Lewis Rat Model Research Articles

Long-term dietary l-arginine supplementation attenuates proteinuria and focal glomerulosclerosis in experimental chronic renal transplant failure

Glomerular endothelial nitric oxide synthase expression is decreased in humans during acute rejection and chronic renal transplant failure (CRTF). This may contribute to vascular damage through changes in the renal hemodynamics and enhanced endothelial adhesion of leukocytes and platelets. Dietary supplementation of l-arginine may increase endothelial NO production, thereby protecting the vascular wall and improving renal hemodynamics. We tested the hypothesis that long-term l-arginine supplementation attenuates the development of CRTF in an experimental model for renal transplantation. In the Fisher 344 to Lewis rat model for renal transplantation, renal function and histology of untreated rats was compared with rats receiving l-arginine in the drinking water (10 g/L), starting 2 days before transplantation. Every 4 weeks systolic blood pressure was measured and serum and urine were collected for measurement of nitrite and nitrate (NO x ), creatinine, and proteinuria. At 34 weeks the histological renal damage was assessed by scoring focal glomerulosclerosis and measurement of alpha-smooth muscle actin (α-SMA) expression. Urinary NO x was significantly increased in treated animals. Proteinuria was significantly lower in l-arginine-treated animals from week 24 onward ( p<0.05). Plasma creatinine and creatinine clearance did not differ between the groups. The focal and segmental glomerulosclerosis (FGS) score (max 400) at week 34 was also significantly lower in treated rats arbitrary U (20±21 vs 61±67 arbitrary U; p<0.05). The expression of α-SMA was lower in l-arginine-treated rats than in untreated rats (1.93±0.8% area surface vs 3.64±2.5% area surface). In conclusion, in this experimental model for CRTF, l-arginine administration significantly reduced FGS and proteinuria, without affecting renal function. Our data suggest that dietary l-arginine supplementation attenuates progression of CRTF and may therefore be an additional therapeutic option in human renal allograft recipients.

Antisperm autoantibody response after unilateral vas deferens ligation in rats: when does it develop?

Unilateral obstruction or injury to the vas deferens can result in significant injury to the contralateral testicle. Antisperm autoantibodies are thought to play a significant role in this phenomenon. It has been reported that early surgical repair of the vas, before the development of antisperm autoantibodies, will prevent any potential damage to the contralateral testicle. This led us to investigate the timing of the antisperm antibody production and to attempt to determine whether antibody production precedes histologic testicular damage in the Lewis rat model. In a controlled study, mature rats were divided into temporal groups, with the experimental animals all receiving a unilateral vasectomy. At postoperative endpoints of 1, 7, 15, or 30 days, blood samples were collected for immunologic assay, and the testicles were harvested for histologic examination. Antibody levels were measured by an immunobead test using goat anti-rat immunoglobulin G (IgG)-coated Sepharose beads; tissue sections were fixed in Bouin solution, embedded in paraffin, and stained with hematoxylin and eosin. There was no statistically significant histologic difference between any of the groups. However, immunologic evaluation revealed a statistically significant increase in immunobead antibody binding in the 30-day group compared to the control groups (P = .02). These data seem to indicate that in this model, antisperm antibody production is not evident until 15-30 days after unilateral injury to the vas deferens occurs, and the development of these antibodies precedes any demonstrable histologic damage to the testicle. If it is correct to infer that human antisperm antibody production will also precede histologic testicular damage, and further, that the onset of the human autoantibody response may vary from several days to weeks, then in cases of suspected or known ductal injury, the clinical monitoring of antisperm antibody levels could enable testicular damage to be predicted prior to its development and thus be avoided.

Normalization of brain death-induced injury to rat renal allografts by recombinant soluble P-selectin glycoprotein ligand.

Donor brain death has been considered a significant risk factor for both early and late organ allograft dysfunction. This central injury not only evokes an upsurge of catecholamines with resultant peripheral tissue vasoconstriction and ischemia but also promotes release of hormones and inflammatory mediators that may also affect the organs directly. One of the resultant influences of these events is the rapid upregulation of the acute-phase adhesion molecules, the selectins. These initiate leukocyte adhesion to vascular endothelium and trigger subsequent cellular and molecular changes in the compromised tissues. An established F344 --> LEW rat model of chronic rejection was used to examine (1) whether the initial inflammatory events that develop within kidney allografts from brain-dead donors could be normalized using a recombinant soluble form of P-selectin glycoprotein ligand and (2) whether amelioration of these early changes would alter the inexorable progression of chronic allograft rejection. Untreated living donor controls experienced unrelenting chronic rejection over time. This complex process was accelerated in brain-dead donor kidneys. Treatment with P-selectin glycoprotein ligand prevented the early inflammatory changes in the transplanted organs and their subsequent (200 d) functional and morphologic manifestations, particularly when the soluble ligand was administered both to the donor before organ removal and to the recipient after engraftment. This strategy of using a naturally occurring selectin ligand to prevent donor-associated chronic graft dysfunction may be of special clinical interest in cadaver donor transplantation.

Experimental Autoimmune Encephalomyelitis in the Wistar Rat: Dependence of MBP-specific T Cell Responsiveness on B7 Costimulation

Experimental autoimmune encephalomyelitis (EAE) is an animal model of human multiple sclerosis that requires the activation of autoreactive T cells for the expression of pathology. EAE has been most frequently studied in the Lewis rat model as well as in several murine models of EAE including the PLJ and B10PL strains. In the present study we describe a novel model of EAE induced in the Wistar rat strain by immunization with guinea pig spinal cord antigens and pertussis toxin (PT). T cell responses were induced to myelin basic protein. Autoreactive T cells could be totally blocked by the in vitro treatment with CTLA4Ig, a protein that blocks the costimulation of autoreactive T cells. The addition of IL-2 could reverse the inhibition seen in vitro with CTLA4Ig. The effects of inhibition of B7 costimulation were also examined by an analysis of cytokine responses and IL-2 receptor on T cells. CTLA4Ig treatment in vitro reduced the expression of IL-2 receptor on T cells, enhanced T cell apoptosis and decreased the synthesis of IL-2, IFN- n and TNF- f . CTLA4Ig treatment had no effect on IL-10 synthesis by T cells, a cytokine implicated in the functions of regulatory T cell subsets. Overall, our studies support the rationale of B7 blocking therapies as a potential treatment for models of multiple sclerosis. The induction of EAE in the Wistar rat provides yet another novel model in which to examine the regulation of T cell autoimmunity.

Differential activation of microglial cells in local and remote areas of IRBP1169-1191-induced rat uveitis.

Using a Lewis rat model of interphotoreceptor retinoid binding protein (IRBP)-induced experimental autoimmune uveitis (EAU) we examined cellular reactions in the optic pathway (retina, choroid, optic nerve, optic tract, colliculus superior, and visual cortex). Two to six animals were studied at days 0, 7, 9, 11, 12, 13, 14, 18 and 22 after immunization by immunohistochemistry with monoclonal antibodies against ED , ED2, OX6, OX22, EMAP II, AIF-1 and W3/13. In the retina, choroid and distal optic nerve increased immunoreactivity to ED1, OX6, OX22, EMAP II, AIF-1 and W3/13 was initially observed at day 9, peaked at days 13-14 and diminished rapidly from day 18 onwards. No changes were seen in the density of ED2-positive resident macrophages. In the optic tract, ED1 and OX6 expression was induced in microglial cells beginning with day 11 and persisted until day 22. AIF-1, EMAP II and ED2 expression was not visibly up-regulated and no lymphocytic infiltrates (OX22-, W3/13-positive cells) were observed. In the central projection fields, no cellular reaction could be found. Thus, cellular response in IRBP-induced rat uveoretinitis is not restricted to the eye. Microglial activation is also seen in the distal optic nerve and optic tract. This remote microglial activation, however, differs in intensity, time course and expression of activation markers, thus indicating different activation cascades. The mild remote microglial activation is probably due to neuronal-microglial interactions resulting from neuronal damage in the retinal ganglion cell layer and nerve fiber layer with consecutive axonal degeneration and not from an inflammatory reaction as seen in the eye.

The role of crosslinking in modification of the immune response elicited against xenogenic vascular acellular matrices.

We have used detergent and enzymatic extraction of natural arteries to produce an acellular matrix vascular prosthesis (AMVP). Implanted as an allograft in a canine model, this AMVP shows excellent handling characteristics, low thromboreactivity, no evidence of aneurysm, and exceptional graft patency in the peripheral vasculature. As a first step in the development of xenograft AMVPs, we processed caprine carotid arteries to AMVP and implanted them as femoral interposition grafts in dogs. Explanted xenografts at 4 weeks showed multifocal mixed inflammatory infiltrates and focal destruction of the medial elastin in the inflammatory foci. To further study the immune response to xenogenic AMVP, we implanted canine-derived AMVPs and fresh canine arteries for 4 weeks in a Lewis rat model. Extraction to AMVP markedly reduced the circulating antibody response to the xenogenic implants; however, histological analysis revealed that both xenograft arteries and AMVPs produced a marked immune response with penetration of mononuclear cells into the media and adventitia. To modify the immune response, we applied three crosslinking techniques to the canine AMVPs: glutaraldehyde, polyglycidyl ether, and carbodiimide. All crosslinkers significantly reduced degradation and cellular infiltration of the prostheses. However, crosslinking neither eliminated the chronic inflammatory response surrounding the implants nor reduced the humoral response to the xenogenic materials.

Osteoprotegerin (OPG) gene therapy in animal models of osteoarticular disease

The soluble decoy receptor osteoprotegerin (OPG) regulates bone resorption by inhibiting osteoclast formation, function and survival. We investigated OPG gene therapy as a means of ameliorating chronic osteoarticular disease using a mouse ovariectomy (OVX) model of estrogen deficiency-induced osteoporosis (Mol Ther 2001, 3:1–9). Young adult female mice injected once with an adenoviral (Ad) vector carrying a human fusion protein combining the OPG ligand-binding and immunoglobulin constant domains (hOPG-Fc) developed serum OPG levels that exceeded the threshold needed for efficacy (as assessed by a marked increase in bone density) for over 12 months. The extent of OPG production and skeletal enhancement was titer-dependent. Mice subjected to OVX or sham surgery and then treated with Ad-hOPG-Fc had significantly more bone volume and fewer osteoclasts in axial and appendicular bones after 4 weeks. In contrast, animals given OVX and either a control vector expressing β-galactosidase or vehicle had significantly less bone than did comparably treated, sham-operated mice. This study confirms that a single Ad gene transfer can produce persistent high-level OPG expression and shows that OPG gene therapy may prove useful in treating osteoporosis. We anticipate that OPG gene therapy will offer similar promise as a bone-sparing agent in chronic arthritis since we have shown previously that injected OPG significantly inhibits skeletal erosion in the Lewis rat model of mycobacterial-induced adjuvant arthritis (Nature 1999, 402:304–309).

T cell-mediated antigen presentation: a potential mechanism of infectious tolerance.

Differentiation of the T cell repertoire and the physiology of T cell-mediated antigen presentation are reviewed in relation to mechanisms of self-tolerance. Recent research has indicated that T cell development is a continual process that optimizes partial recognition of self as a homeostatic set-point. Specific T cell antigen recognition of partial agonists is intrinsically linked to expression of class II MHC glycoproteins on T cells. Even ligands that act as TCR antagonists in IL-2 production assays have sufficient agonistic strength to induce expression of class II MHC glycoproteins on T cells. Thus, the intrinsic self-reactivity of the T cell repertoire may promote T-APC activity in vivo and may explain why thymic and peripheral T cells express low but significant levels of class II MHC glycoproteins. T-APC activity induces extensive apoptosis among responder T cells, causes desensitization among surviving responders, and has been implicated in the adoptive transfer of tolerance in the Lewis rat model of experimental autoimmune encephalomyelitis. Overall, these findings support a relationship between the partial recognition of self MHC ligands, expression of class II MHC glycoproteins on mature peripheral T cells, tolerogenic T cell-mediated antigen presentation, and desensitization of pathogenic self-reactive T cells.

Chronic blockade of angiotensin II action prevents glomerulosclerosis, but induces graft vasculopathy in experimental kidney transplantation.

Long-term renin-angiotensin system blockade is beneficial in a variety of renal diseases. This study examines the long-term (34 weeks) effects of the angiotensin-converting enzyme inhibitor lisinopril and the angiotensin II receptor type I blocker L158,809 in the Fisher to Lewis rat model of chronic renal transplant failure. Treatment in allografted rats with lisinopril or L158,809 was initiated 10 days after transplantation, or at the time when proteinuria exceeded 50 mg/24 h. Untreated allografts and syngrafts served as controls. In contrast to syngrafts, untreated allografts developed proteinuria, hypercholesterolaemia, interstitial damage, and glomerulosclerosis. Lisinopril or L158,809 treatment in allografts starting at day 10 after transplantation completely prevented this, with the exception of interstitial damage, but this treatment also caused a reduction in blood pressure and renal function. Moreover, the intimal surface area of the renal arteries was dramatically increased in allografts treated with either lisinopril or L158,809 compared with untreated allografted rats. Treatment once proteinuria had developed was less effective in preventing glomerulosclerosis, but also caused less intimal expansion. Thus, chronic renin-angiotensin system blockade preserves glomerular morphology in the absence of proteinuria, but enhances intimal hyperplasia and reduces renal function in experimental transplantation. In view of these results, it should be questioned whether such treatment benefits renal transplant patients in the long term.

HIV-Specific CD4(+) and CD8(+) immune responses are generated with a gp120-depleted, whole-killed HIV-1 immunogen with CpG immunostimulatory sequences of DNA.

We examined the adjuvant effects of a synthetic CpG oligodeoxynucleotide immunostimulatory sequence (ISS) using a whole-killed, gp120-depleted HIV antigen (HIV-1 antigen) in a Lewis rat model. We hypothesized that HIV-1-specific CD4(+) T helper (Th) immune responses could be enhanced when an ISS was combined with an HIV-1 antigen in incomplete Freund's adjuvant (IFA). We also reasoned that if such Th responses were sufficient, such a combination might also induce HIV-specific CD8(+) T cell immune responses. Here we demonstrate that the HIV-1 antigen in IFA combined with ISS stimulates both CD4(+) and CD8(+) HIV-specific immune responses as measured by interferon-gamma (IFN-gamma) in the ELISPOT assay. A strong correlation between these CD4(+) and CD8(+) responses was demonstrated. Furthermore, we found that the HIV-1 antigen in IFA with ISS as an adjuvant stimulated strong antibody responses to core antigen (p24). These studies suggest that the combination of the whole-killed, gp120-depleted HIV-1 antigen in IFA with ISS may be an ideal candidate to test in nonhuman primates and in human studies as a preventive HIV-1 vaccine.

Giant cell myocarditis: diagnosis and treatment.

Giant cell myocarditis is a rare but devastating disease that usually affects young otherwise healthy individuals. Associations with thymoma, inflammatory bowel disease, and a variety of autoimmune disorders have been reported. The rate of death or heart transplantation is approximately 70% at 1 year. Data from a Lewis rat model and from observational human studies suggest that giant cell myocarditis is mediated by T lymphocytes and may respond to treatment aimed at attenuating T cell function. Recent findings from the Giant Cell Myocarditis Registry, a clinical and pathologic database from 63 cases of giant cell myocarditis gathered from 36 medical centers, include the following: The sensitivity of endomyocardial biopsy for giant cell myocarditis for patients who undergo transplantation or autopsy is 82 to 85%. Registry subjects who received cyclosporine in combination with steroid, azathioprine, or muromonab-CD3 have prolonged transplant-free survival (12.6 months vs. 3.0 months for no immunosuppression). Post-transplantation survival is approximately 71% at 5 years despite a 25% rate of giant cell infiltration in the donor heart. To confirm and extend these findings, a randomized trial of immunosuppression including muromonab-CD3, cyclosporine, and steroids is underway.

A peptide vaccine that prevents experimental autoimmune myasthenia gravis by specifically blocking T cell help

Myasthenia gravis (MG) and its animal model, experimental autoimmune (EA) MG, are caused by T cell-dependent autoantibodies that react with the nicotinic acetylcholine receptor (AChR) on muscle and interfere with neuromuscular transmission. Thus, selective inactivation of CD4(+) AChR-specific T helper cells should lower AChR Ab levels and ameliorate disease. In the Lewis rat model of EAMG, alpha chain residues 100-116 of the AChR represent the dominant T cell epitope, which is important in helping Ab responses to this autoantigen. In the present report, we have applied a new design technique that requires no knowledge of Ag receptor sequences on errant T cells in order to develop a synthetic peptide vaccine against T cells reactive with the aforementioned T cell epitope. Immunization with the peptide 1) induced polyclonal and monoclonal Ab, which inhibited AChR 100-116 stimulation of AChR-sensitized lymphocytes and recognized Vbeta15 containing T cell receptors on AChR 100-116-specific T cell lines and clones; 2) lowered AChR Ab levels; 3) reduced the loss of muscle AChR; and 4) lessened the incidence and severity of EAMG. These findings suggest a new strategy for the functional abrogation of epitope-specific T cells that could have potential application to human autoimmune diseases.

Cyclosporin A protects striatal neurons in vitro and in vivo from 3-nitropropionic acid toxicity

The neuroprotective properties of cyclosporin A (CsA) are mediated by its ability to prevent mitochondrial permeability transition during exposure to high levels of calcium or oxidative stress. By using the mitochondrial toxin 3-nitropropionic acid (3NP), the present study assessed whether CsA could protect striatal neurons in vitro and in vivo. In vitro, 3NP produced a 20-30% reduction of striatal glutamic acid decarboxylase-immunoreactive (GAD-ir) neurons. A single treatment with CsA protected GAD-ir neurons from 3NP toxicity at lower (0.2 or 1.0 microM), but not at higher (5.0 microM) doses. Similar findings were seen when the cultures were treated twice with cyclosporin. In vivo experiments used the Lewis rat model of Huntington's disease (HD) in which a low 3NP dose was delivered subcutaneously through an osmotic minipump. Rats received unilateral or bilateral intrastriatal saline injections to disrupt the blood-brain barrier (BBB) and facilitate CsA reaching vulnerable neurons. In the first experiment, CsA treated 3NP-lesioned rats displayed significantly more dopamine-and adenosine-3;, 5;-monophosphate-regulated phosphoprotein (DARPP32-ir) neurons ipsilateral to BBB disruption compared to the contralateral intact striatum, indicating that disruption of the BBB maybe necessary for CsA's neuroprotective effects. In the second experiment, stereological counts of DARPP32-ir neurons revealed that CsA protected striatal neurons in a dose-dependent manner following bilateral disruption of the striatal BBB. Rats treated with the higher (15 or 20 mg/kg) but not lower (5 mg/kg) doses of CsA displayed greater numbers of DARRP32-ir striatal neurons relative to vehicle-treated 3NP-lesioned rats. Thus, under conditions in which CsA can gain access to striatal neurons, significant protection from 3NP toxicity is observed. Therefore, CsA or more lipophilic analogues of this compound, may be of potential therapeutic benefit by protecting vulnerable neurons from the primary pathological event observed in HD.

In vivo biocompatibility evaluation of Cibacron blue-agarose.

This study investigated the biocompatibility of Cibacron blue-agarose as a biomaterial for microencapsulation. Cibacron blue-agarose is known to have an affinity for albumin under certain pH conditions and in the proper steric environment. Thus it was postulated that the material's high affinity for host albumin might reduce a secondary immune response and reduce the fibrotic overgrowth that often accompanies transplanted foreign materials. In vivo tests were performed using the Lewis rat model. Both Cibacron blue-agarose and plain agarose disks were prepared, with some disks from each group being pre-exposed to sera from Lewis rats. The disks were transplanted into the peritoneal cavities of Lewis rats. After 115 days the disks were excised. Fibrotic overgrowth was analyzed using light microscopy, and a blind study was used to measure the average growth thickness on each disk. The results demonstrated that all disks developed some fibrotic encapsulation and that the presence of Cibacron blue was not significant in reducing fibrotic overgrowth (p = 0.62). Agarose disks pre-exposed to sera had significantly less average overgrowth than any other group (p = 0. 06).

A Complementary Peptide Vaccine That Induces T Cell Anergy and Prevents Experimental Allergic Neuritis in Lewis Rats

We have developed and described a new method of altering T cell-mediated autoimmune diseases by immunization with the complementary peptide against T cell epitopes. The complementary peptide (denoted NAE 07-06) to the bovine P2 protein, residues 60-70 (denoted EAN 60-70), was tested in the Lewis rat model of experimental allergic neuritis (EAN). Immunization with NAE 07-06 induced polyclonal and monoclonal Abs that inhibited the proliferation of the P2-specific T cell line, stimulated with EAN 60-70, and recognized Vbeta, but not Valpha, of TCRs. Proliferation of T cells treated with anti-NAE 07-06 Abs could be partially restored by treatment with rIL-2, in accordance with an anergy model. A homologous sequence was found between NAE 07-06 and the VDJ junction of the TCR beta-chain from an EAN 60-70-specific T cell line. Rats preimmunized with NAE 07-06 in vivo before EAN induction showed less disease severity clinically and histologically. These data suggest a new therapeutic approach for T cell-mediated autoimmune disorders through the induction of anti-TCR Abs with complementary peptide Ags.

Current Literature: Fas-mediated therapy for autoimmune disease.

Zhou, T., Song, L., Yang, P., Wang, Z., Lui D. and Jope, R.S. (1999) Bisindolylmaleimide VIII facilitates Fas-mediated apoptosis and inhibits T cell-mediated autoimmune diseases Nat. Med. 5, 42–49

Bisindolylmaleimide VIII facilitates Fas-mediated apoptosis and inhibits T cell-mediated autoimmune diseases.

Fas-mediated apoptosis is essential for the elimination of cells, and impaired apoptosis can have severe detrimental consequences. Bisindolylmaleimide VIII potentiated Fas-mediated apoptosis in human astrocytoma 1321N1 cells and in Molt-4T cells, both of which were devoid of apoptosis induced by anti-Fas antibody in the absence of bisindolylmaleimide VIII, and in Jurkat and CEM-6 T cells, which showed slight and moderate apoptotic responses, respectively, to low levels of Fas stimulation. Potentiation of Fas-mediated apoptosis by bisindolylmaleimide VIII was selective for activated, rather than non-activated, T cells, and was Fas-dependent, as it was not observed in T cells from Fas-deficient lpr/lpr mice. Administration of bisindolylmaleimide VIII to rats during autoantigen stimulation prevented the development of symptoms of T cell-mediated autoimmune diseases in two models, the Lewis rat model of experimental allergic encephalitis and the Lewis adjuvant arthritis model. Thus, the use of agents such as bisindolylmaleimide VIII may be therapeutically useful for supporting more effective elimination of detrimental cells through enhancement of Fas-dependent apoptosis signaling.

Late blockade of T cell costimulation interrupts progression of experimental chronic allograft rejection.

Early blockade of T cell-costimulatory activation pathways prevents development of experimental chronic allograft rejection. Ongoing T cell recognition of alloantigen and activation may also play an important role in progression of chronic rejection, but definitive evidence is lacking. We used the fusion protein CTLA4Ig to block CD28-B7 T cell costimulation late after the onset of initial graft injury. Using the F334 into LEW rat model of chronic renal allograft rejection, transplant recipients were treated with a 10-d course of cyclosporine, and a subgroup received a single injection of CTLA4Ig at 8 wk after transplant. Functionally, CTLA4Ig administration prevented development of progressive proteinuria (14.3+/-4.1 mg/24 h versus 41.0+/-12.0 mg/24 h at 24 wk after transplant, P < 0.05). Histologically, graft mononuclear cell infiltration, glomerular hypertrophy, focal and segmental glomerulosclerosis, and intimal vascular hyperplasia were all attenuated in CTLA4Ig-treated animals. Lastly, reverse transcriptase-PCR and immunohistologic studies showed a significant reduction in the intragraft expression of key products of T cell and macrophage activation, and upregulation of what have recently been termed as "protective" genes, including the bcl family members, Bcl-2 and Bcl-xL, and hemoxygenase. Our data are the first to demonstrate that blocking T cell-costimulatory activation late after transplantation, after initial graft injury, prevents progression of chronic allograft rejection supporting the hypothesis that ongoing T cell recognition of alloantigen and activation are key mediators of ongoing chronic allograft rejection.

IMMUNOMODULATORY FUNCTION OF LOW MOLECULAR WEIGHT HYALURONATE DELAYS ALLOGRAFT REJECTION AND IMPROVES SURVIVAL IN A RAT ACUTE KIDNEY REJECTION MODEL.

517 CD44 is an important T cell surface proteoglycan that mediates diverse T- cell functions including adhesion, homing, migration, activation and signal transduction. Its ligands include collagen, fibrinogen and hyaluronate (HA). Since HA can inhibit lymphocyte adhesion and transmigration in vitro, we assessed its immunomodulatory effects in the WF to LEW rat model of acute kidney allograft rejection. Male WF donor kidney was transplanted orthotopically into a male LEW recipient after receiving a unilateral nephrectomy on day O. Contralateral nephrectomy of the naive kidney was performed on day 5. Renal allograft rejection was defined as death due to uremia. Graft survival was estimated using a Kaplan Meier survival plot. Serum creatinine was measured on day 5 and day 30 in the control and experimental group as an index of allograft function. Control animals received either no treatment (n=4) or a low dose regimen of CsA alone (1mg/day s.c.) for 5 days from the day of transplantation (n=13). Experimental animals received CsA(1mg/day, s.c.) in combination with HA (100μg/day, i.p.) for 5 days (n=19). The mean survival of the no-treatment control group was 15±0.9 days while the CsA treated group rejected their graft at 21±4.8 days (mean± SD). FigureCombined HA+CsA treatment resulted in a significant prolongation of graft survival (mean survival 52.7±7.6 days) compared to animals receiving CsA alone. HA treatment alone (day 0-5) did not prolong graft survival (mean 7.5±0.64 days) (n=4). In both CsA and CsA+HA treated group serum creatinine measurements on day 7 were consistently low (0.56±01 1mg/dl vs. 0.85±0.36mg/dl). On day 30, however, the HA+CsA group had a significantly lower serum creatinine as compared to the CsA alone group(1.38±0.8mg/ml vs. 2.9±0.55mg/ml) (p<0.05). In additional experiments animals treated with either CsA alone or combined CsA and HA were harvested on day 7 in order to investigate renal allograft morphology using a modified BANFF scoring system. Each sample was assessed by an independent renal pathologist in a blinded fashion. The CsA group (n=3) had a mean score of 1.66±0.57 whereas in the HA+CsA group two renal allografts showed borderline changes according to BANFF and the third renal allograft showed a normal allograft morphology without any signs of allograft rejection. In conclusion, addition of short term low molecular HA treatment to a low dose CsA regimen significantly improved renal allograft survival in the rodent model of acute allograft rejection. Mechanisms of action include stimulation or blockade of the CD44 receptor, altered migration and adhesion behavior. Further work is required to elucidate the importance of the interaction of low molecular HA in mediating the inhibitory effect on the immune response.

Prevention of late renal changes after initial ischemia/reperfusion injury by blocking early selectin binding.

Increasing clinical evidence suggests that delayed initial function secondary to ischemia/reperfusion injury alone, and particularly in combination with early episodes of acute rejection, reduces kidney allograft survival over time. We investigated changes developing over the long term following a standardized ischemia/reperfusion insult in a Lewis rat model. The left kidney was isolated in a uninephrectomized host and cooled, and the pedicle was clamped for 45 min. Animals were followed for 48 weeks after initial renal injury. Organs were removed serially (4, 8, 16, 24, 32, 40, and 48 weeks) for immunohistology and reverse transcriptase polymerase chain reaction. Progressive proteinuria developed after 8 weeks. By immunohistology, CD4+ leukocytes and ED-1+ macrophages infiltrated the ischemic organs in parallel with up-regulation of major histocompatibility complex class II antigen expression. Because macrophages have been shown to be critical in chronic changes in other models, they were examined primarily in these studies. By reverse transcriptase polymerase chain reaction, macrophage-derived, fibrosis-inducing factors (transforming growth factor-beta, interleukin 6, and tumor necrosis factor-alpha) remained highly and constantly expressed throughout the follow-up period. The long-term influence of initial treatment with the soluble form of P-selectin glycoprotein ligand-1, a soluble ligand for P- and E-selectin, was then examined. All functional and structural changes remained at relative baseline, similar to uninephrectomized controls. These data suggest that blocking the initial selectin-mediated step after ischemia/reperfusion injury, which triggers significant early cellular and molecular events, also reduces later renal dysfunction and tissue damage over time. In part, the findings may be explained by the sparing of functioning nephron units, which if destroyed or compromised by the original insult, may contribute to long-term graft failure. This approach may be important clinically in the transplantation of kidneys from non-heart-beating or marginal donors or organs experiencing prolonged ischemic times.