with faster eGFR decline in APOL1 b 2 2 via binding to and activating 3 integrins on podocytes, high-risk patients ( 2.5 ml/min per 1.73 m per year; which triggers activation of podocyte Rac-1 GTPase and P=0.03), but not in APOL1 low-risk patients. In the 607 cytoskeletal rearrangement (4), effects which may vary on patients in AASK, associations between suPAR and eGFR the basis of the suPAR level, form, and source (e.g.,bone decline in APOL1 high-risk patients were more modest marrow cells) (4,5,22). In our study, no association was (20.6 ml/min/1.73 m2 per year; P=0.01), but higher suPAR found between suPAR and worsening proteinuria overall, was also significantly associated with eGFR decline in the but we did see this association in participants with two APOL1 low-risk population (20.3 ml/min per 1.73 m2 per APOL1 risk alleles. This observation is consistent with the year; P,0.001). In our study, we continued to find strong hypothesis that suPAR can synergize with APOL1 to associations between higher suPAR and clinical outcomes, promote integrin activation through a tripartite complex even after adjustment for rigorously measured markers of and an autophagosomal reaction that leads to podocyte GFR and proteinuria; however, associations with CKD detachment (11). Conversely, we did not find statistically progression, ESKD, and mortality were similar by APOL1 significant interaction between suPAR and the APOL1 risk risk status, likely because of the relatively small differences status for the outcomes of CKD progression, ESKD, or in the magnitude of eGFR decline shown in our previous death. study. In our previous study, we investigated the interaction The association between higher baseline suPAR and all- between APOL1 risk status and eGFR decline in the cause mortality is consistent with other studies in select EmCAB, a clinical cohort of patients undergoing cardiac patient populations, including those with cancer, infection, catheterization, and performed replication analyses in the cardiovascular diseases, and patients with diabetes and AASK cohort (11). In the 487 patients in EmCAB, higher ESKD (23–27). The mechanism underlying the association Clin J Am Soc Nephrol 13: 1013–1021, July, 2018 suPAR in Black Americans with CKD, Luo et al. 1019

Table 3. Results of multivariable Cox regression analyses assessing the associations between baseline serum suPAR concentration and CKD progression, ESKD, worsening proteinuria, and all-cause mortality, after stratification by APOL1 risk status in the African-American Study of Kidney Disease and Hypertension

APOL1 Risk Status Outcome No. of events HRa (95% CI) P Value

High risk: two risk alleles; n=143 CKD progression 81 1.25 (0.93 to 1.68) 0.15 ESKD 69 1.32 (0.94 to 1.84) 0.10 Doubling of UPCR to $220 mg/g 85 1.46 (1.08 to 1.99) 0.02 All-cause mortality 20 1.37 (0.61 to 3.06) 0.44 Low risk: zero or one risk allele; n=464 CKD progression 168 1.42 (1.17 to 1.73) ,0.001 ESKD 106 1.56 (1.20 to 2.04) 0.001 Doubling of UPCR to $220 mg/g 235 1.12 (0.97 to 1.31) 0.13 All-cause mortality 71 1.53 (1.19 to 1.97) 0.001 Not genotyped; n=348 CKD progression 114 1.15 (0.91 to 1.45) 0.24 ESKD 96 1.35 (1.04 to 1.76) 0.03 Doubling of UPCR to $220 mg/g 128 0.87 (0.71 to 1.05) 0.15 All-cause mortality 127 1.17 (0.96 to 1.41) 0.12

Results were adjusted for demographics (age and sex), the African American Study of Kidney Disease and Hypertension trial arm (BP control goal and trial medication), kidney measures (UPCR and measured GFR), and clinical risk factors (history of heart disease, history of smoking, and C-reactive protein) at baseline. CKD progression was defined as doubling of serum creatinine from baseline or ESKD (requiring dialysis or kidney transplantation). Worsening proteinuria was defined as pre-ESKD doubling of 24-hour UPCR to $220 mg/g. suPAR, soluble urokinase-type plasminogen activator receptor; HR, hazard ratio; 95% CI, 95% confidence interval; UPCR, 24-hour urine protein-to-creatinine ratio. aHR per SD higher log-transformed baseline suPAR.

between suPAR and mortality requires additional study, APOL1 high-risk genotype. Future studies should evaluate but it does not appear to be mediated solely by classic the utility of measuring suPAR in clinical practice. inflammation, as associations were robust after adjustment for CRP in our study as well as others (24,27,28). A recent Acknowledgments study of 476 patients with CKD found that associations The authors thank the staff and participants of the African- between suPAR and cardiovascular events were also in- American Study of Kidney Disease and Hypertension for their fl dependent of markers of malnutrition or in ammation (29). important contributions. The strengths of our study include its large sample size, J.C., A.T., L.J.A., and M.E.G. are supported by the National In- large number of events, long follow-up, per-protocol stitute of Diabetes and Digestive and Kidney Diseases grant R01- kidney measurements, and robust results in sensitivity DK108803-02. J.C. and M.E.G. are supported by the National analyses. The study population consisted of Black Amer- Institute of Diabetes and Digestive and Kidney Diseases grant U01- icans with moderate CKD (GFR between 20 and 65 ml/min DK085689-07. C.M.R. is supported by a mentored research scientist 2 per 1.73 m ), a group that suffers disproportionately high development award from the National Institute of Diabetes and risk for ESKD and other adverse outcomes. However, our Digestive and Kidney Diseases (K01 DK107782). T.K.C. is supported study has some limitations. First, our data originated from by the Norman S. Coplon Extramural Grant Program by Satellite participants in a randomized, controlled trial, and levels of Healthcare, a not-for-profit kidney care provider. baseline proteinuria were low (individuals with 24-hour Part of this work was presented as a moderated poster on March . UPCR 2500 mg/g were excluded from AASK). Second, 20, 2018 at the American Heart Association EPI/Lifestyle 2018 only baseline suPAR was measured. Third, because of lack Scientific Sessions in New Orleans, Louisiana. of data, we were unable to describe the participants’ baseline history of heart disease in more detail, or analyze specific Disclosures causes of death. Fourth, like all observational studies, we J.C., A.S.L., and L.A.I. have a provisional patent filed on 8/15/ cannot ascribe causality, and our study is limited by the 2014, “Precise estimation of GFR from multiple biomarkers” (PCT/ potential for unmeasured confounding and imprecise mea- US2015/044567). The technologyis not licensedin whole or inpart to surements. Nonetheless, given the rigorous design of AASK any company. Tufts Medical Center, John Hopkins University, and and its focus on protocol-based ascertainment of kidney mea- Metabolon Inc. have a collaboration agreement to develop a product sures and outcomes, the influence of confounding and mea- to estimate GFR from a panel of markers. T.K.C. previously owned surement errors was minimized to the greatest extent possible. stock in Pfizer Pharmaceuticals. A.S.L. reports funding to Tufts In conclusion, higher suPAR was associated with CKD Medical Center for research and contracts with the National progression, ESKD, and all-cause mortality in Black Amer- Institutes of Health, National Kidney Foundation, Amgen, and icans with hypertension-attributed CKD, independently of Siemens. L.A.I. reports funding to Tufts Medical Center for research APOL1 risk status, GFR, and proteinuria. This suggests that and contracts with the National Institutes of Health, National suPAR may be a promising marker and/or mediator of risk Kidney Foundation, and Reata Pharmaceuticals, and consulting in Black Americans with and without CKD. Interestingly, agreements with Tricedia Inc. and Omeros Corp. J.R. is cofounder the association between baseline suPAR and worsening and shareholder of TRISAQ, a biopharmaceutical company that proteinuria was present only among patients with the develops suPAR products. 1020 Clinical Journal of the American Society of Nephrology

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