Supporting Information for:
Shillingford JM, Murcia NS, Larson CH, Low SH, Hedgepeth
R, Brown N, Flask CA, Novick AC, Goldfarb DA, Kramer-Zucker
A, Walz G, Piontek KB, Germino GG, Weimbs T (2006) The mTOR
pathway is regulated by polycystin-1 and its inhibition reverses
renal cystogenesis in polycystic kidney disease.
Proceedings
of the National Academy of Sciences of the USA 103, 5466-5471
Supporting Figure 6
Supporting Figure 7
Supporting Table 1
Fig. 6. The N-terminal cytoplasmic domain of PC1 induces cell cycle arrest and apoptosis. (A and B)
Identical numbers of MDCK cells stably transfected for the
membrane-anchored N-terminal cytoplasmic domain of PC1 (NTM-PC1) were
cultured in the presence or absence of DOX for 48 h. (A) Numbers
of cells attached after this time were quantified and are expressed in
arbitrary units. Note that induction of expression of NTM-PC1 resulted
in a significant decrease of cell numbers. (B) DNA of detached
cells was stained with DAPI to reveal typical condensed and fragmented
apoptotic nuclei in NTM-PC1-expressing cells (+DOX). (C) The DNA
of attached cells was isolated and analyzed by agarose-gel
electrophoresis. Note that induction of NTM-PC1 expression results in
DNA-laddering, indicative of apoptosis. (D and E) Cell cycle analysis of attached cells was carried out by FACS in induced and noninduced NTM-PC1 cells. (D) Note that at 24 h, cells exhibited a significant increase in G1/G0 cell cycle and a significant reduction of cells in S-phase. (E) The fraction of apoptotic cells is significantly increased after induction of expression of NTM-PC1 for 24 and 48 h. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig. 7. Analysis of renal volume changes by MRI scanning. Vehicle- and rapamycin-treated orpk-rescue (Tg737orpk/orpk;TgRsq) mutant mice were subjected to MRI scanning at day 150, day 164, and day 178. The maximum slice volume for each kidney was determined by using the analysis program OSIRIX. The outline of each kidney at each time point was traced as shown and is represented as a merged image. Note the visual increase in maximal slice volume in vehicle-treated orpk-rescue mutant mice vs. the decrease in maximal slice volume in rapamycin-treated orpk-rescue mutant mice over the time course of the study. Cystic structures, evident as black areas within the kidney parenchyma, are significantly fewer in rapamycin-treated orpk-rescue mutant mice vs. vehicle-treated orpk-rescue mutant mice. (Scale bar, 3 mm.)
Table 1. Calculated CT renal volume of native polycystic kidneys in kidney transplant patients receiving rapamycin vs. nonrapamycin immunosuppression
|
Rapamycin Treatment |
||||||
|
Patient |
Kidney |
Initial renal volume, ml |
Final renal volume, ml |
Volume change, ml |
Volume % change |
Follow-up, months |
|
1 |
A |
1,081 |
639 |
–442 |
–40.9% |
23 |
|
B |
1,069 |
736 |
–333 |
–31.2% |
23 |
|
2 |
C |
623 |
535 |
–88 |
–14.1% |
44 |
|
D |
974 |
757 |
–217 |
–22.3% |
44 |
|
3 |
E |
1,288 |
937 |
–351 |
–27.3% |
11 |
|
F |
1,788 |
1,344 |
–444 |
–24.8% |
11 |
|
4 |
G |
1,802 |
1,572 |
–230 |
–12.8% |
14 |
|
Averages |
1,232 |
931 |
–301 |
–24.8% |
24 |
|
|
Analysis |
Total % Volume Change: |
–24.8% ± 9.7% (P =.001) |
||||
|
% Volume Change per Month: |
–1.4% ± 0.8% (P =.005) |
||||
|
Nonrapamycin treatment |
||||||
|
Patient |
Kidney |
Initial renal volume, ml |
Final renal volume, ml |
Volume change, ml |
Volume % change |
Follow-up, months |
|
5 |
M |
1,621 |
1,406 |
–215 |
–13.3% |
22 |
|
N |
1,174 |
1,251 |
77 |
6.6% |
22 |
|
6 |
O |
464 |
459 |
–5 |
–1.1% |
69 |
|
P |
735 |
575 |
–160 |
–21.8% |
69 |
|
7 |
Q |
1,949 |
1,689 |
–260 |
–13.3% |
20 |
|
Averages |
1,189 |
1,076 |
–113 |
–8.6% |
40 |
|
|
Analysis |
Total % volume change |
–8.6% ± 11.2% (P = 0.16) |
||||
|
% Volume change per month: |
–0.3% ± 0.4% (P = 0.22) |
||||
Volumes of native polycystic kidneys in human transplant patients were calculated by using an accepted ellipsoid volume formula from dimensions measured retrospectively on patient CT scans. Initial volumes were calculated from CT scans performed no more than 12 months prior to 5 months after receiving a kidney transplant and initiating immune-suppression therapy. Final volumes of these same native kidneys were also measured retrospectively from a CT scan performed at least 11 months after transplantation. The follow-up period between the two scans was measured and is shown as follow-up in months. Patients who received rapamycin as part of their immune suppression were placed in the rapamycin treatment group and those that did not were placed in the nonrapamycin treatment group. Volume changes for the kidneys were then analyzed statistically within groups by using a paired t-test and between the two groups by using an unpaired t-test.