Debra A. Hullett

RS-61443 : A NEW, POTENT IMMUNOSUPPRESSIVE AGENT

The immunosuppressive activity of RS-61443, a semisynthetic derivative of mycophenolic acid, was examined in 33 canine renal allografts. Initial studies established that triple therapy consisting of 20 mg/kg RS-61443 in combination with 5 mg/kg cyclosporine and 0.1 mg/kg methylprednisolone was the optimal combination to prevent graft rejection. The median survival time was 8.1 +/- 1.2 days in dogs without treatment (n = 5), 8.5 +/- 1.7 days in the treatment control group (CsA 5 mg/kg, MP 0.1 mg/kg; n = 6), 36.0 +/- 9.6 days with RS-61443 monotherapy (40 mg/kg; n = 6); and 122.4 +/- 38.75 days with triple therapy (n = 16). Graft prolongation was statistically significant when compared with controls (P less than 0.05 and 0.002, respectively). Six recipients in the triple therapy group survived over 150 days without major adverse effects. Long-term administration of RS-61443 (20 mg/kg/day) did not cause nephrotoxicity, hematotoxicity, or hepatotoxicity, with the exception of a slight elevation of the alkaline phosphatase levels. Gastrointestinal symptoms including gastritis, diarrhea, and anorexia were common, especially under 40 mg/kg RS-61443 monotherapy, and appeared to be dose-related. Despite its immunosuppressive activity, an increased susceptibility to bacterial or viral infections was not observed. Histological studies of the kidney grafts revealed slight interstitial cell infiltration without vascular or glomerular damage.

Prolongation of allograft survival by 1,25-dihydroxyvitamin D3.

BACKGROUND 1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, is now believed to play a significant role in the immune responses, both in vitro and in vivo, preventing the development of several autoimmune diseases. These studies suggest that 1,25-dihydroxyvitamin D3 may be effective in prolonging allograph survival. METHODS To test the hypothesis that 1,25-dihydroxyvitamin D3 would prolong allograft survival, neonatal heart grafts were transplanted to allogeneic recipients receiving either 19-nor-1,25-dihydroxyvitamin D2 (200 ng/day) or 1,25-dihydroxyvitamin D3 (50 ng/mouse/day) orally through the diet. The efficacy of 1,25-dihydroxyvitamin D3 in prolonging graft survival in a vascularized model was determined by heterotopic ACI to Lewis heart transplants. RESULTS The provision of exogenous 1,25-dihydroxyvitamin D3 or an analog, 19-nor-1,25-dihydroxyvitamin D2, to mice markedly prolonged the survival of neonatal mouse heart allografts. Similar results were obtained with a vascularized heterotopic heart transplant model in rats. Cyclosporine at a maximum 25 mg/kg dose for mice proved less effective than 1,25-dihydroxyvitamin D3. Graft survival in mice differing at class I and class II loci (B10.A(4R) --> C57BL/10) increased from 13.0+/-1.1 days to 51.0+/-5.6 days and was significantly better than cyclosporine monotherapy (33.2+/-3.6). Rat heart survival in a high responder strain combination (ACI --> Lewis) increased from 6.2+/-0.3 to 25.2+/-2.8 days. The increased survival of the transplants brought about with 1,25-dihydroxyvitamin D3 was not accompanied by hypercalcemia in rats. CONCLUSION These results suggest that 1,25-dihydroxyvitamin D3 can be used as an effective agent in preventing graft rejection.

Epithelial-to-mesenchymal transition and oxidative stress in chronic allograft nephropathy.

Epithelial‐to‐mesenchymal transition (EMT) and oxidative stress contribute to kidney tissue fibrosis in various forms of native kidney disease. However, their role in chronic allograft nephropathy (CAN) remains somewhat uncertain. To address this question, kidney transplants were performed in 3‐month‐old rats, using the Fisher 344 → Lewis model of CAN. Six‐month posttransplant, kidney allografts displayed significant tubular atrophy, interstitial fibrosis and vascular wall thickening. Allograft recipients had significantly higher levels of serum creatinine (4.7 ± 1.3 versus 0.59 ± 0.08 mg/dL, p = 0.03) and proteinuria (380 ± 102 versus 30.2 ± 8 mg/dL, p = 0.04) compared to syngeneic grafts. Semiquantitative PCR, immunoblot and immunohistochemical analyses demonstrated increased α‐smooth muscle actin (α‐SMA) mRNA and protein levels coupled with reduced E‐cadherin mRNA and protein immunoreactivity, confirming the presence of CAN‐associated EMT. Allograft α‐SMA levels were increased as early as 1–2 weeks posttransplant. Immunohistochemical studies for collagen type I and III, superoxide anion (O2−), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) confirmed that tubular O2−, eNOS and iNOS, and interstitial collagen I, III and O2− levels were significantly increased in CAN‐associated EMT. In conclusion, these observations suggest that CAN‐associated EMT may be a link between oxidative stress and allograft fibrosis.

1,25-Dihydroxyvitamin D3 prolongs graft survival without compromising host resistance to infection or bone mineral density

BACKGROUND Recently, we have shown that 1,25-dihydroxyvitamin D3 prolongs graft survival in mice and rats when the donor and recipient differ at two or more major histocompatability loci. Among the most serious side effects encountered with the currently available transplantation antirejection drugs are an increased susceptibility to infection and decreased bone mineralization. Our results suggest that 1,25-dihydroxyvitamin D3 prolongs graft survival without these side effects of bone loss and susceptibility to infection. METHODS We compared the ability of 1,25-dihydroxyvitamin D3-treated, nontreated, or cyclosporine (CsA)-treated mice to resist infection with Candida albicans and herpes simplex virus-1. To determine bone density, femurs were collected from nontreated, 1,25-dihydroxyvitamin D3-treated (50 ng/mouse/day), or CsA-treated (25 mg/kg/day) mice, and bone ash was determined. RESULTS Here we show that 1,25-dihydroxyvitamin D3 treatment does not increase the susceptibility of the host to fungal or viral infection. Furthermore, CsA causes bone loss, whereas 1,25-dihydroxyvitamin D3 actually increases bone mass. CONCLUSIONS The use of 1,25-dihydroxyvitamin D3 and its analogs to increase transplant survival will avoid bone loss and opportunistic infection, two important disadvantages of the most widely used transplant antirejection drugs--CsA and the glucocorticoids.

Monocyte Chemotactic Protein-1 Expression Is Associated With the Development of Vein Graft Intimal Hyperplasia

Infiltration of immunologically active cells into vein grafts is concomitant with the development of intimal hyperplasia (IH) and often leads to obliterative stenosis and graft failure. Previous work has demonstrated the prolonged presence of monocytes and macrophages in vein grafts. The stimuli attracting these macrophages remain unidentified. Monocyte chemotactic protein-1 (MCP-1), a potent and specific chemokine for monocytes/macrophages, is secreted by smooth muscle cells, endothelial cells, fibroblasts, and leukocytes, all of which are present in grafted veins. In this study, we examined the temporal profile of MCP-1 gene expression in rat vein grafts by using reverse transcription-polymerase chain reaction (PCR) and immunohistochemistry. Epigastric vein-to-femoral artery bypass grafts were microsurgically placed and harvested at various time points after grafting. Histological analysis confirmed the consistent development of IH. PCR was performed and relative levels of MCP-1 quantified by autoradiography. Our results show that MCP-1 mRNA levels increased 28-fold by 4 hours after grafting and up to 117-fold by 1 week. After this time MCP-1 mRNA levels decreased; nonetheless, even at 8 weeks after grafting, message levels remained elevated 7-fold above baseline. Immunoreactive MCP-1 protein and ED1+ macrophages were detected at all time points; the degree of immunostaining correlated with MCP-1 mRNA levels. Our results support the hypothesis that upregulation of MCP-1 gene expression in vein grafts results in the recruitment of monocytes and tissue macrophages to the vein wall, which leads to IH. The correlation between monocyte/ macrophage infiltration and IH suggests a critical role for these cells in IH development.

Nox-2 Is a Modulator of Fibrogenesis in Kidney Allografts

We studied the role of classical phagocytic NADPH oxidase (Nox) in the pathogenesis of kidney allograft tubulointerstitial fibrosis. Immunofluorescence studies showed that Nox‐2 and p22phox (electron transfer subunits of Nox) colocalized in the tubulointerstitium of human kidney allografts. Tubular Nox‐2 also colocalized with α‐SMA in areas of injury, suggestive of epithelial‐to‐mesenchymal transition (EMT). Interstitial macrophages (CD68+) and myofibroblasts (α‐SMA+) expressed Nox‐2 while graft infiltrating T cells (CD3+) and mature fibroblasts (S100A4+) were Nox‐2−. These results were confirmed in the Fisher‐to‐Lewis rat kidney transplant model. Areas of tubulitis were associated with Nox‐2 and α‐SMA, suggestive of EMT. Immunoblot analyses showed that Nox‐2 upregulation was associated with oxidative stress (nitrotyrosine) and fibrogenesis (α‐SMA and phospho‐Smad2) at 3 weeks and 6 months. Allografts treated with Nox inhibitors (DPI or apocynin) for 1 week showed reduced fibronectin and phospho‐Smad2 and increased E‐cadherin levels. Cyclosporine A, TGF‐β1 and angiotensin II increased Nox‐2 mRNA levels 2‐ to 7‐fold in vitro (NRK52E cells). Treatment with specific Nox inhibitors (DPI or apocynin) prevented the downregulation of E‐cadherin and upregulation of fibronectin transcripts. In aggregate, these studies suggest that Nox‐2 is involved in the pathogenesis of allograft tubulointerstitial fibrosis via activation transcription factor Smad2, EMT and myofibroblasts.

Xenogeneic transplantation of porcine islets: an overview.

The extreme demand for human organs or tissues for transplantation has driven the search for viable alternatives. Pigs are considered a possible source of tissue for a number of reasons including shared physiology, plentiful supply, short gestation, and, more recently, the generation of transgenic animals. Porcine islets show promise as a source of islets for the treatment of type 1 diabetes mellitus. Porcine islets regulate glucose levels in the same physiologic range as humans, and porcine insulin has been used for years as an exogenous source of insulin for glucose control. In this review, we discuss the advantages and disadvantages of the use of adult or neonatal porcine islets, the immunologic challenges facing transplantation of xenogeneic islets, and the concerns regarding transmission of infectious agents between species. Porcine islets isolated from both adult and neonatal pigs are capable of restoring euglycemia in experimental animal models of diabetes. Adult islets are more difficult to isolate, whereas neonatal islets have great proliferation potential but require several weeks to function posttransplantation. Xenogeneic islets are susceptible to complement-mediated lysis after the binding of preformed natural antibodies and cellular immunity involving both macrophages and CD4+ T cells. In addition, the potential for transmission of porcine endogenous retroviruses, porcine cytomegalovirus, and porcine lymphotropic herpesvirus type 1 are all concerns that must be addressed. Despite the challenges facing xenotransplantation, the extreme need for donor organs and tissues continues to drive progress toward overcoming the unique issues associated with transplantation between species.

HSP27 is involved in the pathogenesis of kidney tubulointerstitial fibrosis

We hypothesized that heat shock protein 27 (HSP27), a small heat shock protein with actin-remodeling properties, is involved in the pathogenesis of kidney tubulointerstitial fibrosis. We first examined its expression in the rat unilateral ureteral obstruction (UUO) model of kidney fibrosis and epithelial-to-mesenchymal transition (EMT). Immunoblot analyses showed that UUO resulted in significant upregulation of TGF-beta1, alpha-smooth muscle actin (alpha-SMA), total and phosphorylated HSP27, and phosphorylated p38MAPK. Immunofluorescence studies showed that HSP27 costained with TGF-beta1, alpha-SMA, and E-cadherin in areas of tubulointerstitial injury. We next attempted to translate these studies in an in vitro model of EMT using rat proximal tubular epithelial cells (NRK52E). TGF-beta1 (20 ng/ml) treatment resulted in EMT (upregulation of alpha-SMA and downregulation of E-cadherin) and significant upregulation of total and phosphorylated HSP27 and p38MAPK after 3 days. Real-time PCR analyses showed that HSP27, vimentin, and fibronectin increased whereas E-cadherin mRNA levels decreased. Double-staining immunofluorescence studies showed intracytoplasmic colocalization of HSP27 with both F-actin and E-cadherin in cells undergoing EMT. HSP27 overexpression by transient transfection significantly increased E-cadherin while decreasing E-cadherin repressor Snail levels. In aggregate, these studies show that HSP27 is involved in the pathogenesis of TGF-beta1-induced EMT and chronic tubulointerstitial fibrosis. HSP27 overexpression may delay injury by upregulating E-cadherin through downregulation of Snail.

A Retrospective Evaluation of 1,25-Dihydroxyvitamin D3 and Its Potential Effects on Renal Allograft Function

Background/Aims: 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) plays an important role in regulating immunologic responsiveness in addition to its effects on bone metabolism. This is potentially beneficial in the transplant setting. Animal studies have demonstrated the utility of 1,25-(OH)2D3 in prolonging allograft survival. Therefore, we evaluated the effects of 1,25-(OH)2D3 (oral calcitriol) in human renal transplant recipients. Methods: A case-control study was undertaken assessing the effects of calcitriol on transplant function. The effect of calcitriol on renal function was analyzed using general linear mixed modeling of the change in slope of serum creatinine (Scr) prior to and following the start of calcitriol therapy. Results: There was a significant increase in baseline Scr (p < 0.001) prior to starting calcitriol. Following initiation of calcitriol, there was a deceleration in the rate of loss of graft function (p = 0.031 at day 300 of therapy). Graft survival was also prolonged in calcitriol-treated patients compared to a control population with evidence of chronic allograft nephropathy but no calcitriol therapy (p < 0.03). Overall, there were no adverse or harmful effects related to calcitriol therapy. 1,25-(OH)2D3 therapy was associated with (1) a deceleration in the rate of loss of renal function in transplant recipients with more than one year of allograft function, and (2) no significant change in allograft function early after transplantation. Conclusion: These data suggest that short- and long-term prospective trials evaluating 1,25-(OH)2D3 or 1,25-(OH)2D3 analogs in human kidney transplantation are warranted. Such trials may help us elucidate mechanism and duration of action, as well as safety issues related to these novel immunomodulatory agents.

Heat shock protein 27 in chronic allograft nephropathy: a local stress response.

Background. Heat shock protein (HSP) 27 plays a cytoprotective role through its antioxidant, antiapoptotic, and actin-stabilizing properties during cell stress. The authors hypothesized that HSP27 is involved in chronic allograft nephropathy (CAN), a chronic state of inflammation and stress. Methods. The authors used the Fisher 344-to-Lewis model of CAN. Transplants were performed in 3-month-old recipient rats. HSP27 mRNA and protein levels were determined using semiquantitative polymerase chain reaction, microarray (stress-toxicity, GEArray) analyses, gene sequencing, immunoblotting, and immunohistochemical analyses at 10 days and 6 months posttransplant. P38 mitogen-activated protein kinase (MAPK), manganese (Mn) superoxide dismutase (SOD), copper-zinc (CuZn) SOD, FasL, Bax, hypoxia-inducible factor (HIF)-1&agr;, and CD3+ lymphocytes were studied in parallel as selective biomarkers of oxidative stress (OS), apoptosis, hypoxia, and graft-infiltrating immune cells. Results. Six months after transplantation, kidney allografts displayed histologic and functional features of CAN, including tubular atrophy, interstitial fibrosis, glomerulosclerosis, and increased proteinuria and serum creatinine levels. HSP27 mRNA and protein levels in CAN were reduced by 50% and 85%, respectively (P=0.04). Immunohistochemical analyses revealed a “shift” in HSP27 from the medulla to the cortex in allografts with CAN. Bax, phosphorylated p38-MAPK, HIF-1&agr;, and MnSOD followed a parallel relocation pattern. CD3+ lymphocyte density and tubular FasL expression were also greater in the cortex of allografts with CAN. Time-course analyses revealed that most of these changes were present as early as 10 days posttransplant. Conclusions. The shift of HSP27 from the medulla to the cortex, combined with greater CD3+, p38-MAPK, Bax, FasL, HIF-1&agr;, and MnSOD immunoreactivity in this area of the kidney, likely represents an allograft-level response to CAN-related OS-hypoxia.