Elaine M. Bailey

Computerized histomorphometric assessment of protocol renal transplant biopsy specimens for surrogate markers of chronic rejection.

BACKGROUND Chronic transplant rejection has emerged as the commonest cause of long-term renal allograft failure, and early identification of those grafts at risk could allow the targeting of specific therapies aimed at delaying this process. This study explores the usefulness of quantitative immunohistochemistry in defining biopsy-based surrogate markers of allograft damage. METHODS A consecutive series of 52 renal transplant recipients immunosuppressed with cyclosporine were studied. Needle core transplant biopsies were performed at 1, 3, and 6 months postoperatively. Immunostaining for collagen III, and smooth muscle actin, tenascin, and infiltrating leukocytes was performed using an indirect immunoperoxidase technique. The interstitial area stained (%) was measured using a semiautomatic image analysis system. The results were related to glomerular filtration rates (GFR) measured at 6, 12, and 24 months after transplantation using rank correlation coefficients. RESULTS The area fraction of immunostained collagen III correlated with 6-month GFR (r=-0.42, P=0.005) and was predictive of 12-month GFR (r=-0.32, P=0.03). An area fraction of immunostained collagen III of >40% at 6 months was associated with a significantly lower GFR at 24 months, compared with a percentage area of < or =40% (31+/-4 versus 45+/-4 ml/min/1.73 m2, P=0.01). Furthermore, a collagen III of >40% at 6 months identified patients who were at risk of progressive deterioration in graft function. CONCLUSIONS Grafts with poorer long-term function can be predicted using 6-month protocol biopsy specimens immunostained for collagen III. This should prove to be a useful ad interim surrogate marker of allograft damage in studies addressing the effects of new immunosuppressive agents on the development of chronic rejection.

Vascular endothelial growth factor mRNA expression in minimal change, membranous, and diabetic nephropathy demonstrated by non-isotopic in situ hybridisation.

AIM: To investigate vascular endothelial growth factor (VEGF) mRNA expression in glomerular disease in the context of heavy proteinuria. METHODS: Non-radioisotopic in situ hybridisation was performed using a cocktail of 12 deoxyoligonucleotides complementary to VEGF mRNA labelled during solid phase synthesis with 2,4-dinitrophenyl. Archival renal biopsies were studied from cases of minimal change nephropathy, membranous nephropathy, diabetic nephropathy, and controls, matched for age, sex, race, and storage time. Hybrid detection used NBT/BCIP colorimetric development. RESULTS: More VEGF mRNA positive glomerular cells per unit cross sectional diameter were seen in minimal change nephropathy (mean (SEM), 19.35 (1.5)) compared with controls (12.6 (1.73)), p < 0.01. In contrast, fewer were seen in diabetic nephropathy (5.93 (0.97)) compared with controls (9.97 (1.25)), p < 0.03. Analysis of membranous nephropathy (10 (1.62)) showed no difference from controls (10.98 (1.51)), NS. In addition, in minimal change nephropathy there was a significant correlation between 24 hour protein excretion at the time of biopsy and the number of VEGF mRNA cells per glomerulus (r = 0.08, p = 0.01). CONCLUSIONS: Using non-radioisotopic in situ hybridisation, VEGF mRNA is almost exclusively expressed by visceral glomerular epithelial cells. Abnormal numbers of cells are seen in both minimal change and diabetic nephropathy. As VEGF exists in a number of functionally distinct isoforms, further study of qualitative VEGF isoform expression in diagnostic groups is indicated.

Exaggerated systemic antibody response to mucosal Helicobacter pylori infection in IgA nephropathy.

Numerous studies in the literature report aberrant immune responsiveness in immunoglobulin A (IgA) nephropathy. However, all these studies investigate immune responses invoked by an artificially engineered antigen challenge. For the first time in IgA nephropathy, we report the systemic humoral responses generated as part of an active mucosal immune response against a common environmental pathogen, Helicobacter pylori (Hp). We studied 22 patients with IgA nephropathy and 9 controls without renal disease who were shown to be infected with Hp, using a 13C-urea breath test. Hp antigen-specific enzyme-linked immunosorbent assays were established to measure the anti-Hp IgA, IgG, and IgA and IgG subclass antibody levels. In addition, anti-Hp responses in the monomeric and polymeric (pIgA) fractions of serum IgA were measured after separation by gel filtration high-performance liquid chromatography. IgA nephropathy was associated with both a greater rate of IgA anti-Hp seropositivity (P < 0.05) and a more pronounced IgA anti-Hp antibody response (P < 0.01). In almost all cases, IgA anti-Hp was IgA1, and more than 90% was polymeric. There was no difference in the frequency of IgG anti-Hp seropositivity, but patients produced a much greater IgG anti-Hp response (P < 0.01). In addition, the IgG subclass responses were markedly different, with IgG1 predominant in controls and IgG2 and IgG3 the major subclasses produced in IgA nephropathy. We have shown an exaggerated systemic antibody response to mucosal infection caused by Hp in patients with IgA nephropathy, predominantly consisting of pIgA1, IgG2, and IgG3. This suggests that in IgA nephropathy, not only is pIgA1 production poorly controlled, but regulation of IgG isotype switching in response to mucosal pathogens is also deranged.

Visceral glomerular epithelial cell DNA synthesis in experimental and human membranous disease.

Background: Membranous nephropathy (MN) is a ‘non-proliferative’ glomerulonephritis. However, visceral glomerular epithelial cell (vGEC) proliferating cell nuclear antigen staining and increased glomerular histone mRNA in passive Heymann nephritis (PHN), suggest that vGECs may enter the cell cycle and undergo DNA synthesis. We used in situ hybridisation for histone mRNA, an S-phase specific marker, to investigate this possibility and identify the cellular origin of histone mRNA in PHN and MN. Methods: PHN was induced in 16 Sprague-Dawley rats. There were 8 saline/serum controls. 12 animals were sacrificed on days 5 and 10. Renal biopsies from 10 proteinuric cases with MN and matched controls were studied. Results: Day-5 Heymann animals demonstrated more S-phase cells/glomerulus than controls (0.53 ± 0.09 vs. 0.195 ± 0.045; p < 0.01). Glomerular S-phase cells were also increased in patients compared to controls (0.24 ± 0.07 vs. 0.04 ± 0.018; p < 0.03). In both experimental and human MN, the peripheral location and morphology of glomerular histone mRNA-positive cells was typical of vGECs. Conclusion: The results in PHN indicate that vGECs recently injured with antibody and complement enter into the cell cycle and undergo DNA synthesis. The S-phase vGECs in MN may indicate the persistence of immune injury. Whether or not this process leads to cell replication is open to question.