Association of kidney transplant failure and antibodies against MICA.

Despite the progress in renal transplantation, acute rejection and graft failure still occur and chronic rejection continues to be the main problem in long-term allograft survival. Although kidney transplant rejection has been linked to anti-HLA antibodies, not all patients with failed kidney transplants have anti-HLA antibodies, indicating that other loci may be involved. Sera of 63 patients who experienced kidney rejection were compared against sera of 82 patients with functioning transplants. Sera were examined for IgG and IgM anti-HLA Class I and II antibodies. They were also tested by cytotoxicity against panels of 26 endothelial cell lines, 8 MHC class I chain-related gene A (MICA) recombinant cell lines, and 28 B lymphoblast cell lines. Among patients whose transplants failed, 65% had anti-HLA antibodies compared with 45% of those with functioning kidneys (p < 0.05). Similarly, among those whose transplants failed, 41% had anti-endothelial cell antibodies in contrast to 22% in functioning patients (p < 0.05). Among patients whose grafts failed, 52% had anti-MICA antibodies versus 21% of those with functioning grafts (p < 0.001). Eleven patients with failed grafts and 32 with functioning grafts were negative for all of the above. However, 6 of the former and 7 of the latter showed positive cytotoxicity against B lymphoblasts (p < 0.05). Taking all antibodies together, 92% of patients with graft failure had antibodies as opposed to 70% of patients with functioning grafts (p < 0.001). We postulate that antibodies against HLA, MICA, endothelial cells, and B lymphoblasts could be independently involved in the slow process of chronic graft failure.

Correction of the mineralization defect in hyp mice treated with protease inhibitors CA074 and pepstatin.

Increased expression of several osteoblastic proteases and MEPE (a bone matrix protein) occurs in X-linked hypophosphatemic rickets (hyp). This is associated with an increased release of a protease-resistant MEPE peptide (ASARM peptide), a potent inhibitor of mineralization. Cathepsin B cleaves MEPE releasing ASARM peptide and hyp osteoblast/osteocyte cells hypersecrete cathepsin D, an activator of cathepsin B. Our aims were to determine whether cathepsin inhibitors correct the mineralization defect in vivo and whether hyp-bone ASARM peptide levels are reduced after protease treatment. Normal littermates and hyp mice (n = 6) were injected intraperitoneally once a day for 4 weeks with pepstatin, CAO74 or vehicle. Animals were then sacrificed and bones plus serum removed for comprehensive analysis. All hyp mice groups (treated and untreated) remained hypophosphatemic with serum 1,25 vitamin D3 inappropriately normal. Serum PTH was significantly elevated in all hyp mice groups relative to normal mice (P = 0.0017). Untreated hyp mice had six-fold elevated levels of serum alkaline-phosphatase and two-fold elevated levels of ASARM peptides relative to normal mice (P < 0.001). In contrast, serum alkaline phosphatase and serum ASARM peptides were significantly reduced (normalized) in hyp mice treated with CA074 or pepstatin. Serum FGF23 levels remained high in all hyp animal groups (P < 0.0001). Hyp mice treated with protease inhibitors showed dramatic reductions in unmineralized osteoid (femurs) compared to control hyp mice (Goldner staining). Also, hyp animals treated with protease inhibitors showed marked and significant improvements in growth plate width (42%), osteoid thickness (40%) and cortical area (40%) (P < 0.002). The mineralization apposition rate, bone formation rate and mineralization surface were normalized by protease-treatment. High-resolution pQCT mineral histomorphometry measurements and uCT also confirmed a marked mineralization improvement. Finally, the growth plate and cortical bone of hyp femurs contained a massive accumulation of osteoblast-derived ASARM peptide(s) that was reduced in hyp animals treated with CA074 or pepstatin. This study confirms in vivo administration of cathepsin inhibitors improves bone mineralization in hyp mice. This may be due to a protease inhibitor mediated decrease in proteolytic degradation of the extracellular matrix and a reduced release of ASARM peptides (potent mineralization inhibitors).

Frequency of MIC antibody in rejected renal transplant patients without HLA antibody.

Antibodies to MICA and MICB antigens were sought in the sera of 139 kidney transplant recipients. MICA*001, *002, *007, *008, and MICB*002 antigens were produced in Escherichia coli and then tested using enzyme-linked immunosorbent assay plates. Among 35 normal sera, 6% had MIC antibodies, and among 14 sera from pregnant women, 21% had MIC antibodies. Among 34 patients with functioning transplants with human leukocyte antigen (HLA) antibodies, 24% had MIC antibodies, and 19% of 32 patients without HLA antibodies had MIC antibodies. Among 46 patients who lost grafts with HLA antibodies, 26% had MIC antibodies, and among 27 failed patients without HLA antibodies, 37% had MIC antibodies. We conclude that antibodies to MIC are produced in the course of immunization by pregnancies and kidney transplants. They also occurred more frequently in rejected patients (30%) than in those with functioning grafts (21%).

Serial ten-year follow-up of HLA and MICA antibody production prior to kidney graft failure.

The role of HLA antibodies in chronic allograft rejection was examined utilizing a unique resource of sera collected annually and stored over a 12-year period from patients with rejected or retained grafts. In patients selected for not having preformed HLA antibodies, 679 postoperative serial serum samples from 39 patients who rejected their grafts and 26 with functioning grafts were tested for HLA Class I and Class II antibodies by flow cytometry and for MICA antibodies by cytotoxicity on recombinant cell lines. HLA antibodies were found in 72% of patients who rejected grafts, compared to 46% with functioning transplants (p<0.05). In addition, the incidence of IgG HLA plus MICA antibodies was higher (77%) among those with failed transplants than those with functioning transplants (42%) (p<0.01). Finally, if patients with IgM anti-HLA antibodies were included, 95% of the 39 patients who rejected their grafts had HLA or MICA antibodies, compared to 58% with functioning grafts (p<0.01). Patients who rejected transplants had HLA and MICA antibodies more frequently than those with functioning grafts. These antibodies found in the peripheral circulation, were not necessarily donor-specific, but their association with failure is consistent with a causality hypothesis.

Increased cathepsin D release by Hyp mouse osteoblast cells.

The X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is caused by loss-of-function mutations of PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) leading to rachitic bone disease and hypophosphatemia. Available evidence today indicates that the bone defect in XLH is caused not only by hypophosphatemia and altered vitamin D metabolism but also by factor(s) locally released by osteoblast cells (ObCs). The identity of these ObC-derived pathogenic factors remains unclear. In our present study, we report our finding of a prominent protein in the culture media derived from ObC of the hypophosphatemic (Hyp) mice, a murine homolog of human XLH, which was identified as the murine procathepsin D (Cat D). By metabolic labeling studies, we further confirmed that Hyp mouse ObCs released greater amount of Cat D into culture media. This increased Cat D release by Hyp mouse ObCs was unlikely to be due to nonspecific cell damage or heterogeneous cell population and was found to be associated with an increased Cat D expression at the protein level, possibly due to a reduced Cat D degradation. However, we were not able to detect a direct effect of PHEX protein on Cat D cleavage. In support of the involvement of Cat D in mediating the inhibitory effect of Hyp mouse ObC-conditioned media on ObC calcification, we found that exposure to Cat D inhibited ObC (45)Ca incorporation and that inhibition of Cat D abolished the inhibitory effect of Hyp mouse-conditioned media on ObC calcification. In conclusion, results from our present study showed that Hyp mouse ObCs release a greater amount of Cat D, which may contribute to the inhibitory effect of Hyp mouse ObC-conditioned media on ObC mineralization.

Altered cathepsin D metabolism in PHEX antisense human osteoblast cells.

X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is caused by loss-of-function mutations of PHEX gene in osteoblast cells, leading to rachitic bone disease and hypophosphatemia. Available evidence today indicates that the bone defect in XLH is caused not only by hypophosphatemia and altered vitamin D metabolism, but also by locally released osteoblastic mineralization inhibitory factor(s), referred to as minhibin. In our present study, we found that suppression of PHEX expression by PHEX antisense in human osteoblast cells caused an increase in cathepsin D expression at protein, but not mRNA, levels. This was associated with a decrease in cathepsin D degradation and an increased cathepsin D release into culture media. Our results also showed that lowering cathepsin D activity in antisense cell conditioned media abolished their inhibitory effect on osteoblast cell calcification, suggesting the involvement of cathepsin D in mediating the minhibin activity of the antisense cell conditioned media.