Supplementary material Ann Rheum Dis

Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312 Supplementary material Ann Rheum Dis

Vanarsa et al, Supplementary Table S2 Supplementary Table S2: Patient cohort used for the ELISA validation of urine biomarker candidates Variable All SLE Inactive Active Non-Renal Active Renal (N=62) (N=17) (N=16) (N=29) Age (years) 32.9±9.5 32.8±7.5 42.1±11.3 27.9±4.5 Gender Female, n(%) 62 (100.0%) 17 (100.0%) 16 (100.0%) 29 (100.0%) Ethnicity African-American 58 (93.5%) 16 (94.1%) 13 (81.3%) 29 (100.0%) Caucasian 4 (6.5%) 1 (5.9%) 3 (18.7%) 0 (0.0%) Clinical Assessment SLEDAI 5.9±4.2 0.4±0.8 7.7±3.0 8.1±3.0 rSLEDAI 2.4±2.9 0±0 0±0 5.1±2.1 PGA 1.3±0.8 0.6±0.7 0.8±0.4 2.0±0.6 Laboratory Metrics WBC (X109/L) 6.9±3.3 7.1±2.7 4.3±1.4 8.3±3.6 HGB (g/dl) 11.0±1.5 11.3±1.5 11.3±1.1 10.7±1.7 Platelets (X109/L) 284.0±95.8 235.9±78.9 263.5±62.1 323.2±106.0 C3 (mg/dl) 95.3±34.7 116.5±31.0 87.8±37.5 88.3±31.1 C4 (mg/dl) 18.7±10.2 22.0±8.7 14.9±8.9 19.1±11.1 ESR (mm/h) 43.9±29.2 34.1±26.3 47.9±22.0 47.2±33.5 UrPrCrRatio (mg/mg) 1.2±1.9 0.2±0.1 0.1±0.1 2.5±2.4 Serum Creatinine (mg/dl) 0.9±0.4 0.9±0.5 0.8±0.2 1.0±0.4 Anti-DNA, n(%) 32 (51.6%) 2 (11.8%) 11 (68.8%) 19 (65.5%) Medications Prednisone, n(%) 38 (61.3%) 9 (52.9%) 7 (43.8%) 22 (75.9%) Plaquenil, n(%) 51 (82.3%) 13 (76.5%) 13 (81.3%) 25 (86.2%) Mycophenolate Mofetil, n(%) 32 (51.6%) 8 (47.1%) 9 (56.3%) 15 (51.7%) Tacrolimus, n(%) 1 (1.6%) 0 (0.0%) 1 (6.3%) 0 (0.0%) Azathioprine, n(%) 7 (11.3%) 1 (5.9%) 2 (12.5%) 4 (13.8%) Cyclophosphamide, n(%) 2 (3.2%) 0 (0.0%) 0 (0.0%) 2 (6.9%) Methotrexate, n (%) 2 (3.2%) 0 (0.0%) 1 (6.3%) 1 (3.4%) Abbreviations used: PGA, physician global assessment; UrPrCr ratio, urine protein to creatinine ratio.

Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312 Supplementary material Ann Rheum Dis

Supplementary Table S3: Well documented biomarker proteins rediscovered by the screen Molecule FC SLE/HC p value (t test) Supplementary References Albumin 2.0 0.075 S3, S4 ALCAM 1797.9 0.072 S47, S48 Angiostatin 160.9 0.105 S29 Calreticulin 4.6 0.1 S44 CD163 N/A* 0.08 S10, S11 CD30 20.5 0.008 S51 CXCL16 15.9 0.008 S20, S21 Cystatin S 3.0 0.185 Cystatin SA 1.7 0.529 Cystatin SN 0.4 0.313 E-Selectin 3.9 0.002 S37 Ferritin 187.0 0.001 S49, S50 FOLR1 1.4 0.517 ICAM-1 6.8 0.019 S1, S2 IL-12p40 N/A* 0.3 S35, S36 IL-15 4.1 0.47 S13, S14 IL-6 0.1 0.126 S5, S6 IP-10 43.1 0.013 S22, S23 I-TAC 2.5 0.137 Leptin 3.1 0.024 S12 Lipocalin-1 3.1 0.055 Lipocalin-2 3.2 0.012 S28 L-Selectin 16.7 0.013 MCP-1 7.8 0.089 S7 MCSF 1.5 0.344 S15, S16 MCSF R 51.9 0.008 MIP-3a 49.8 0.13 S27 MIP-3b 8.8 0.04 S45 OPG 52.0 0.002 S33, S34 OPN 7.2 0.004 S18, S19 PF4 43.1 0.109 S40, S41 RAGE 10.7 0.1 S26 RANTES 16.7 0.01 S42, S43 Resistin 8.1 0.015 S46 SDF-1a 9.1 0.249 S32 TFPI 397.8 0.027 S8, S9 TNF RI 3.7 0.038 S17 TNF RII 3.2 0.025 TREM-1 21.7 0.099 S38, S39 TWEAK 4.4 0.105 S30, S31 TWEAK R 1.5 0.743 uPA 3.7 0.004 VCAM-1 49.0 0.068 S24, S25 N/A, not available. * indicates FC was not calculated due to average of healthy control is 0.

Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312 Supplementary material Ann Rheum Dis

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Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312 Supplementary material Ann Rheum Dis

Vanarsa et al, Supplementary Table S4 Supplementary Table S4: 64 proteins elevated in SLE urine (p<0.05; FC>5) compared to HC urine, based on array-based screening of 1000 proteins Protein HC (mean/median) SLE (mean/median) Fold Change1,2 Protein HC (mean/median) SLE (mean/median) Fold Change1,2 Angiostatin 186/0 29924/10002 160.9**** IL-2 Rg 1187/823 8913/8386 7.5*** Angiotensinogen 21406/0 124646/48697 5.8** Integrin alpha 5 862/343 45548/27891 52.8** ANGPTL4 9/5 4854/1416 531.4*** JAM-C 1172/1398 7781/5574 6.6*** Arylsulfatase A 9907/7500 316891/62772 31.9*** LAIR1 1230/902 11999/9333 9.8* B2M 1409/723 8313/7122 5.9*** LAIR2 1161/631 7317/3680 6.3** BMP-5 199/0 10640/9088 53.5**** LAMP1 232/145 4340/2434 18.7* BMP-7 24/0 2140/2048 89.8**** L-Selectin 463/0 7719/6177 16.7*** CA2 548/122 2896/1397 5.3** Lumican 1444/1291 7524/5291 5.2*** Cathepsin H 1306/634 37240/23604 28.5**** MCSF R 62/11 3202/2283 51.9*** CD14 747/348 3936/2893 5.3* Mesothelin 2272/939 18240/19942 8*** CD36 634/662 9165/8422 14.5*** MIF 455/53 13200/11869 29.1**** CD7 2029/549 27565/28075 13.6**** NCAM-1 468/234 4223/4156 9.1** CLEC-2 16/0 3974/1691 246.7** Nectin-1 290/2 3485/1485 12.1* Contactin-3 4016/1860 37244/19731 9.3* NG2 446/485 3763/3382 8.4** Epiregulin 270/27 5992/1630 22.2* OPN 4811/4605 34614/27041 7.2**** FABP1 41899/44000 430436/69278 10.3* OSM R beta 15/0 3232/3502 213.6** FCRL5 459/0 3842/3247 8.4** Pappalysin-2 236/82 11194/2567 47.5* Ferritin 2157/1663 403335/330017 186.9**** Pepsinogen II 338/6 2200/1004 6.5** Fetuin A 3780/1057 34496/6353 9.2** PRX2 568/290 65014/8810 114.5*** FOLR2 72/43 2155/1381 29.8**** Resistin 4438/3671 35972/15065 8.1*** Galectin-7 1996/102 12168/9789 6.1** S100A8 182/11 2314/1105 12.7*** GP73 1715/792 14283/11643 8.3*** Serpin A4 164/23 2390/1271 14.5** GUSB 1721/552 11889/8393 6.9* Siglec-11 31/0 2464/60 79.6* htPAPP-A 120/0 3216/2779 26.7*** Siglec-5 77/29 2996/760 38.7*** ICAM-1 467/67 3168/1952 6.8* SLITRK5 428/126 3576/3065 8.4*** ICAM-2 3430/1466 25972/21732 7.6**** SPINK1 3540/770 20199/9218 5.7** IFNab R2 7/0 77625/2506 11089.3 *** TGFb1 65/0 17653/13896 271.4*** IGFBP-1 853/421 5494/3694 6.4*** TPP1 398/0 6951/2529 17.5** IGFBP-3 279/58 9040/5750 32.4*** Troponin C 148/0 3541/2862 23.9*** IGFBP-6 176/26 13841/5193 78.5**** TSP-1 184/25 3619/3470 19.7*** IL-1 F9 77/0 2596/1299 33.7** uPAR 3261/1903 23305/14128 7.1*** IL-2 Rb 358/38 4652/2158 12.9* VCAM-1 671/0 32852/6995 48.9*** 1. Indicated are the statistical significance p-values as determined by Mann Whitney U test (*, p <0.05; **, p <0.01; ***, p<0.001; ****, p<0.0001). 2. Values in italics have a q-value of <0.05, after multiple testing correction. 3. Mean/Median units = pg/mg

Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312 Supplementary material Ann Rheum Dis

Vanarsa et al, Supplementary Table S5 Supplementary Table S5. ELISA kits used for validation of candidate biomarkers selected from the protein array screening assay Protein ELISA Manufacturer Urine Diltion Result/Notes Angiostatin N/A N/A Not pursued, as extensive literature already exists. ANGptl4 RayBioTech 1 in 2 Selected for further validation in an independant cohort Arylsulfatase A N/A N/A Same cluster as Cathepsin H;hence not pursued BMP-5 RayBioTech 1 in 5 Commercial ELISA Kit did not work. BMP-7 RayBioTech 1 in 5 Commercial ELISA Kit did not work. Cathepsin H RayBioTech 1 in 5 Commercial ELISA Kit did not work. CD40 R&D Systems 1 in 2 Commercial ELISA Kit did not work. CD6 RayBioTech 1 in 2 Commercial ELISA Kit did not work. CLEC-2 RayBioTech 1 in 2 Commercial ELISA Kit did not work. EGF-R R&D Systems 1 in 2 Commercial ELISA Kit did not work. Epiregulin N/A N/A Same cluster as PDGFRb;hence not pursued FABP6 N/A N/A Same cluster as htPAPP-A;hence not pursued Ferritin N/A N/A Not pursued, as extensive literature already exists. FOLR2 RayBioTech 1 in 2 Selected for further validation in an independant cohort Glypican RayBioTech 1 in 2 Selected for further validation in an independant cohort htPAPPA-A RayBioTech 1 in 2 Commercial ELISA Kit did not work. IGFP-3 N/A N/A Not pursued, as extensive literature already exists. IGFP-6 N/A N/A Not pursued, as extensive literature already exists. IL-1 N/A N/A Same cluster as PDGFRb;hence not pursued Integrin alpha 5 N/A N/A Same cluster as Siglec-5;hence not pursued LAMP1 N/A N/A Same cluster as Siglec-5;hence not pursued L-selectin RayBioTech 1 in 20 Selected for further validation in an independant cohort MCSF-R RayBioTech 1 in 2 Commercial ELISA Kit did not work. OSM R beta N/A N/A Same cluster as ANGPTL4;hence not pursued Pappalysin-2 N/A N/A Same cluster as CLEC-2;hence not pursued PD-ECGF N/A N/A Same cluster as Siglec-6;hence not pursued PDGFRb RayBioTech 1 in 2 Selected for further validation in an independant cohort PRX2 R&D Systems 1 in 10 Selected for further validation in an independant cohort Siglec-5 RayBioTech 1 in 2 Commercial ELISA Kit did not work. TGFB-1 RayBioTech 1 in 2 Selected for further validation in an independant cohort TPP-1 RayBioTech 1 in 2 Selected for further validation in an independant cohort TRAIL R1 N/A N/A Same Cluster as CD40;hence not pursued Troponin C N/A N/A Same cluster as L-selectin;hence not pursued TSP-1 RayBioTech 1 in 2 Selected for further validation in an independant cohort VCAM-1 N/A N/A Not pursued, as extensive literature already exists.

Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312 Supplementary material Ann Rheum Dis

Vanarsa et al, Supplementary Table S6

Supplementary Table S6: Urinary biomarker concentrations in validation cohort, comparing AR vs ANR patients1

Cr normalized Urine biomarker in pg/mg, mean (median) AR vs ANR

Molecule HC Inactive SLE ANR AR All SLE Specificity AUC AR/ANR2 PPV NPV Angptl4 6.4(5.9) 11.5(9.9) 1.6(0.2) 30.1(13.6) 17.6(9.8) 0.88 0.96**** 0.94 1.00

FOLR2 0.1(0) 0.2(0) 133.7(8.4) 37.3(5.4) 19.6(0) 0.93 0.72** 0.94 0.53

Glypican5 2.3(0) 11.3(3.7) 70.9(3.) 15.5(8.8) 28.6(3.5) 0.75 0.52 0.81 0.50

L-selectin 1.3(0) 5(0.9) 2.8(1) 35.4(15) 18.6(3.5) 0.88 0.86**** 0.93 0.70

PDGFRβ 119(20.7) 515.7(56.6) 166.2(10.4) 904(114.2) 607.1(62.8) 0.69 0.67* 0.81 0.58

PRX2 0.3(0.2) 1.3(0.4) 0.7(0.2) 2.6(1) 1.7(0.5) 0.69 0.65 0.80 0.52

TGFβ1 35.3(28.5) 58.1(2.1) 44.2(0) 202(45.4) 121.8(13.7) 0.75 0.78*** 0.85 0.71

TPP1 762.2(336.3) 1146.9(0) 8.4(0) 6643.8(2837.2) 3423.6(15.2) 1.00 0.84*** 1.00 0.64

TSP-1 7.4(2.1) 14.7(10.5) 46.9(16.3) 65.8(44.4) 46.8(32.6) 0.63 0.73** 0.81 0.67

1. This is an independent cohort from that used for the initial screen, N=78. (HC=16, Inactive SLE=17, ANR=16, AR=29). 2. Indicated are the statistical significance p-values (*, p <0.05; **, p <0.01; ***, p<0.001; ****, p<0.0001).

Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312 Supplementary material Ann Rheum Dis

Vanarsa et al, Supplementary Table S7

Supplementary Table S7: Urinary biomarker concentration in validation cohort, with AR vs Inactive SLE comparisons1 Cr normalized Urine biomarker in pg/mg, mean (median) AR vs Inactive SLE AUC Molecule HC Inactive SLE ANR AR All SLE Specificity AR/Inactive SLE2 PPV NPV Angptl4 6.4(5.9) 11.5(9.9) 1.6(0.2) 30.1(13.6) 17.6(9.8) 0.91 0.71* 0.92 0.54

FOLR2 0.1(0) 0.2(0) 133.7(8.4) 37.3(5.4) 19.6(0) 1.00 0.74**** 1.00 0.51 Glypican5 2.3(0) 11.3(3.7) 70.9(3.) 15.5(8.8) 28.6(3.5) 0.82 0.52 0.84 0.42 L-selectin 1.3(0) 5(0.9) 2.8(1) 35.4(15) 18.6(3.5) 0.80 0.84**** 0.91 0.73 PDGFRb 119(20.7) 515.7(56.6) 166.2(10.4) 904(114.2) 607.1(62.8) 0.84 0.63 0.83 0.40 PRX2 0.3(0.2) 1.3(0.4) 0.7(0.2) 2.6(1) 1.7(0.5) 0.53 0.61 0.70 0.51 TGFβ1 35.3(28.5) 58.1(2.1) 44.2(0) 202(45.4) 121.8(13.7) 0.91 0.72** 0.91 0.54

TPP1 762.2(336.3) 1146.9(0) 8.4(0) 6643(2837) 3423.6(15.2) 0.94 0.74** 0.90 0.52 TSP-1 7.4(2.1) 14.7(10.5) 46.9(16.3) 65.8(44.4) 46.8(32.6) 0.82 0.94**** 0.92 0.70 1. This is an independent cohort from that used for the initial screen, N=78. (HC=16, Inactive SLE=17, ANR=16, AR=29). 2. Indicated are the statistical significance p-values (*, p <0.05; **, p <0.01; ***, p<0.001; ****, p<0.0001).

Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312 Supplementary material Ann Rheum Dis

Supplementary Table S8: Demographics of Biopsy- Concurrent LN samples

Variable Samples (N=20) Age (years) 39.1±11.9 Gender, n(%) Female 19(95%) Male 1(5%) Race, n(%) African American 10(50%) Caucasian 2(10%) Hispanic 8(40%)

UrPrCr ratio (mg/mg) 2.5±1.3 LN Class (renal biopsy) I 1(5%) II 2(10%) III(±V) 9(45%) IV(±V) 3(15%) V 5(25%) SLEDAI 9.7±4.2 Data are presented as n (%) or average ±SD as appropriate UrPrCr ratio, urine protein to creatinine ratio

Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312 Supplementary material Ann Rheum Dis

Supplementary Table S9: Demographics of patients with other (non-LN) chronic kidney diseases

Diabetic nephropathy HTN nephropathy FSGS Others (n=14) (n=11) (n=9) (n=13)

Age(years) 44.7±12.9 42.8±12.0 14.2±13.744.1±11.5 Ethnicity, n(%) Hispanic 7(50%) 3(27%) 2 (22%) 7 (54%) Caucasian 0(0%) 0(0%) 3 (33%) 1 (8%) African American 6(43%) 8(72%) 3 (33%) 5 (38%) Asian 1(7%) 0 (0%) 1(11%) 0 (0%) Female, n(%) 6(43%) 7(64%) 5 (55%) 6 (46%) CKD stages, n (%) 1 0(0%) 0 (0%) 3 (33%) 2 (15%) 2 3(21%) 1(9%) 1 (11%) 4 (31%) 3 5(36%) 3(27%) 2 (22%) 3 (23%) 4 4(29%) 3(27%) 2 (22%) 0 (0%） 5 2(14%) 4(36%) 1 (11%) 4 (31%) Laboratory metrics UrPrCr ratio (mg/mg) 5.2±4.9 7.5±8.0 2.3±1.7 4.8±1.5 Serum Creatinine(mg/dl) 2.7±1.4 2.8±1.8 2.2±2.0 2.3±1.8 UrPrCR ratio, Urine protein to creatinine ratio; CKD,Chronic Kidney disease

Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312 Supplementary material Ann Rheum Dis

Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312 Supplementary material Ann Rheum Dis

Vanarsa K, et al. Ann Rheum Dis 2020;0:1–13. doi: 10.1136/annrheumdis-2019-216312