α-SMPDL3B

α-β-actin

α-HA

- +- +- + PNGaseF

empty

vecto

r

HA-SMPDL3

B

SMPDL3BA

55 kD

55 kD

BDAPI HA-SMPDL3B

C

AF647

unstained

mouse IgG-AF647

rituximab-AF647

D156ELWKPW161

20P

Sup Fig.1

AF647

AF4

88

unfixed

4% PFA

0100

00

unfixed

4% PFA

unstained mouse IgG-AF488 mouse IgG-AF647 anti-HA-AF488 Rituximab-AF647anti-HA-AF488

Rituximab-AF647

empty vector

HA-SMPDL3B

unstained mouse IgG-AF488 mouse IgG-AF647 anti-HA-AF488 Rituximab-AF647anti-HA-AF488

Rituximab-AF647

A

B

0100

00

0.399.7

00

0100

00

0.199.9

00

8.691.4

00

0100

00

2.797.3

00

3.097.0

00

0.199.9

00

91.88.2

00

91.88.2

00

0100

00

0100

00

0.299.8

00

050.4

049.6

2.297.8

00

0.950.3

1.347.5

0100

00

0100

00

0.899.2

00

044.7

055.3

79.320.7

00

35.09.5

43.312.2

AF647

AF4

88

Sup Fig.2

AF647

AF4

88

unfixed

4% PFA

unstained mouse IgG-AF488 mouse IgG-AF647 anti-HA-AF488 Rituximab-AF647anti-HA-AF488

Rituximab-AF647B

0.299.8

00

0100

00

0.699.4

00

037.7

062.3

4.395.7

00

1.638.0

2.957.5

0.199.9

00

0.199.9

00

1.698.4

00

0.136.3

063.6

64.635.4

00

22.217.5

38.821.5

A

α-SMPDL3B

α-HA

empt

y ve

ctor

HA

-SM

PD

L3B

SM

PD

L3B

55 kD

55 kD

α-β-actin

Sup Fig.3

WT

IL-4 KO

0

2

4

6

8

IL-2

(pg/mL)

WT

IL-4 KO

0

500

1000

IL-1

0(pg/mL)

WT

IL-4 KO

0

20

40

60

80

100

IL-4

(pg/mL)

*

WT

IL-4 KO

0

2

4

6

TNF-

a(pg/mL)

WT

IL-4 KO

0

500

1000

1500

2000

IL-6

(pg/mL)

Sup Fig. 4

1

Patient ID Age Gender Race Medical history Pathologic findings of kidney biopsy Urine protein at time of biopsy

Clinicalpathologic diagnosis

Immunosuppression at time of biopsy

pSTAT6 staining Comments

1 64 Male AA NS, HTNDiffuse foot process effacement with no electron

dense deposits; arteriolar thickening; 2+ glomerular IgG, 1+ glomerular C1q by IF

24 hour urine protein of 8.1

gramsMCD No ++ Recurrent presentation

(prednisone responsive)

2 12 Male W NS Diffuse foot process effacement; minimal IgM and C1q deposition; no deposits on EM UPC 352 MCD No ++ Incident presentation

(prednisone responsive)

3 5 Male W NS Diffuse foot process effacement; negative IF UPC 431 MCD No +Incident presentation (prednisone responsive); good response to rituximab

4 14 Female South Asian NS Diffuse foot process effacement; minimal IgM deposition; no deposits on EM UPC 831 MCD No + Incident presentation

(prednisone responsive)

5 11 Male W NS Diffuse foot process effacement; minimal IgM deposition; no deposits on EM UPC 755 MCD No + Incident presentation

(prednisone responsive)

6 11 Male W NS Diffuse foot process effacement; mild IgM deposition; no deposits on EM UPC 368 MCD No + Incident presentation

(prednisone responsive)

7 26 Female W NS Diffuse foot process effacement, trace C3, otherwise negative UPC 783 MCD No + Incident presentation

(prednisone responsive)

8 26 Female Afro-caribbean NS Diffuse foot process effacement; negative IF UPC 151 MCD No + Incident presentation

(prednisone responsive)

9 4 Female W NSMultifocal foot process effacement with few

globally sclerosed glomeruli; 1+ glomerular IgG, 2+ glomerular IgM, 1+ glomerular C’3 by IF

24 hour urine protein of 1.52

gramsMCD No + Recurrent presentation

(prednisone responsive)

10 10 Male W NSMultifocal foot process effacement, mild

mesangial proliferation; 1+ glomerular C’3, no glomerular Ig by IF

24 hour urine protein of 22

gramsMCD No + Incident presentation

(prednisone responsive)

11 28 Female W NS, obesity, HTN, asthma

Widespread foot process effacement; 1+ glomerular IgG, no C’3 by IF

24 hour urine protein of 12.4

gramsMCD No - Incident presentation

(prednisone responsive)

12 4 Male W NS Diffuse foot process effacement; negative IF24 hour urine protein of 6.2

gramsMCD No -

Incident presentation (prednisone resistant, cyclosporine sensitive)

13 20 Male W NS Diffuse foot process effacement; minimal mesangial hypercellularity; negative IF

24 hour urine protein of 4.6

gramsMCD No - Incident presentation

(prednisone responsive)

14 50 Male W NS Diffuse foot process effacement; negative IF UPC of 8 MCD No - No data

15 31 Female AA NS, AKI, CKDDiffuse foot process effacement, mild to

moderate tubular injury; trace mesangial IgG and IgM by IF

UPC of 18 MCD No - No data

16 52 Female W Proteinuria, HTN, NSAID use

Diffuse foot process effacement, focal interstitial inflammation with eosinophils; negative IF 3+ by dipstick MCD, possible due

to NSAIDs No - No data

17 40 Female W NS Diffuse foot process effacement, mild acute tubular injury; 1+ mesangial IgM by IF

24 hour urine protein of 18

gramsMCD No - No data

18 12 Male West African NS Diffuse foot process effacement; mild IgM with minimal C1q, C3 deposition; no deposits on EM UPC 84 MCD No - Incident presentation

(prednisone responsive)

2

19 12 Female W NS Diffuse foot process effacement; mild IgM with minimal C1q, C3 deposition; no deposits on EM UPC 321 MCD No - Incident presentation

(prednisone responsive)

20 13 Male

Columbian (W + Native

Indian admixture)

NS Diffuse foot process effacement; mild IgM with minimal C1q, C3 deposition; no deposits on EM UPC 867 MCD No - Incident presentation

(prednisone responsive)

21 12 Female West African NS Diffuse foot process effacement; mild IgM with minimal C1q, C3 deposition; no deposits on EM UPC 321 MCD No - Incident presentation

(prednisone responsive)

22 66 Male W NS, HTN Diffuse foot process effacement; negative IF24 hour urine protein of >3.5

gramsMCD No - No data

23 53 Female W NS Diffuse foot process effacement, minimal mesangial C1q, otherwise negative UPC 943 MCD No - Incident presentation

(prednisone responsive)

24 48 Female W NS Diffuse foot process effacement; mild IgM deposition; no deposits on EM UPC 389 MCD No - Incident presentation

(prednisone responsive)

25 54 Male Afro-caribbean NS

Diffuse foot process effacement with mild mesangial proliferative lesion and mild amount of mesangial C3, IgM and C1q; no deposits on

EM

UPC 760 MCD No - Incident presentation (prednisone responsive)

26 45 Female W NSDiffuse foot process effacement, mild tubular atrophy and interstitial fibrosis; 1+ mesangial

IgM and C3 by IF

24 hour urine protein of 7 grams MCD No - No data

27 19 Female W NS Diffuse foot process effacement; minimal C1q, C3 deposition; no deposits on EM UPC 475 MCD No - Incident presentation

(prednisone responsive)

28 21 Male W NS Diffuse foot process effacement; mild IgM deposition; no deposits on EM UPC 449 MCD No - Incident presentation

(prednisone responsive)

29 3 Male W NS Multifocal foot process effacement; negative IF24 hour urine protein of 4.72

gramsMCD No - Recurrent presentation

(prednisone responsive)

pSTAT6 staining key

++ Prominent nuclear staining with no cytoplasmic background

+ Enhanced nuclear staining with cytoplasmic background staining present- No nuclear or cytoplasmic staining

AKI: Acute kidney injury

CKD: Chronic kidney disease

IF: Immunofluorescence

UPC: Urine protein-to-creatinine ratio

AA: African-American

W: White

NS: Nephrotic syndrome

HTN: Hypertension

MCD: Minimal change disease

Supplemental Table 1. Clinical characteristics of minimal change disease patients screened for glomerular phosphoSTAT6.