18 Z$S0X$q}
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number 9 'IDbe{
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The
sVP2$?
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at to)Pl}9QkK
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The kLbo |p"cT
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 y|YhDO
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, K555z+,'e
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive )`zfDio-1V
control for Ca2+ induction. The data thus obtained was analyzed with the software Win ;o.,vQF*
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on }:+P{
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 ~PC S_
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were /
!A&z4;D
harvested and fractionated as detailed above, and the S-100 fractions were assessed by kZS&q/6A*
immunoblotting for presence or absence of annexin I. 2|LkCu)~,"
Mouse bone marrow transduction and transplantation. t8-LPq
Retrovirus-mediated transduction of mouse bone marrow cells was done by published JP 8v2)
p
methods49. Prestimulated low-density bone marrow cells were infected with high-titer i%9xt1
c_
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated H/O v8|
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing xiQ;lE
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by ~kPZh1n`
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) [T#9#3
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone pw1&WP&?3
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis 1 *$-.
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of uJ/&!q<3
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was WhR'MkfL
confirmed by immunoblot analysis. PiwI.c
Determination of renal morphology Q
GDfX
_
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an .R{P%r
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, FcZ)_m6m
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner A+F@JpV
for morphologic alterations, as previously detailed 9~WjCa*,&
Patient population #me'1/z
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) ?TVR{e:
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per ;yg
9{"O
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) 1\+d 5Q0
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. !RwOUCk
The Mayo Institutional Review Board and the Research Ethical Board, University Health #:3~I
Network, University of Toronto approved the study protocol. All patients gave written fQOh%i9n5
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or Smlf9h
&
19 QAh6!<.;@
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 Fr ryZe=
months were not included in the study. Patients with active infection, diabetes, or a xu7Q^F#u
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), O<?z\yBtS^
medications, malignancies) were also excluded. ,BG
L|5?3z
Treatment Rck k
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of "kT?9&