J. A. Bruijn

Expression of glomerular extracellular matrix components in human diabetic nephropathy: decrease of heparan sulphate in the glomerular basement membrane.

SummaryDiabetic nephropathy is characterized by albuminuria which proceeds to overt proteinuria. The highly negatively stained HS side chain of heparan sulphate proteoglycan (HSPG) is a major determinant of the charge-dependent permeability of the GBM. We set out to study the presence of HS and HSPG in the GBM of patients with diabetic nephropathy using newly developed monoclonal antibodies, and to compare HSPG expression to the expression of other previously investigated glomerular extracellular matrix compounds. Immunohistochemically, glomerular extracellular matrix components were analysed in 14 renal biopsies of patients with diabetic nephropathy and compared with those of normal control subjects. Monoclonal antibodies used were: JM403 against the HS side chain of GBM HSPG and JM72 against the HSPG-core protein. Also, a polyclonal antiserum (B31) against human GBM-HSPG-core protein was used. Additionally, antibodies were used against collagen types I, III, IV and against α1(IV)NC, α3(IV)NC and fibronectin. Staining was scored for intensity and for staining pattern by four independent observers who had no previous knowledge of the sample origin. No glomerular staining was seen for collagen type I. Collagen type III was present in some diabetic nodules. Anti-collagen type IV showed a decreased GBM staining in patients with diabetic nephropathy (p = 0.04). With anti-α1(IV)NC no changes in GBM staining intensity were observed; with anti-α3(IV)NC brilliant GBM staining was seen in both groups. Increased mesangial staining (p = 0.003) was seen with anti-collagen type IV in biopsies with nodular lesions. No differences were observed for fibronectin although it was abundantly present in the mesangial area of biopsies from patients with diabetic nephropathy. In biopsies with mesangial expansion and in biopsies with diabetic nodules, we observed a decreased GBM (p = 0.001) HS side chain staining (JM403) without changes in HSPG-core protein staining (JM72,B31). The HS staining pattern regularly changed from a linear to a more granular and irregular pattern. In patients with a creatinine clearance of more than 15 ml/min, the intensity of GBM HS staining showed an inverse correlation with the rate of proteinuria (r = -0.85, p = 0.004), suggesting a functional relationship. The decreased HS staining in the GBM may reflect the potentially disrupted charge barrier in diabetic nephropathy.

Hypotheses on the Etiology of Antineutrophil Cytoplasmic Autoantibody–Associated Vasculitis: The Cause Is Hidden, but the Result Is Known

The first description of what is now known as antineutrophil cytoplasmic autoantibody-associated necrotizing vasculitis appeared more than 140 yr ago. Since then, many aspects of the pathogenic pathway have been elucidated, indicating the involvement of antineutrophil cytoplasmic autoantibodies, but why antineutrophil cytoplasmic autoantibodies are produced in the first place remains unknown. Over the years, many hypotheses have emerged addressing the etiology of antineutrophil cytoplasmic antibody production, but no exclusive factor or set of factors can so far be held responsible. Herein is reviewed the most influential hypotheses regarding the causes of antineutrophil cytoplasmic antibody-associated vasculitis with the aim of placing in an epidemiologic background the different hypotheses that are centered on environmental and genetic influences.

The clinical significance of allospecific antibodies against endothelial cells detected with an antibody-dependent cellular cytotoxicity assay for vascular rejection and graft loss after renal transplantation.

Serum samples of 64 consecutive patients who underwent renal transplantation in our institution were examined for the presence of antibody-dependent cellular cytotoxicity (ADCC) activity against endothelial cells (EC). From each patient serum samples were obtained immediately before transplantation and 1 week, 1 month and 1 year thereafter. The results were evaluated in the context of tests to measure donor-specific humoral immunity against lymphocytes and monocytes, and related to parameters of presensitization, graft survival, and histology. Sera from 10 patients were positive for ADCC on a panel of HLA-typed endothelial cells. In 8 patients sera were already positive before transplantation and remained positive thereafter. In 4 patients a positive crossmatch with donor T and B cells and monocytes could be observed after transplantation. In only one patient were these crossmatches positive before transplantation. A significant correlation was found between ADCC positivity and vascular rejection (P=0.015); in addition graft survival was significantly better in the ADCC negative group vs. the positive group (P=0.0004). These data demonstrate the significance of allospecific anti EC antibodies for the occurrence of vascular rejection and graft loss after renal transplantation.

Regulation of glomerular epithelial cell production of fibronectin and transforming growth factor-beta by high glucose,not by angiotensin II

Accumulation of matrix proteins is a prominent feature of diabetic nephropathy. Glomerular visceral epithelial cells (GVECs) are important contributors to extracellular matrix (ECM) production in the glomerulus. Factors involved with increased accumulation of ECM proteins are high glucose, angiotensin II (ANG II), and transforming growth factor (TGF)-β. Therefore, we investigated the effects of high glucose and ANG II on fibronectin and TGF-β production by human GVECs in vitro. We found that ANG II had no effect on the production of fibronectin and TGF-β by GVECs. Using reverse transcriptase–polymerase chain reaction analysis, no ANG II receptor could be detected on these cells. However, high glucose induced a twofold increase in fibronectin (P < 0.01) and a three- to sixfold increase in TGF-β (P < 0.001) production. Similar results were obtained by analyzing the mRNA levels of fibronectin (increased 2.7-fold) and TGF-β (increased 3.5-fold). Addition of increasing concentrations of rTGF-β to control cells resulted in increased fibronectin production. Neutralizing antibodies against TGF-β significantly reversed the increase in fibronectin protein and mRNA caused by high glucose back to control levels. We conclude that high glucose concentrations stimulate the synthesis of fibronectin and that this effect is mediated by induction of TGF-β. These results suggest that in diabetic nephropathy, high glucose levels play a role in changing the matrix composition of the glomerular basement membrane through induction of TGF-β. Our results indicate that a contribution to this process by an effect of ANG II on GVECs seems unlikely.

Interstitial rejection, vascular rejection, and diffuse thrombosis of renal allografts : Predisposing factors, histology, immunohistochemistry, and relation to outcome

Histological and immunohistochemical analyses were made of biopsy specimens from 50 consecutive patients who experienced putative graft rejection. The mean age of the patients was 44.5 years (range, 17-69 years) and 26 were men. There were 67 evaluable allograft specimens, which were grouped according to the histological diagnosis: group 1, acute tubulointerstitial rejection (n = 42); group 2, acute vascular rejection (n = 18); and group 3, diffuse thrombosis (n = 7). Over a follow-up period of 21-57 months, the mean number of rejection episodes was 1.7, 2.8, and 3.3 in groups 1, 2, and 3, respectively. Allograft loss occurred in 7 out of 30, 10 out of 16, and 4 out of 4 patients in groups 1, 2, and 3, respectively. The following histological parameters differed significantly (P < 0.05) among the groups: interstitial edema, congestion of peritubular capillaries, glomerular thrombosis, and glomerular ischemia (group 3 > group 2 > group 1). Interstitial bleeding was seen more often in group 2 and 3 tissues than in group 1 specimens (P < 0.01). Immunohistochemical analyses showed that vascular rejection was associated with WT14 staining for monocytes and macrophages around the tubuli and with interstitial deposition of complement factor 3. With regard to serology, positive anti-endothelial cell antibody-dependent cellular cytotoxicity was associated with vascular rejection and thrombosis of the graft in all patients tested, and with graft loss in 75%. Pre-existent positive anti-IgG immunofluorescence on peritubular capillaries in pretransplant biopsy specimens incubated with patient serum was found in only 3 of the 50 patients, but was associated with graft loss in 2 of the 3. Cytomegalovirus infection was associated with a higher percentage of graft loss. There were significant intergroup differences in panel reactive antibodies before transplantation (P < 0.001), with higher titers in groups 2 and 3. The findings in relation to interstitial rejection are compatible with cellular rejection, while the data on vascular rejection support a humorally mediated pathogenesis.

Glomerular hypertrophy precedes albuminuria and segmental loss of podoplanin in podocytes in Munich-Wistar-Frömter rats

Focal segmental glomerulosclerosis (FSGS) is a common cause of end-stage renal disease. Albuminuria is a risk factor for FSGS and is influenced by environmental, genetic, and sex-specific factors. Podocytes play a central role in the development of albuminuria, but the precise relationship between early glomerular and podocyte-associated damage and albuminuria is unclear. Furthermore, experimental findings demonstrate a sex difference in development of albuminuria and FSGS. We investigated the early glomerular changes in male Munich-Wistar-Frömter (MWF) rats, which spontaneously develop albuminuria, and male albuminuria-resistant spontaneously hypertensive rats (SHR). In addition, since female MWF rats are protected from overt proteinuria and progressive renal disease, we compared the phenotypic changes in podocytes during early development of albuminuria in male and female MWF rats. In male MWF rats, glomerular hypertrophy preceded the onset of albuminuria and was greater than in male SHR. Albuminuria developed starting at 6 wk of age and coincided with focal and segmental loss of podoplanin, increased expression of desmin, entrapment of albumin in affected podocytes, and focal and segmental foot process effacement at the ultrastructural level. Other podocyte-associated molecules, such as nephrin and zonula occludens 1, were unaffected. Early glomerular hypertrophy and podocyte damage did not differ between male and female MWF rats. Our data show for the first time that albuminuria in male and female MWF rats is preceded by glomerular hypertrophy and accompanied by focal and segmental loss of podoplanin when FSGS was not yet present.

Apoptosis of acinar cells in pancreas allograft rejection

BACKGROUND Recently it has been recognized that apoptosis of target cells may occur during liver and kidney allograft rejection and is probably induced by infiltrating cells. Pancreas rejection is also characterized by a cellular infiltrate, however, the occurrence of apoptosis has not been investigated. We assessed whether pancreas rejection was associated with apoptosis of target cells and an influx of granzyme B (GrB)-positive or CD68-positive cells. METHODS Eighteen pancreas biopsies (10 of 18 with rejection) from 15 patients with a pancreas-kidney transplantation were stained with the in situ end-labeling method for apoptosis, and for CD3, GrB, and CD68. RESULTS Significantly more apoptotic acinar cells were found in biopsies with rejection when compared with biopsies without rejection. No difference was observed between the groups for GrB+ or CD68+ cells. CONCLUSION We conclude that pancreas rejection is associated with apoptosis of acinar cells, but not with an increased influx of GrB+ cells or macrophages.

T-Cell Receptor V-Gene Usage in T-Cell Lines Propogated from Graft-Infiltrating T Lymphocytes in Needle Biopsies of Rejecting Renal Allografts

Needle biopsies taken from renal allografts of 42 renal transplant patients were tested for in vitro propagation of graft-infiltrating T-lymphocytes (GITL). From 30 out of 42 needle biopsies T-lymphocyte cell lines could be established. There was a significant correlation between in vitro outgrowth of T cells and histological signs of graft rejection. The majority of GITL cell lines displayed cytotoxicity both against donor proximal tubular epithelial cells (PTEC) and PHA stimulated donor splenocytes in an MHC class I restricted fashion. However, six cell lines were only cytotoxic against donor PTEC which is suggestive of recognition of tissue-specific antigens by these GITL derived T-cell lines. Analysis to the level of diversity of the T-cell receptor repertoire by PCR with TCRBV-family specific-oligonucleotides of a selection of these GITL derived cell lines revealed that the majority of the T-cell lines tested were polyclonal in nature on the basis of TCRBV gene family usage. A clear dominance of TCRBV genes was observed in only three GITL derived T-cell lines. There was no apparent correlation with the diversity of the TCRBV repertoire of GITL derived T-cell lines and the number of HLA-mismatches between the recipient and donor derived graft. Furthermore, the time interval between the transplantation and biopsy sampling did not contribute to the level of diversity of the TCRBV repertoire nor the tissue-specificity of these GITL derived T-cell lines.

Involvement of Minor Histocompatability Antigens in the Rejection of an HLA Identical Renal Transplant from a Living Related Donor

Graft-infiltrating T-lymphocytes (GITL) were isolated from two successive biopsies of a patient who had received an HLA-identical kidney from his brother. Both T-cell lines were highly cytotoxic against cultured proximal tubular epithelial cells (PTEC) and PHA stimulated peripheral blood lymphocytes (PBL), both of donor origin. In addition paired samples of PBL isolated at the time of the second rejection and cultured in a similar fashion as GITL displayed cytotoxicity against PTEC, although to a lesser extent. Cytotoxicity was not due to LAK activity since HLA typed target cells were specifically lysed. By using PBL from several members of the patient’s family as target it was demonstrated that cytotoxicity was related to the haplotype HLAA25, B18, CW7. Susceptibility to lysis was inherited independently from the HLA haplotypes as would be expected of minor histocompatibility antigens (mH). Using a panel of HLA typed PBL not related to the patient, a single HLA specificity could not be demonstrated. Cytotoxicity was T cell mediated and MHC class I restricted as could be shown by inhibition experiments. Adhesion between GITL and PTEC was almost completely dependent on the LFA-1/ICAM-1 adhesion pathway. The diversity of the T-cell receptor repertoire in both T-cell lines was reduced on the basis of usage of certain TCRBV gene families in comparison to paired control PBL. However, the T-lymphocyte repertoire of GITL propagated from the second biopsy was more extensive when compared to the repertoire of GITL propagated from the first biopsy. These results demonstrate that mH antigens could play a critical role in renal allograft rejection in HLA-identical siblings and that they are expressed on PTEC. Furthermore the T-cell response against the renal allograft seems to be mediated initially by T cells with a restricted TCRBV gene family usage. As the rejection process progresses, the diversity of the T lymphocytes which accumulate into the renal allograft increases.