Christopher Y. Nguan

Promotion of cell proliferation by clusterin in the renal tissue repair phase after ischemia-reperfusion injury

Renal repair begins soon after the kidney suffers ischemia-reperfusion injury (IRI); however, its molecular pathways are not fully understood. Clusterin (Clu) is a chaperone protein with cytoprotective functions in renal IRI. The aim of this study was to investigate the role of Clu in renal repair after IRI. IRI was induced in the left kidneys of wild-type (WT) C57BL/6J (B6) vs. Clu knockout (KO) B6 mice by clamping the renal pedicles for 28-45 min at the body temperature of 32°C. The renal repair was assessed by histology and confirmed by renal function. Gene expression was examined using PCR array. Here, we show that following IRI, renal tubular damage and Clu expression in WT kidneys were induced at day 1, reached the maximum at day 3, and significantly diminished at day 7 along with normal function, whereas the tubular damage in Clu KO kidneys steadily increased from initiation of insult to the end of the experiment, when renal failure occurred. Renal repair in WT kidneys was positively correlated with an increase in Ki67(+) proliferative tubular cells and survival from IRI. The functions of Clu in renal repair and renal tubular cell proliferation in cultures were associated with upregulation of a panel of genes that could positively regulate cell cycle progression and DNA damage repair, which might promote cell proliferation but not involve cell migration. In conclusion, these data suggest that Clu is required for renal tissue regeneration in the kidney repair phase after IRI, which is associated with promotion of tubular cell proliferation.

Reduction of Foxp3-expressing regulatory T cell infiltrates during the progression of renal allograft rejection in a mouse model

BACKGROUND Regulatory T (Treg) cells are the immune suppressors in the maintenance of immune homeostasis and tolerance to self and non-self antigens, and may have therapeutic potential in the treatment of transplant rejection in patients. However, Treg cell development and action are poorly understood in transplantation. In this study, the association of CD4(+)Foxp3(+) infiltrates within renal allograft tissue with graft survival was investigated in a mouse model. METHODS Kidney donors from C57BL/6J mice (H-2(b)) were transplanted to bilaterally nephrectomized Balb/c recipient mice (H-2(d)). Treg cells were examined with FACS and immunohistochemical staining. RESULTS Here we showed that without any immunosuppressive regimen, kidney allografts were mostly rejected from 20 to 60 days after transplantation. During the progression of allograft rejection Foxp3(+) Treg phenotype infiltrates were significantly diminished, while intragraft expression of TGF-beta1, IL-6, IL-17 and IL-23 was up-regulated. The regulatory function of CD4(+)CD25(+) infiltrates was confirmed by their suppressive activity in mixed lymphocyte reaction. Further in vitro studies indicated that primary renal tubular epithelial cell (TEC) cultures produced high levels of IL-6 in response to allogeneic lymphocyte or IL-17 stimulation, and neutralization of IL-6 increased CD4(+)CD25(+)Foxp3(+) cells in co-cultures with TEC. CONCLUSION Diminution of Foxp3(+) Treg infiltrates associates with renal allograft rejection, and neutralization of IL-6 activity enhances Foxp3(+) Treg cell differentiation. Our findings suggest that increase in intragraft IL-6 may down-regulate infiltrating Foxp3(+) Treg cells.

Reduction of cold ischemia–reperfusion injury by graft-expressing clusterin in heart transplantation

BACKGROUND Cold ischemia-reperfusion injury (IRI) is a major factor for early graft dysfunction and is associated with rejection episodes in heart transplantation. Clusterin (CLU) is a cytoprotective protein with chaperone activity. This study was designed to examine the impact of donor-expressing CLU on cold IRI. METHODS Donor hearts from wild-type C57BL/6J (H-2(b); B6 WT) vs CLU knockout C57BL/6J (H-2(b); B6 KO) mice were stored at 4°C for 8 hours, followed by heterotopic transplantation to B6 WT mice. The functional recovery of heart grafts was determined by scoring palpation, and tissue injury was determined by release of creatine kinase (CK) and lactate dehydrase (LDH) and also by histology. RESULTS Heart cells constitutively expressed CLU, and mature CLU protein was localized mostly in the endothelium as well as on the cell surface of cardiac myocytes. As compared with CLU-deficient hearts, WT hearts were more resistant to cold injury during cold preservation, and had a better functional recovery after prolonged cold preservation and transplantation. The improved graft function of CLU-expressing grafts correlated significantly with reduced neutrophil infiltration and cardiac injury, including myocytic apoptosis and necrosis. Furthermore, in vitro examination showed that ectopic expression of CLU in cultured myocytes increased cell membrane stability after exposure to cold temperature and prevented cell death. CONCLUSIONS CLU expression renders donor hearts resistance to cold IRI in transplantation, suggesting that upregulation of CLU expression in donor hearts may have potential for protecting heart grafts from cold IRI.

Renal tubular epithelial cells as immunoregulatory cells in renal allograft rejection

Kidney transplantation is the best therapeutic option for patients with end-stage kidney disease. However, the long-term survival of kidney transplants still remains relatively poor even under potent immunosuppression. Thus, it is necessary to further review the pathogenesis of renal allograft failure. Here, we discuss the potential impact of activated resident tubular epithelial cells (TECs) on infiltrating leukocyte responses on renal allograft failure. Immunohistochemical staining or in situ hybridization of renal allograft biopsies shows that activated TECs produce inflammatory cytokines and may act as nonprofessional antigen-presenting cells in the response to stimulation from leukocyte infiltration. Further experimental studies confirm that by a feedback loop, activated TECs positively or negatively regulate the destructive activity of infiltrating leukocyte through (1) alteration of leukocyte activation, proliferation, differentiation, and migration to the graft through secretion of cytokines and chemokines; and (2) direct regulation of infiltrating T-cell function through cell-cell contact. Specifically targeting kidney factors has significant impact on renal graft damage or kidney disease. This review suggests that graft TECs can regulate intragraft immune responses, and modulation of specific graft TEC responses as a therapeutic strategy may benefit long-term survival of kidney transplants.

Halofuginone suppresses T cell proliferation by blocking proline uptake and inducing cell apoptosis

Inactivation of T cells is a widely used strategy for immunosuppression. Halofuginone (HF) is an antiprotozoal agent for treating parasites in veterinary medicine, and has been demonstrated to inhibit collagen type 1 synthesis, T helper 17 cell differentiation and cytokine production in activated T cells. The present study was designed to examine the biological effects of HF against T cell receptor and interleukin (IL)-2 stimulated T cell proliferation. T cell proliferation in cultured murine splenocytes was determined by methylthiazol tetrazolium assay. Cell apoptosis was mainly determined by fluorescence-activated cell sorting with Annexin-V and 7-aminoactinomycin D staining. Here, we showed that HF significantly suppressed T cell proliferation in naïve splenocyte cultures in response to alloantigen or anti-CD3 antibody (IC₅₀, 2-2.5 nM; P<0.0001), or in activated T cell cultures in response to IL-2 (IC₅₀, 16 nM; P<0.0001) in a dose-dependent manner. HF did neither attenuate IL-2 production in anti-CD3 antibody activated T cells nor disrupt STAT5 signaling in IL-2-stimulated T cells, but its anti-T cell proliferation was correlated with an increase in cell apoptosis and a decrease in proline uptake in culture medium. Further experiments showed that proline supplement in cell culture medium significantly prevented HF-mediated suppression of T cell proliferation and cell apoptosis. In conclusion, these data suggest that HF interferes with proline incorporation or uptake, resulting in apoptosis via amino acid starvation response in T cells in the response to antigen/mitogen or IL-2 stimulation.

Immunosuppression without calcineurin inhibition: optimization of renal function in expanded criteria donor renal transplantation

Abstract:  Introduction:  To assess the efficacy of calcineurin inhibitor (CNI)‐free immunosuppression vs. calcineurin‐based immunosuppression in patients receiving expanded criteria donor (ECD) kidneys.

Early but not late allograft nephrectomy reduces allosensitization after transplant failure

INTRODUCTION Allosensitization is a significant obstacle to retransplantation for patients with primary renal graft failure. METHODS We assessed the impact of allograft nephrectomy (Group I) and weaning of immunosuppression (Group II) on percent panel reactive antibody (%PRA) at various time points after graft failure in 132 patients with a median follow-up of 47 months. Of these, 68% had allograft nephrectomy while 32% were placed on the waiting list and were either taken off immunosuppression, left on prednisone or on low-dose immunosuppressive therapy. RESULTS When groups were stratified into early (<6 months) and late (>6 months) graft failure, patients who had transplant nephrectomy for early failure demonstrated a decline in %PRA from 46% at time of graft failure to 27% at last follow-up (p = 0.02); conversely, %PRA continued to rise in Group II experiencing early allograft failure. Both Groups I and II patients with late graft failure maintained elevated %PRA at last follow-up. CONCLUSION Allograft nephrectomy may play a role in limiting allosensitization in patients with early but not late graft failures.

Requirement of clusterin expression for prosurvival autophagy in hypoxic kidney tubular epithelial cells

Cellular autophagy is a prosurvival mechanism in the kidney against ischemia-reperfusion injury (IRI), but the molecular pathways that activate the autophagy in ischemic kidneys are not fully understood. Clusterin (CLU) is a chaperone-like protein, and its expression is associated with kidney resistance to IRI. The present study investigated the role of CLU in prosurvival autophagy in the kidney. Renal IRI was induced in mice by clamping renal pedicles at 32°C for 45 min. Hypoxia in renal tubular epithelial cell (TEC) cultures was induced by exposure to a 1% O2 atmosphere. Autophagy was determined by either light chain 3-BII expression with Western blot analysis or light chain 3-green fluorescent protein aggregation with confocal microscopy. Cell apoptosis was determined by flow cytometric analysis. The unfolded protein response was determined by PCR array. Here, we showed that autophagy was significantly activated by IRI in wild-type (WT) but not CLU-deficient kidneys. Similarly, autophagy was activated by hypoxia in human proximal TECs (HKC-8) and WT mouse primary TECs but was impaired in CLU-null TECs. Hypoxia-activated autophagy was CLU dependent and positively correlated with cell survival, and inhibition of autophagy significantly promoted cell death in both HKC-8 and mouse WT/CLU-expressing TECs but not in CLU-null TECs. Further experiments showed that CLU-dependent prosurvival autophagy was associated with activation of the unfolded protein response in hypoxic kidney cells. In conclusion, these data suggest that activation of prosurvival autophagy by hypoxia in kidney cells requires CLU expression and may be a key cytoprotective mechanism of CLU in the protection of the kidney from hypoxia/ischemia-mediated injury.

Hyperbranched Polyglycerol as a Colloid in Cold Organ Preservation Solutions

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation.

Calibration and Stereo Tracking of a Laparoscopic Ultrasound Transducer for Augmented Reality in Surgery

Laparoscopic ultrasound is a useful adjunct for guidance in minimally invasive surgery. Tracking the location of the ultrasound transducer relative to the laparoscope would enable an augmented reality overlay of subsurface anatomical features on the surgeon’s field of view. The accuracy of tracking is a critical aspect for such augmented reality guidance. We propose stereo tracking of visible markers on a new “pick-up” laparoscopic ultrasound transducer and a direct transformation of the ultrasound image into the coordinates of a stereo laparoscope. We also suggest that ultrasound calibration be performed using a separate stereo camera system with a wide baseline. Such calibration is shown to improve point reconstruction accuracy from 3.1 mm to 1.3 mm.