Protein Kinase C-δ Mediates Kidney Tubular Injury in Cold Storage–Associated Kidney Transplantation

Abstract

BackgroundKidney injury associated with cold storage is a determinant of delayed graft function and the long-term outcome of transplanted kidneys, but the underlying mechanism remains elusive. We previously reported a role of protein kinase C-δ (PKCδ) in renal tubular injury during cisplatin nephrotoxicity and albumin-associated kidney injury, but whether PKCδ is involved in ischemic or transplantation-associated kidney injury is unknown.MethodsTo investigate PKCδ’s potential role in injury during cold storage–associated transplantation, we incubated rat kidney proximal tubule cells in University of Wisconsin (UW) solution at 4°C for cold storage, returning them to normal culture medium at 37°C for rewarming. We also stored kidneys from donor mice in cold UW solution for various durations, followed by transplantation into syngeneic recipient mice.ResultsWe observed PKCδ activation in both in vitro and in vivo models of cold-storage rewarming or transplantation. In the mouse model, PKCδ was activated and accumulated in mitochondria, where it mediated phosphorylation of a mitochondrial fission protein, dynamin-related protein 1 (Drp1), at serine 616. Drp1 activation resulted in mitochondrial fission or fragmentation, accompanied by mitochondrial damage and tubular cell death. Deficiency of PKCδ in donor kidney ameliorated Drp1 phosphorylation, mitochondrial damage, tubular cell death, and kidney injury during cold storage–associated transplantation. PKCδ deficiency also improved the repair and function of the renal graft as a life-supporting kidney. An inhibitor of PKCδ, δV1-1, protected kidneys against cold storage–associated transplantation injury.ConclusionsThese results indicate that PKCδ is a key mediator of mitochondrial damage and renal tubular injury in cold storage–associated transplantation and may be an effective therapeutic target for improving renal transplant outcomes.

Proximal Tubule p53 in Cold Storage/Transplantation-Associated Kidney Injury and Renal Graft Dysfunction

“…Basic research (6,7) and clinical trial (8) revealed that inhibition of proximal tubular cell death with caspase inhibitors during cold storage improves graft function and histology. Moreover, we (9) and others (10)(11)(12)(13) identified intrinsic mitochondrial pathway of apoptosis in cold storage/transplantation kidney injury. Despite these findings, the cellular and molecular mechanism of cold storage/transplantation kidney injury remains elusive and there is an urgent need for effective renoprotective approaches.…”

“…The kidneys from donor mice were transplanted into syngeneic recipients following previously described procedures with minor modifications (9,21). The donor mice were anesthetized with pentobarbital (50 mg/kg intraperitoneally).…”

“…Immunohistochemistry staining for KIM-1 was performed as previously described (9). Tissue was exposed to goat KIM-1 antibody (Novus, AF1750) and ImmPRESS-AP (Alkaline Phosphatase) horse anti-goat IgG.…”

“…For in vitro experiments, we took advantage of cold storage/rewarming model to mimic the ATP-depletion and replenishment in vivo (9,11,27,28). Rat kidney proximal tubular cells (RPTCs) were cold (4 • C) stored in UW solution followed by incubation in full culture medium at 37 • C for rewarming.…”

“…To examine the therapeutic potential of p53 inhibition for transplanted kidneys, we established a life-supporting model, in which the recipient original kidney was removed on day 5 after transplantation so that the transplanted kidney became the life-supporting kidney as described in our recent work (9). DMSO or pifithrin-α was given before donor harvest, included in UW solution during cold storage and every other day after surgery as illustrated in Figure 8A (drug administration detailed in materials and methods).…”