Bcl-2 in Postischemic Donor Kidneys
selected against fibroblasts through to the D-valine
supplement in the growth media. Cultures were usu-
Cecka JM (2002). Does early injury have lasting impact?
Presented at the ASTS Second Annual Winter Symposium,
Bench to Bedside, Ischemia and Reperfusion Injury in Clinical
Transplantation, January 25–27, Miami Beach, FL.
ally incubated at 37° C and 5% CO ; for all proposed
2
experiments, cell monolayers 3 to 6 days of age were
used. Viability of cells was determined by light micros-
copy after trypan blue staining.
Cohen JJ (1993). Apoptosis. Immunol Today 14:126–130.
Cuttle L, Zhang XJ, Endre ZH, Winterford C, and Gobe GC
Tubular cells were characterized using proximal tubu-
lar markers such as HEA (Miltenyi Biotec, Gladbach,
Germany), anti-CD13 antibodies (Aminopeptidase-N;
Dako), angiotensin AT1 and AT2 receptor (Santa
Cruz Biotechnology, Santa Cruz, California). and
negative control staining with fibroblast-specific an-
tibody (Dianova, Hamburg, Germany) followed by
flow cytometric analysis.
RNA samples from cells derived from five different
nephrectomy specimens were pooled to obtain a
representative cross-section of human tubular gene
expression in healthy tubular epithelial cells. All exper-
iments were performed with the same calibrator RNA,
which was stored in aliquots at Ϫ80° C. GAPDH
served as internal standard.
(
2001). Bcl-X(L) translocation in renal tubular epithelial cells in
vitro protects distal cells from oxidative stress. Kidney Int
9:1779–1788.
5
Gavrieli Y, Sherman Y, and Ben-Sasson SA (1992). Identifi-
cation of programmed cell death in situ via specific labeling
of nuclear DNA fragmentation. J Cell Biol 119:493–501.
Gjertson DW (2000). Impact of delayed graft function and
acute rejection on kidney graft survival. Clin Transpl :467–
4
80.
Gobe G, Zhang XJ, Willgoss DA, Schoch E, Hogg NA, and
Endre ZH (2000). Relationship between expression of Bcl-2
genes and growth factors in ischemic acute renal failure in
the rat. J Am Soc Nephrol 11:454–467.
Groenewoud AF and Thorogood J (1993). Current status of
the Eurotransplant randomized multicenter study comparing
kidney graft preservation with histidine-tryptophan-
ketogluterate, University of Wisconsin, and Euro-Collins so-
lutions. The HTK Study Group. Transplant Proc 25:1582–
Quantification of gene expression was performed
according to the delta Ct method (⌬Ct /⌬Ct ), as
2
1
described elsewhere (Jeyaseelan et al, 2001). Briefly, a
ratio was calculated between the expression of the
gene of interest to the expression of the internal
1
585.
standard (GAPDH) in the calibrator (⌬Ct ) and in the
Hall BM, Tiller DJ, Duggin GG, Horvath JS, Farnsworth A,
May J, Johnson JR, and Sheil AG (1985). Post-transplant
acute renal failure in cadaver renal recipients treated with
cyclosporine. Kidney Int 28:178–186.
1
probe (⌬Ct ). Because the cDNA doubles in every
2
Ct
PCR cycle, 2⌬ yields the x-fold expression of the
gene of interest compared with calibrator. The intra-
individual coefficient of variation was below 1% in the
calibrator samples and 15 Ϯ 9% in the microdissected
donor kidney biopsy samples.
Hariharan S, Johnson CP, Bresnahan BA, Taranto SE, McIn-
tosh MJ, and Stablein D (2000). Improved graft survival after
renal transplantation in the United States, 1988 to 1996.
N Engl J Med 342:605–612.
Statistical Analysis
Jeyaseelan K, Ma D, and Armugam A (2001). Real-time
detection of gene promoter activity: Quantitation of toxin
gene transcription. Nucleic Acids Res 29:E58–8.
Continuous variables were described by mean Ϯ SD,
and correlations were calculated using the Spearman
coefficient. The Kruskall-Wallis test or the Wilcoxon
rank-sum test was used for the comparison between
three or two groups, respectively. A p value Ͻ 0.05
was considered statistically significant. The SAS Sys-
tem V8.1 (SAS Institute Inc., 2000; Cary, North Caro-
lina) was used for statistical analysis. All experiments
were performed in triplicates, and the mean value was
used.
Koo DD, Welsh KI, McLaren AJ, Roake JA, Morris PJ, and
Fuggle SV (1999). Cadaver versus living donor kidneys:
Impact of donor factors on antigen induction before trans-
plantation. Kidney Int 56:1551–1559.
Lieberthal W and Levine JS (1996). Mechanisms of apoptosis
and its potential role in renal tubular epithelial cell injury. Am J
Physiol 271:F477–F488.
Oberbauer R, Rohrmoser M, Regele H, Muhlbacher F, and
Mayer G (1999). Apoptosis of tubular epithelial cells in donor
kidney biopsies predicts early renal allograft function. J Am
Soc Nephrol 10:2006–2013.
Acknowledgement
The authors acknowledge the expert technical as-
sistance of Mrs. Nicole Huttary with sample adminis-
tration and Drs. Pfaller and Gstraunthaler with the
isolation of the primary human tubular epithelial cells.
Oberbauer R, Schwarz C, Regele HM, Hansmann C, Meyer
TW, and Mayer G (2001). Regulation of renal tubular cell
apoptosis and proliferation after ischemic injury to a solitary
kidney. J Lab Clin Med 138:343–351.
Ojo AO, Hanson JA, Meier-Kriesche H, Okechukwu CN,
Wolfe RA, Leichtman AB, Agodoa LY, Kaplan B, and Port FK
(2001). Survival in recipients of marginal cadaveric donor
kidneys compared with other recipients and wait-listed trans-
plant candidates. J Am Soc Nephrol 12:589–597.
References
Baer PC, Tunn UW, Nunez G, Scherberich JE, and Geiger H
(
1999). Transdifferentiation of distal but not proximal tubular
epithelial cells from human kidney in culture. Exp Nephrol
:306–313.
7
Olsen S, Burdick JF, Keown PA, Wallace AC, Racusen LC,
and Solez K (1989). Primary acute renal failure (“acute tubular
necrosis”) in the transplanted kidney: Morphology and patho-
genesis. Medicine (Baltimore) 68:173–187.
Burns AT, Davies DR, McLaren AJ, Cerundolo L, Morris PJ,
and Fuggle SV (1998). Apoptosis in ischemia/reperfusion
injury of human renal allografts. Transplantation 66:872–876.
Laboratory Investigation • July 2002 • Volume 82 • Number 7 947