Tadashi Oikawa

Significance of histochemical expression of Hanganutziu-Deicher antigens in pig, baboon and human tissues

THERE is increasing interest in the clinical application of xenotransplantation. This interest has been sparked predominantly by the severe shortage of donors. In addition, xenotransplantation might be useful for avoiding the recurrence of viral disease in the transplanted organ. The humoral barrier leading to the immune response is well known for clinical practice of xenotransplantation; that is, xenoreactive natural antibodies to the graft endothelium. Human xenoreactive natural antibodies are directed predominantly against Gala1–3Gal (a-Gal antigen), a saccharide expressed in cells of lower mammals and New World monkeys, but not in cells of Old World monkeys, apes, or humans. Anti–a-Gal antibody plays a major role in hyperacute rejection (HAR) after pig-to-human xenotransplantation. Antibody binding to this sugar, expressed as a modification of endothelial cell membrane glycoproteins and glycolipids, activates complement by the classical pathway. Many investigators believe that pig-to-human xenotransplantation will become a common clinical application and that the pig-to-baboon model represents a good experimental design for study. Hanganutziu–Deicher (HD) antibodies have been known as antibodies detectable in patients with serum sickness. In 1924, the Romanian pathologist Hanganutziu noticed that a serum taken from a patient who received antitetanus horse serum abnormally and strongly agglutinated sheep red blood cells. This agglutination was not sheep-specific, as red blood cells from the horse, guinea-pig, rabbit, calf, and pig expressed similar titers. In 1926, Deicher replicated these results. The antibodies reacting broadly with heterologous antigens were called heterophilic antibodies. HD antibodies are heterophilic antibodies as Forssman antibodies and Paul–Bunnell antibodies. Higashi et al and Merrick et al found that antigens recognized by HD antibodies are gangliosides containing N-glucolylneuramic acid (NeuGc). Gangliosides are sialic acid–containing glycophospholipids present on the surface of all mammalian cells. Sialic acids are a group of nine carbohydrates of two main types: N-acetylneuraminic acid (NeuAc) and N-glucolylneuramic acid. NeuAc is expressed ubiquitously, whereas, NeuGc, the hydroxylated form of NeuAc, is not present in birds or humans. HD antigen is widely distributed in mammalian species with the exception of humans. Some antipig antibodies are directed against epitopes other than the a-Gal antigen. Grafting animals into human patients should be followed by an increase in HD antibodies. Therefore, it is possible that non–a-Gal antibody can cause rejection, even if a-Gal epitopes are successfully removed from donor pig organs. The purpose of this study is to elucidate the significance of HD antigen as non–a-Gal epitopes in clinical xenotransplantation.

Complement fragment C4d deposition in peritubular capillaries in acute humoral rejection after ABO blood group-incompatible human kidney transplantation.

Background. Acute humoral rejection (AHR) is the most important risk factor for early graft loss in ABO-incompatible (ABO-i) kidney transplantation (RTx). The pathogenesis and diagnostic criteria for AHR after ABO-i RTx remain unclear. Complement fragment C4d deposition in peritubular capillaries (PTC), which is a sensitive indicator for activation of the classical complement pathway, was studied to establish the pathologic diagnostic indicator of AHR. Methods. Forty-four graft biopsy specimens from 19 patients with ABO-i living donors were analyzed within 90 days after RTx. Nineteen biopsy specimens with acute rejection after ABO-compatible (ABO-c) living-related RTx were used as controls. Diffuse and bright C4d deposition in PTC was considered significantly positive. Results. All of 8 recipients with AHR showed significantly positive C4d in PTC in the ABO-i group, but 9 of 11 recipients without AHR were negative. In the ABO-c RTx group, 16 of 19 recipients were negative for C4d in PTC. The prevalence of C4d in PTC was significantly higher in ABO-i RTx (P <0.05). Conclusions. C4d deposition is valuable as a specific and sensitive indicator for AHR, even of mild severity, in ABO-i RTx.

Proximal tubular angiotensinogen in renal biopsy suggests nondipper BP rhythm accompanied by enhanced tubular sodium reabsorption

Objectives: The renal capacity for sodium excretion is impaired by a reduction in the glomerular ultrafiltration coefficient and by enhancement of the fractional tubular sodium reabsorption (FRNa), leading to a nondipper circadian blood pressure (BP) rhythm. Angiotensin II in the systemic circulation can be easily filtered across the glomerular capillary walls and stimulates renal proximal tubular angiotensinogen (PT-AGT) production, leading to the activation of intrarenal angiotensin II, which is known to augment the FRNa in animal models. Methods: We performed an immunohistochemical investigation to determine the contribution of PT-AGT to enhancement of FRNa and the nondipper circadian BP rhythm in 40 consecutive patients with primary IgA nephropathy (IgAN). Results: Immunostaining for PT-AGT was increased in the IgAN patients compared with control individuals (P = 0.04), and correlated directly with the FRNa (r = 0.39, P = 0.01) and the night/day ratio of BP (r = 0.38, P = 0.02), but not creatinine clearance (r = −0.008, P = 0.9). The night/day ratio of BP was determined by both creatinine clearance (r = −0.36, P = 0.03) and FRNa (r = 0.47, P = 0.006). Conclusion: Tubular sodium reabsorption is stimulated by intrarenal angiotensin II, as indicated by PT-AGT, and contributes to the genesis of the nondipper BP rhythm. Further studies are needed to evaluate whether or not treatment to prevent sodium retention is useful for patients who exhibit increased PT-AGT in renal biopsies.

Clinicopathological Findings of Bucillamine-Induced Nephrotic Syndrome in Patients with Rheumatoid Arthritis

This paper describes pathological and clinical investigations of glomerular lesions in bucillamine-induced nephropathy by analyzing biopsy materials from 9 patients with rheumatoid arthritis (RA). There was no specific predisposition for nephrotic syndrome induced by bucillamine in clinical profiles related to age, onset, duration of disease, sex, activity and dose of bucillamine. In light-microscopic, electron-microscopic and immunofluorescent findings, the characteristic changes were similar to those of idiopathic membranous glomerulonephritis (MGN). After discontinuance of bucillamine, the nephrotic syndrome improved slowly with or without corticosteroid therapy. Results confirmed that the most common lesion of nephrotic syndrome associated with bucillamine therapy in RA is MGN. We recommend that corticosteroid therapy should be restricted to cases with severe proteinuria.

Angiotensin-Converting Enzyme Inhibitor and Anemia in a Patient Undergoing Hemodialysis

We have observed a polycythemic patient with poly-cystic kidney disease (PCKD) and hypertension who was treated with enalapril maleate, which is a long-acting angiotensinconverting enzyme inhibitor (ACEI). In this patient, we studied the suppression of erythropoietin (Ep) synthesis by an ACEI. A 51-year-old man first noted the abdominal tumor in 1978. Included in his family history was the fact that his elder brother and elder sister had PCKD, which is characterized by autosomal dominant heredity and progressive renal failure. He consulted a physician for the abdominal tumor in 1978, and he was discovered to have multiple bilateral renal cysts without hepatic or pancreatic cysts. The diagnosis of PCKD was based on family history and computertomographic (CT) findings. In October 1984, he was transferred to our hospital for treatment of chronic renal failure (CRF). He was diagnosed as having end-stage CRF and operated upon for an arteriovenous fistula. His renal function slowly deteriorated. In April 1985, he started hemodialysis (HD) therapy. After HD treatment, his condition improved rapidly and his hematocrit (Ht) increased (18–25%). In 1988, Ht exceeded 40%. Because renal tumor with secondary polycythemia is well known [1], he was examinated by CT. There was no difference between the prior study and the follow-up study, which revealed multiple renal cysts occupying the renal parenchyma and no evidence of a malignant tumor. As the cencentration of Ep was 86.6 mU/ml and no renal tumor was found, we concluded that the polycythemia was caused by PCKD per se.

Acute humoral rejection of kidney allografts in patients with a positive flow cytometry crossmatch (FCXM)

The patients with a positive flow cytometry crossmatch (FCXM) are categorized as a high‐risk group causing hyperacute or accelerated acute rejection after kidney transplantation. According to the successful results of ABO‐incompatible renal transplantation, we have performed the living related transplant operations in the recipients with positive FCXM for donor T cells, but having a negative complement‐dependent lymphocytotoxic reaction test. We have followed the clinical course of 4 FCXM‐positive patients, and 2 of them have developed acute humoral rejection. We report the strategies for FCXM‐positive living kidney transplantations and the characteristics of pathological findings of acute humoral rejection in FCXM‐positive renal transplants. We have had few episodes of acute humoral rejection in ABO‐incompatible kidney transplantations under immunosuppressive regimens, including cyclophosphamide, but 2 patients of 4 with FCXM‐positive kidney transplantations developed acute humoral rejections. The differences in immunosuppressive regimen between ABO‐incompatible and FCXM‐positive kidney transplantations concern anti‐lymphocyte globulin (ALG) and splenectomy. We have not performed splenectomy and ALG administration in FCXM‐positive kidney transplantations. Severe acute rejection episodes have been experienced on post‐operative days 7 and 9 in 2 of 4 FCXM‐positive recipients. The early acute rejection episodes were clinically and pathologically diagnosed as typical humoral rejections. We have examined an immunofluorescent study to prove the diagnosis of humoral rejection in FCXM‐positive kidney transplantations; both immunoglobulin M and C3 were positive for the whole course of humoral rejection. The 2 patients with acute humoral rejection recovered after treatment with double filtration plasmapheresis or plasma exchange to remove their anti‐donor antibodies. The gold standard of success in FCXM‐positive kidney transplantations is to suppress the production and reduce the level of anti‐donor antibodies after transplant operations.

Enzymatic removal of αGal antigen in pig kidneys by ex vivo and in vivo administration of endo-β-galactosidase C

Xenotransplantation using the pig as a donor species is considered to be a promising solution to the serious shortage of organ donors. Both hyperacute and acute vascular rejection (AVR) are believed to be associated with xenoreactive antibody binding to αGal epitopes on the pig vascular endothelial cells. Thus, suppression of this antigen‐antibody reaction would appear essential for successful long‐term xenograft survival. The purpose of this study was to examine the efficacy of ex vivo and in vivo administration of recombinant endo‐β‐galactosidase C (EndoGalC which, in previous in vitro studies, has been proven to digest αGal antigens completely) on αGal epitopes expressed in pig kidneys. Excised pig kidneys were perfused with University of Wisconsin solution containing EndoGalC and preserved for 4 h. After cold storage, the pig kidney was transplanted into another pig. Ex vivo perfusion and cold storage with EndoGalC reduced αGal epitope expression on vascular endothelial cells to an undetectable level. However, αGal antigens began to be expressed again as early as 1 day after transplantation. The digestion of αGal epitopes by EndoGalC did not cause any damage to the kidney graft. EndoGalC was intravenously administered to two pigs (15 kg), without causing any serious adverse effect. Twelve hours later, >98% of αGal antigens on pig red blood cells (RBCs) had been digested. Immunohistochemical study revealed almost complete elimination of αGal expression on vascular endothelial cells of the kidney graft 4 and 8 h after in vivo administration, but reappearance within 24 h. EndoGalC was administered to a baboon after an interval of 2 months. The second administration did not result in any serious toxicity or reduction in efficacy. These results suggest that ex vivo and in vivo administration of EndoGalC is simple and useful in removing αGal epitopes from pig organs. As the effect of EndoGalC is temporary, multiple in vivo administrations of EndoGalC would be required to inhibit the reappearance of αGal epitopes. Alternatively, transgenic techniques of introducing the gene for EndoGalC into the donor organ might permanently prevent αGal expression.

Chronic cyclosporin nephropathy: long‐term effects of cyclosporin on renal allografts

Takeda A, Uchida K, Haba T, Tominaga Y, Katayama A, Yoshida A, Oikawa T, Morozumi K. Chronic cyclosporin nephropathy: long‐term effects of cyclosporin on renal allografts. Clin Transplantation 2001: 15 (Supplement 5): 22–29. ©Munksgaard, 2001.

Potential value of high‐dose mizoribine as rescue therapy for ongoing acute humoral rejection

Mizoribine (MZ) inhibits the proliferation of lymphocytes selectively via inhibition of inosine monophosphate dehydrogenase, like mycophenolate mofetil (MMF). The clinical dosage of MZ (2–5 mg/kg) is much lower than that of MMF (20–60 mg/kg). The purpose of this study was to examine whether high‐dose MZ would be effective for treatment of acute humoral rejection. Renal transplantation was performed in a different pig strain combination. Group 1 (n = 2) received no treatment. Group 2 (n = 4) received cyclosporine microemulsion (6 mg/kg) and prednisolone (0.1 mg/kg) as baseline immunosuppression. Groups 3 (n = 4), 4 (n = 4) and 5 (n = 4) were additionally treated with MZ for rescue therapy, 30, 10 and 3 mg/kg, respectively, immediately after rejection was observed. All pigs developed acute vascular rejection between days 4 and 8. Complete reversal of acute rejection including reduction of elevated serum creatinine, suppression of anti‐donor antibody production and pathological finding, was obtained in 3/4 (group 3), 1/4 (group 4) and 0/4 (group 5). Rescue with high‐dose MZ (30 mg/kg) reversed ongoing acute humoral rejection. Such a high dose of MZ was tolerable for pigs. However, leukocytopenia was observed when MZ trough level was maintained over 10 μg/ml. Treatment with high‐dose MZ would be applicable to a clinical trial, if blood level is carefully monitored.

A case report of recurrence of mixed cryoglobulinemic glomerulonephritis in a renal transplant recipient.

Takeda A, Ootsuka Y, Suzuki T, Yamauchi Y, Tsujita M, Kawaguchi T, Horike K, Oikawa T, Goto N, Nagasaka T, Watarai Y, Uchida K, Morozumi K. A case report of recurrence of mixed cryoglobulinemic glomerulonephritis in a renal transplant recipient.
Clin Transplant 2010: 24 (Suppl. 22): 44–47.