Calpain inhibitor-1 reduces renal ischemia/reperfusion injury in the rat.

BACKGROUND: Activation of the cysteine protease calpain has been implicated in renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of calpain inhibitor-1 (Cal I-1) in an in vivo model of renal I/R injury. METHODS: Male Wistar rats were administered Cal I-1 (10 mg/kg, IP) 30 minutes before undergoing bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Plasma concentrations of urea, creatinine, Na(+), gamma-glutamyl transferase (gamma GT), aspartate aminotransferase (AST) and urinary Na(+), glutathione S-transferase (GST), and N-acetyl-beta-D-glucosaminidase (NAG) were measured for the assessment of renal dysfunction and I/R injury. Creatinine clearance (C(Cr)) and fractional excretion of Na(+) (FE(Na)) were used as indicators of glomerular and tubular function, respectively. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of neutrophil infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). RESULTS: Cal I-1 significantly reduced I/R-mediated increases in urea, creatinine, gamma GT, AST, NAG, and FE(Na) and significantly improved C(Cr). Cal I-1 also significantly reduced kidney MPO activity and MDA levels. Cal I-1 also reduced histologic evidence of I/R-mediated renal damage and caused a substantial reduction in the expression of iNOS and COX-2, both of which involve activation of nuclear factor-kappa B (NF-kappa B). CONCLUSIONS: : These results suggest that Cal I-1 reduces the renal dysfunction and injury associated with I/R of the kidney. We suggest that the mechanism could involve the inhibition of I/R-mediated activation of NF-kappa B.

Tempol, a membrane-permeable radical scavenger, reduces oxidant stress-mediated renal dysfunction and injury in the rat.

BACKGROUND: The generation of reactive oxygen species (ROS) contributes to the pathogenesis of renal ischemia-reperfusion injury. The aim of this study was to investigate the effects of tempol in (1) an in vivo rat model of renal ischemia/reperfusion injury and on (2) cellular injury and death of rat renal proximal tubular (PT) cells exposed to oxidant stress in the form of hydrogen peroxide (H2O2). METHODS: Male Wistar rats underwent bilateral renal pedicle clamping for 45 minutes followed by reperfusion for six hours. Tempol (30 mg/kg/h), desferrioxamine (DEF; 40 mg/kg/h), or a combination of tempol (30 mg/kg/h) and DEF (40 mg/kg/h) were administered prior to and throughout reperfusion. Plasma concentrations of urea, creatinine, Na+, gamma-glutamyl transferase (gammaGT), aspartate aminotransferase (AST), and urinary Na+ and N-acetyl-beta-D-glucosaminidase (NAG) were measured for the assessment of renal function and reperfusion injury. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of polymorphonuclear (PMN) cell infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of nitrotyrosine and poly(ADP-ribose) synthetase (PARS). Primary cultures of rat PT cells were incubated with H2O2 (1 mmol/L for 4 h) either in the absence or presence of increasing concentrations of tempol (0.03 to 10 mmol/L), DEF (0.03 to 10 mmol/L), or a combination of tempol (3 mmol/L) or DEF (3 mmol/L). PT cell injury and death were determined by evaluating mitochondrial respiration and lactate dehydrogenase (LDH) release, respectively. RESULTS: In vivo, tempol significantly reduced the increase in urea, creatinine, gammaGT, AST, NAG, and FENa produced by renal ischemia/reperfusion, suggesting an improvement in both renal function and injury. Tempol also significantly reduced kidney MPO activity and MDA levels, indicating a reduction in PMN infiltration and lipid peroxidation, respectively. Tempol reduced the histologic evidence of renal damage associated with ischemia/reperfusion and caused a substantial reduction in the staining for nitrotyrosine and PARS, suggesting reduced nitrosative and oxidative stress. In vitro, tempol significantly attenuated H2O2-mediated decrease in mitochondrial respiration and increase in LDH release from rat PT cells, indicating a reduction in cell injury and death. Both in vivo and in vitro, the beneficial actions of tempol were similar to those obtained using the Fe2+ chelator DEF. However, coadministration of DEF and tempol did not produce any additional beneficial actions against renal ischemia/reperfusion injury or against oxidative stress-mediated PT cell injury/death. CONCLUSION: Our results suggest that the membrane-permeable radical scavenger, tempol, reduces the renal dysfunction and injury associated with ischemia/reperfusion of the kidney.

Expression of beta 1 integrins in IgA nephropathy.

AIM: To compare the expression of beta 1 integrins in renal biopsies from patients with IgA nephropathy with that found in normal human kidney. METHODS: Thirty renal biopsies from patients with IgA disease plus six control specimens were stained with monoclonal antibodies directed against the alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha v, and beta 1 integrin chains using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. The intensity of integrin expression was graded semiquantitatively by a pathologist unaware of the antibody used. RESULTS: Glomerular crescents stained strongly for alpha 3, alpha v, and beta 1, but integrin expression was greatly reduced or absent in fibrotic glomeruli. There were no alterations in the intensity of mesangial cell staining for any of the integrins tested. There was accentuated staining for the alpha 2, alpha 5, alpha v, and beta 1 chains in areas of interstitial scarring plus alpha 2, alpha 3, alpha v, and beta 1 on damaged tubules. Inflammatory cells expressed alpha 4, alpha 5, and beta 1. CONCLUSIONS: In IgA nephropathy the interstitium is the main site of altered beta 1 integrin expression. Glomerular crescents also express several beta 1 integrins, but we found no differences in the intensity of integrin expression on mesangial cells. Altered beta 1 integrin expression may play a role in tubulointerstitial scarring in IgA disease. Thus modulation of integrin expression might attenuate this process.

Glomerular epithelial and mesangial cell culture and characterization.

The advent of in vitro culture techniques has allowed us to culture homogeneous populations of glomerular mesangial and epithelial cells to aid our understanding of the development of glomerular disease at the cellular level. Advances in our knowledge of the pathogenic mechanisms have made it clear that the response of intrinsic glomerular cells to external stimuli plays an important role in glomerular injury (1, 2). Glomerular cells from several mammalian species have been isolated, propagated, and in some instances, cloned (3-5).

Restrictive dermopathy: a disorder of skin differentiation with abnormal integrin expression.

Clinical features and histological findings in two sibs who died from restrictive dermopathy in the neonatal period are described. Fibroblasts cultured from a skin biopsy from the second sib and fibroblasts from normal neonatal skin were studied using monoclonal antibodies to visualise integrin subunits by immunocytochemistry. Restrictive dermopathy fibroblasts displayed increased expression of the alpha-1 and alpha-2 subunits of integrin, those responsible for collagen binding. The increase was not matrix dependent. Integrins may play an important role in tissue differentiation, and our findings support the hypothesis that restrictive dermopathy is a disorder of skin differentiation.

Immune responses to noninherited maternal RT1A antigens in inbred rats.

Humoral responses to non-inherited maternal class I antigens (class I NIMAs) were assessed in 3 groups of inbred rats expressing the RT1u phenotype. Group 1 consisted of the progeny of (AO X DA)F1 X PVG matings; their haplotype was RT1u/c and their non-inherited maternal haplotype RT1a. Group 2 were the progeny of (AO X LEW)F1 X PVG matings the haplotype of which was also RT1u/c, but their non-inherited maternal haplotype was RT1l. Group 3 comprised 8 (AO X PVG)F1 (RT1u/c) hybrids. All rats received 2 intravenous blood transfusions (0.5 ml) from male DA (RT1Aa) donors on days 0 and 7. They were bled at weekly intervals for 6 weeks and again at 20 weeks after the first transfusion. Alloantibody responses to RT1Aa were assessed by an indirect hemagglutination assay (IHA)* and by a 51Chromium-release complement-dependent red cell cytotoxicity assay. All groups exhibited vigorous anti-class I antibody responses to the DA transfusions. No significant differences were detected, however, in antibody titers between the groups either by IHA or CDC or in the rates of decay of antibody titers up to week 20. In addition no blocking activity was found in sera obtained on day 0 from group 1 animals and tested for antiidiotypic antibody activity to cytotoxic anti-RT1Aa antibodies. In order to assess whether suppressor activity had been activated by the initial transfusions, in the animals in which class I NIMA was RT1Aa, all groups were rechallenged with a DA transfusion at week 20. All animals exhibited vigorous anamnestic responses to this challenge and no significant differences were detected between groups. In order to determine whether cellular tolerance to the noninherited maternal haplotype was present in group 1 animals, proliferative responses were assessed by one-way mixed lymphocyte cultures, using DA lymph node stimulator cells. No significant differences were detected in proliferative or kinetic responses between lymph node cells from rats the noninherited maternal haplotype of which was RT1a or from naive (AO X PVG)F1 hybrids. Peak proliferative responses to DA cells in rats the noninherited maternal haplotype of which was RT1l were similar, but maximal on day 4 as opposed to day 3. Hence in these inbred rat strains no evidence of humoral tolerance to class I NIMAs was detected. In addition there was no evidence of cellular tolerance to the noninherited maternal MHC haplotype.

Influence of antiidiotypic antibody activity on renal transplant outcome.

The presence of cytotoxic HLA antibodies (Ab1) against donor lymphocytes in pretransplant sera is almost always associated with rapid rejection of the renal transplant. We have investigated the possibility that antiidiotypic antibodies (Ab2) to cytotoxic HLA antibodies might modulate the immune response and favorably influence renal allograft outcome. The role of antibodies (Ab3) which potentiate the cytotoxic effect of Ab1 was also studied. Pretransplant sera from 63 patients were tested for inhibitory or potentiating activity in the short antiidiotypic assay. Inhibitory activity was detected in 30 patients and in 28 the transplant survived more than a year. Of patients without antibody activity 11 of 17 had grafts surviving more than one year, and of those showing potentiating activity 11 of 16 were functioning at a year. The difference in transplant survival between the first group and the other two groups was statistically significant (P less than 0.05). There was no significant difference in survival rates between the latter two groups. Potentiating activity is therefore not an independent predictor of transplant failure, whereas the presence of antiidiotypic antibody activity did correlate with improved allograft survival.

Reduction of sensitization induced by blood transfusion in the rat by monoclonal antibodies.

Primary and secondary alloantibody responses were monitored in (AOxPVG)F1 hybrid rats after three transfusions of DA blood; the initial transfusion was either untreated or pretreated with monoclonal antibody directed to class I antigens or other cell surface markers. Mean antibody activity in recipient sera against class I DA antigens was significantly decreased by pretreatment with the monoclonal antibodies. The most marked suppression was associated with pretreatment by antibodies to the four major nonoverlapping epitopes of the RT1Aa antigen. Subsequent transfusions of DA blood failed to stimulate a secondary response. Crossreactivity of the alloantibody reactivity with BDIX antigens was diminished by pretreating the transfusions with rat anti-RT1A antibodies and, to a lesser extent, with a mouse monoclonal antibody (OX-18) to a common class I determinant. Monoclonal antibody pretreatment had no effect on the humoral response to class II DA antigens. These studies indicate that blood transfusions pretreated with monoclonal antibodies induce a less-potent cytotoxic humoral immune response and that reactivity is most effectively suppressed by completely masking the class I antigen. This technique may prove of clinical value in preventing the sensitization caused by blood transfusions in potential transplant recipients.

Monoclonal antibodies to cultured human glomerular mesangial cells. I. Reactivity with haematopoietic cells and normal kidney sections.

The aim of this study was to produce monoclonal antibodies to cultured human glomerular mesangial cells in order to obtain specific markers for these cells and to aid the study of their function. Using standard monoclonal antibody techniques, 29 hybridomas producing antibodies directed to cultured mesangial cells were obtained. Most of these antibodies were not reactive with normal or neoplastic haematopoietic cell lines by flow cytometry. Fourteen of the 29 culture supernatants bound to various components of normal human kidney sections stained by the alkaline phosphatase/anti-alkaline phosphatase (APAAP) method. Ten of these supernatants reacted with components within the glomerulus, with six binding to the mesangium. These studies suggest that (1) mesangial cells in culture may show significant de-differentiation, because most supernatants which reacted with mesangial cells in culture did not do so in tissue sections; (2) antibodies reactive with haematopoietic cells may not detect the majority of immunogenic surface antigens on cells in tissues; and (3) some of the antibodies which we have produced may prove to be useful markers for mesangial cells in glomerular disease.

The effect of cyclosporin administered during a third-party blood transfusion protocol on humoral immune responses.

Antigen pre-treatment in animals undoubtedly prolongs graft survival. In man, however, routine pre-transplantation blood transfusions have recently become controversial, principally because of the adverse effect of transfusion-induced sensitisation on graft survival rates. We have monitored the effect of cyclosporin administered during a planned programme of third-party blood transfusions on the development of both cytotoxic and anti-idiotypic antibodies. A total of 24 patients were recruited to the study; ten received cyclosporin with blood transfusions (BT) (group 1), 14 received BT alone (group 2). Anti-HLA antibodies developed in 3 of 9 patients in group 1 and 8 of 12 patients in group 2 (P less than 0.05). Anti-idiotypic antibody activity was detectable in 9 of 9 patients in group 1 and 7 of 12 patients in group 2 (P less than 0.006). The mechanism by which cyclosporin can prevent an anti-HLA antibody response and promote an anti-idiotypic response is unclear.

Cyclosporin A and the humoral response to blood transfusion in the rat: definition of antigens which suppress alloantibody formation.

1. Blood transfusions improve renal allograft survival rates, but may induce antibodies which are directed to class I major histocompatibility complex antigens and mediate hyperacute transplant rejection. A model to study the development of such antibodies was developed in inbred strains of rats. 2. The influence of transplantation antigens shared between an initial course of blood transfusions, given with cyclosporin A, and a subsequent antigenic challenge (blood transfusion), given without cyclosporin A, on alloantibody responses to class I major histocompatibility complex antigens was then investigated. 3. Cyclosporin A administration prevented the development of alloantibodies to class I major histocompatibility complex antigens during the initial transfusion period. 4. After the challenge transfusions, alloantibody responses to class I major histocompatibility complex antigens were suppressed when class I major histocompatibility complex or minor histocompatibility antigens were shared between the initial and final transfusions. 5. This suppression only extended to third party class I antigens when minor histocompatibility complex antigens were shared between the initial and final transfusions. Sharing of class I or class II antigens had no effect on alloantibody responses to third party class I antigens coexpressed on the same cell. 6. These studies suggest that cyclosporin A given with blood transfusions may prevent clinical sensitization while permitting the development of suppressor activity, mediated by shared minor histocompatibility complex determinants, to a broad range of potential donor antigens.

Renal transplantation: cyclosporin A and antibody development after donor-specific transfusion.

The survival of a one haplotype, mismatched living-related renal allograft is improved by donor specific transfusion (DST) before transplantation although the mechanism is unclear. The major risk of DST is sensitization of the recipient to donor lymphocytes precluding transplantation. Fifty prospective recipients of a living related transplant received either DST with cyclosporin A (group I) or DST alone (group II). Persistent donor sensitization precluding transplantation occurred in no patients in group I but in six in group II (P less than 0.05). Ten of 14 of those who developed donor cytotoxicity had previously been pregnant or received greater than or equal to 10 third party transfusions compared with 11 of 36 without such a history (P less than 0.05). Alloantibodies detected by a cellular ELISA developed following DST in 29% patients and antiidiotypic antibodies detected by the short antiidiotypic assay (SAA) in 36%; antiidiotypic activity occurred more frequently in those given cyclosporin A (P less than 0.02). Potentiating activity in the SAA which occurred in sera from six patients after DST had no influence on transplant outcome. Persistent sensitization, particularly in potential transplant recipients who have been pregnant or received many transfusions, can be prevented by giving cyclosporin A with DST; the mechanisms of this effect may be the induction of antiidiotypic antibodies. Both alloantibodies and antiidiotypic antibodies are induced by DST and may protect a subsequent renal allograft from the specific donor.

Immunological studies of trophoblast antigens: no evidence for human leucocyte antigen (HLA) linkage.

Parental disparity for trophoblast-lymphocyte crossreactive (TLX) antigens may promote successful pregnancy. A TLX antigen system has been defined on peripheral blood lymphocytes by heteroantisera. More recently, we have reported additional activity against antigens on B lymphocytes alone termed trophoblast-B lymphocyte crossreactive (TBX) antigens. In the present study we have investigated ten TLX sera in order to determine if their target antigens are linked to the human leucocyte antigen (HLA) gene complex. The sera showed no selective activity when tested against target B lymphocytes from ten normal donors. Cytotoxic activity of TLX antisera against peripheral blood lymphocytes from six normal donors was not reduced when the class I HLA antigens of the target cells were blocked with a monoclonal antibody (PA 2.6). Similarly the cytotoxic activity of both TBX antisera against B lymphocytes from six normal donors was not decreased when class II HLA antigens were blocked by a monoclonal antibody (FMC 4). Within a family the cytotoxic activity of the TLX antisera was absorbed equally by lymphocytes from siblings who shared neither HLA haplotype. Antibody content in TLX and TBX antisera is not directed toward the classically defined HLA class I or class II antigens and is not linked to the HLA gene complex.

Alloantibody and transferable suppressor activity induced by cyclosporine and blood transfusions in the rat.

The effect of cyclosporine on the alloantibody response to blood transfusion was investigated in inbred strains of rats by IHA and CELISA; recipient animals differed from the donors at the class I (RT1A) or both class I and class II (RT1B) antigens of the major histocompatibility complex. Alloantibody titers stimulated in high responder PVGu/c animals by blood transfusions were attenuated by cyclosporine; this effect was not demonstrated in low responder PVGc rats, as alloantibody titers decreased after further blood transfusions whether or not cyclosporine was given. Cyclosporine not only reduced the initial IgM response but suppressed the subsequent production of IgG. Splenocytes from rats receiving cyclosporine and blood transfusions from donors that differed from the recipients at the class I antigen were effective in suppressing the subsequent antibody response to blood transfusion. When blood transfusions from donors which differed from the recipients at both class I and class II antigenic loci were given after splenocyte transfer, a greater degree of immunosuppression was detected than if the transfusion donor differed only at the class I locus. These data suggest that the sensitization produced by blood transfusions and the persistence or decline of the alloantibody response depend upon the responder status of the recipient. Blood transfusions given with cyclosporine are capable of inducing suppressor activity that is transferable in spleen homogenates. Subsequent alloantibody responses are influenced by the class I and class II disparities of the donor and recipient animals. If these results can be extrapolated to clinical practice, cyclosporine should be given with pretransplant blood transfusions to prevent sensitization, and the transfusion donor should differ from the recipient at both class I and class II antigenic loci.