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.