Table 1.
Demographic information for 24 perfusates obtained from perfusion of human kidneys destined for transplantation.
Fig 1.
Comparison of injury markers and total protein in perfusate from DBD (n = 17) versus cDCDD (n = 9) human donors.
A: NGAL activity B: LDH activity C: Cytochrome c oxidase activity D: Total protein level Error bars represent Standard Error of the Mean (SEM). * p<0.05 using Student’s T test. DBD = Donation after Brain Death cDCDD = controlled Donation after Circulatory Determination of Death LDH = lactate dehydrogenase NGAL = neutrophil gelatinase associated lipocalin
Fig 2.
Comparison of MMP-9 and MMP-2 levels in perfusates from kidneys from DBD (n = 15) and cDCDD (n = 9) human donors.
Error bars represent Standard Error of the Mean (SEM). * p<0.05 using Student’s T test. DBD = Donation after Brain Death cDCDD = controlled Donation after Circulatory Determination of Death MMP = matrix metalloproteinase.
Fig 3.
Matrix Metalloproteinase levels and injury markers in perfusate from clinically transplanted kidneys with DGF (n = 5) and without DGF (n = 19).
A: MMP-9 level B: MMP-2 level C: NGAL levelD: LDH activity Error bars represent Standard Error of the Mean (SEM). * p<0.05 using Student’s T test. DGF = Delayed Graft Function MMP = matrix metalloproteinase NGAL = neutrophil gelatinase associated lipocalin.
Fig 4.
Light microscopy (H&E stain) of representative rat kidney at initial flush and after 22 hours of machine cold perfusion (n = 5 per time point).
Fig 5.
Injury markers in the perfusate of rat kidney with warm ischemic injury undergoing machine cold perfusion at 5, 10, and 22 hours (n = 4–7 each).
A: NGAL levelB:LDH activityC: Cytochrome c oxidase activity Error bars represent Standard Error of the Mean (SEM). * p<0.05 using Student’s T test. LDH = lactate dehydrogenase NGAL = neutrophil gelatinase associated lipocalin.
Fig 6.
Comparison of MMP-9 and MMP-2 levels at 5, 10, and 22 hours in perfusates from rat kidney with warm ischemic injury undergoing machine cold perfusion (n = 7).
Insert: representative zymography with MMP-9 and MMP-2 activitiesError bars represent Standard Error of the Mean (SEM). * p<0.05 compared to 5 hours using Student’s T test.
Fig 7.
Representative electron micrographs of rat kidneys and comparison of mitochondrial damage at the time of retrieval and after 22 hours of machine cold perfusion. (5K magnification).
A: Clear arrows = mitochondrial swelling and loss of detail at 22 hours Dark arrows = swelling and detachment of the tubular endothelium at 22 hours. B (n = 4). Left panel = cytochrome c oxidaseRight panel = percent undamaged outer membrane of mitochondria. Error bars represent Standard Error of the Mean (SEM). * p<0.05 using Student’s T test.
Fig 8.
MMP-2 expression in perfusate from rat kidney cold perfused with KPS-1 solution with MMP-2 siRNA added (n = 4).
Insert: a representative zymography for MMP-2. Error bars represent Standard Error of the Mean (SEM). * p<0.05 using Student’s T test. MMP = matrix metalloproteinase.
Fig 9.
LDH activity and Cytochrome c oxidase level in perfusate of rat kidney with warm ischemic injury at 5, 10, and 22 hours perfused with KPS-1 with scrambled siRNA versus KPS-1 with siRNA 50 nM (n = 4–7 each).
A: LDH activity. B: Cytochrome c oxidase level. Error bars represent Standard Error of the Mean (SEM). * p<0.05 compared to 5 hours using Student’s T test. # p<0.05 compared to 22 hours using Student’s T test.
Fig 10.
Effect of doxycycline on LDH, cytochrome c oxidase, and total protein release.
A: Confirmation of inhibition of MMP-2 and MMP-9 activity by doxycycline using zymography. B: LDH activity in perfusate of rat kidney with warm ischemic injury at 5, 10, and 22 hours perfused with KPS-1 with and without doxycycline (n = 7 each). C: Cytochrome c oxidase level in perfusate of rat kidney with warm ischemic injury at 5, 10, and 22 hours perfused with KPS-1 with and without doxycycline (n = 7 each). D: Total protein release in perfusate of rat kidney with warm ischemic injury at 5, 10, and 22 hours perfused with KPS-1 with and without doxycycline (n = 6 each). Error bars represent Standard Error of the Mean (SEM). * p<0.05 compared to 5 hours using Student’s T test.# p<0.05 compared to 22 hours using Student’s T test.KPS-1 = Kidney Preservation Solution 1MMP = matrix metalloproteinase.
Fig 11.
NGAL in perfusate of rat kidney with warm ischemic injury at 22 hours with KPS-1 and KPS-1 supplemented with doxycycline and MMP-2 siRNA compared to control (5 hours) (n = 4).
Error bars represent Standard Error of the Mean (SEM). * p<0.05 compared to 5 hours using Student’s T test.# p<0.05 compared to 22 hours using Student’s T test.KPS-1 = Kidney Preservation Solution 1MMP = matrix metalloproteinaseNGAL = neutrophil gelatinase associated lipocalin.
Fig 12.
Level of undamaged mitochondria measured by the levels of cytochrome c oxidase activity in rat kidney tissue after initial flush and following 22 hours of machine cold perfusion with KPS-1, KPS-1 with doxycycline, and KPS-1 with MMP-2 siRNA (n = 4).
Error bars represent Standard Error of the Mean (SEM). * p<0.05 compared to 0 hours using Student’s T test.# p<0.05 compared to 22 hours using Student’s T test.KPS-1 = Kidney Preservation Solution 1MMP = matrix metalloproteinase.
Fig 13.
Experimental ProtocolKPS-1 = Kidney Perfusion Solution -1 MMP = matrix metalloproteinase.