Renal allograft rejection: beta-chemokine involvement in the development of tubulitis.

BACKGROUND: Tubulitis is a defining feature of renal allograft rejection. Graft dysfunction may result from damage inflicted on tubular epithelial cells by intratubular cytotoxic T lymphocytes. Graft cells are known to produce chemokines during acute rejection, but it is not known whether changes in expression of specific chemokines can influence the composition of the intratubular lymphocyte population. We examined expression of individual chemokines in biopsy sections showing different pathological rejection grades. METHODS: Sections from Banff-graded transplant biopsies were examined for the presence of beta-chemokines (MCP-1, MIP-1alpha, MIP-1beta, and RANTES) by immunofluorescence and semiquantitative confocal laser scanning microscopy. RESULTS: Beta-chemokines were expressed predominantly at the basolateral surface of tubular epithelial cells. Expression of MCP-1 and MIP-1beta was significantly higher in sections showing grade 2 rather than grade 1 acute rejection. RANTES and MIP-1alpha showed no significant variation in level of expression between rejection grades. CONCLUSIONS: Beta-chemokines are expressed by tubular epithelial cells during acute rejection. Consistent expression of RANTES and MIP-1alpha suggests a general role in recruiting T lymphocytes. However, MCP-1 and MIP-1beta may play a more subtle role in recruitment of specific T-cell subsets, such as Th1 cells, during acute cellular rejection.

Focal changes in nerve, muscle and connective tissue in normal and unstable human bladder.

OBJECTIVE: To compare and quantify, in a morphological study, the changes that occur in the connective tissue elements (elastin and collagen), muscle fibre diameters and nerve densities between normal, idiopathic and neuropathic bladders. MATERIALS AND METHODS: Bladder tissue was obtained from 27 patients undergoing cystectomy for carcinoma, from 12 with idiopathic instability and from seven neuropathic patients who were undergoing ileocystoplasty. A combination of histochemical and immunohistochemical techniques were used to detect detrusor muscle, connective tissue and nerve profiles in the bladder tissue. RESULTS: In both idiopathic and neuropathic bladder tissue the structural changes were highly punctate. From the density of nerve profiles, three areas were defined: (i) apparently unaffected normal fascicles with a high density of nerves, no hypertrophy of the muscle and no infiltration of elastin and collagen. The nerve density in these areas was similar to that in normal bladder tissue. (ii) Fascicles with a low density of nerve profiles, muscle hypertrophy but no connective tissue infiltration. (iii) Areas with few nerve profiles, muscle hypertrophy and extensive elastin and collagen infiltration within the fascicles. The mean (sem) density of nerve profiles in control tissue was 752 (53) nerves/mm2 and in the idiopathic bladders was 905 (91), 81 (20) and 74 (38) nerves/mm2 in the three defined areas, respectively. In the neuropathic tissues the nerve profile densities were 672 (249), 57 (23) and 37 (28) nerves/mm2, respectively. Fibre diameter, elastin and collagen content and nerve density were measured in normal and unstable bladder tissue using these three defined areas. The mean (sem) fibre diameter was 6.81 (0.52) in normal bladder; in idiopathic bladder tissue the fibre diameters in the three areas were 6.72 (0.62), 7.06 (0.62) and 7.34 (1.15) micrometer, respectively, and in neuropathic bladders were 6.75 (0.62), 8.24 (0.62) and 9.35 (0.62) micrometer, respectively. The relative areas of elastin were 0.79 (0.70), 0.56 (0.45) and 18.3 (4.1)% for the control, normal and affected areas of the neuropathic bladders, respectively, and the relative areas of collagen were 3.5 (1.3), 6.15 (3.6) and 15.7 (5. 0)%, respectively. The pattern was similar in idiopathic bladders. CONCLUSION: These observations suggest that the primary defect in the idiopathic and neuropathic bladders is a loss of nerves accompanied by a hypertrophy of the cells. These changes may continue with further hypertrophy of the cells and an increased production of elastin and collagen within the muscle fascicles.

Beta-chemokine expression and distribution in paraffin-embedded transplant renal biopsy sections: analysis by scanning laser confocal microscopy.

Previous immunohistochemical and in situ hybridisation studies have shown that, in tubulitis associated with acute cellular rejection of human renal allografts, intratubular T cells proliferate and are fully activated in situ. In the immunohistochemical study reported here we have attempted to establish some understanding of the involvement of the beta-chemokines RANTES, MCP-1, MIP-1alpha and MIP-1beta in recruiting T cells to the intratubular site. Paraffin-embedded routine biopsy sections were treated for conventional indirect immunofluorescence to detect the selected chemokines. Scanning laser confocal microscopy was used to provide a measure of fluorescence intensity resulting from binding of FITC-labelled secondary antibody. Cells expressing chemokines could be identified and, within the limits of the staining method, it was possible to obtain a semi-quantitative assessment of individual chemokine activity at different points in biopsy sections by constructing a profile of fluorescence intensity. High concentrations of chemokines (especially RANTES, MIP-1beta and/or MIP-1alpha) were localised to the basolateral surface of tubular epithelial cells (TEC). MCP-1 was also consistently present but at a lower level than RANTES except in one case identified as BANFF category 3. There was diffuse distribution of chemokines in the interstitial matrix and low intensity fluorescence outlined some endothelial cells of peritubular venules and interstitial fibroblast-like cells. Our results suggest a mechanism for specific chemotactic recruitment of inflammatory cells by TEC-produced chemokines.

Renal allograft rejection--in situ demonstration of cytotoxic intratubular cells.

A nonisotopic in situ hybridization method to detect perforin mRNA was developed in cytospin preparations of IL-2-stimulated normal human lymphocytes and applied to formalin-fixed acutely rejected renal transplant material. Individual cells expressing perforin mRNA were localized in severely damaged tubular areas, and a number of these cells appeared to be located inside the tubular basement membrane in close association with tubular epithelial cells. Immunoperoxidase staining in acetone-fixed cryostat sections of acutely rejected kidney confirmed that a considerable proportion of infiltrating cells was CD8+; many of these were in an intratubular location. In addition, perforin protein was identified in individual cells in similar locations to perforin mRNA-positive cells. Again, some intratubular cells were identified. Our findings illustrate that these cells can be fully activated with definite cytotoxic potential. Previously we have demonstrated that T lymphocytes proliferate within the tubular compartment during tubulitis, a characteristic condition in acute renal allograft rejection, and that there is associated tubular epithelial cell proliferation. In this study we think that we have further clarified the consequences of invasion of tubules by lymphoid cells. Our in situ hybridization method in rapid and convenient and may be applied to archival material.

In situ lymphoproliferation in renal transplant biopsies.

A double immunohistochemical labelling procedure in paraffin-embedded renal tissue is reported in which CD3 was targeted as a T cell marker and Ki67 as a marker of cell proliferation. Proliferating and quiescent T cells were unequivocally identified in situ, and their precise location within the kidney was clarified by the use of periodic acid-Schiff counterstaining to outline the basement membranes. Proliferating tubular epithelial cells were also clearly identified. The results showed that T lymphocytes proliferate within the tubular compartment during acute renal allograft rejection. Preliminary evaluation of the method in routine transplant biopsies indicated significant correlations between histologically defined rejection grade and mean intratubular T lymphocytes per tubular cross section and between proliferation of tubular epithelial cells and of intratubular T lymphocytes. The associated tubular epithelial cell proliferation may be a response to local damage.

Anti-glomerular basement membrane glomerulonephritis in the mouse: the role of macrophages.

An immunohistochemical study was undertaken on fixed, paraffin-embedded mouse kidney in order to elucidate the role and significance of infiltrating macrophages in a mouse model of anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). Tissue was available representing the full gamut of histological features seen in this model. The mouse macrophage-specific antigen F4/80 was detected in tissue sections of glomerulonephritic kidney and the pattern and extent of staining was compared with normal mouse kidney. In glomerulonephritic kidney, an increase in the number of F4/80-positive cells was evident in close proximity to and surrounding Bowman's capsule of those glomeruli which were severely damaged, with extensive fibrin deposition and well developed cellular crescents. F4/80-positive cells did not feature in the glomerular tuft or in the region of the parietal epithelium of Bowman's capsule even when extensive cellular crescents were present. Breaks in Bowman's capsule were not demonstrated. We conclude that F4/80-positive macrophages are not a major constitutive cell type of developing crescents in this mouse model of anti-GBM GN but, by virtue of their peri-glomerular localization, may be involved in the destructive process, perhaps producing signalling molecules which contribute to the inflammatory reaction.

In vitro bromodeoxyuridine labelling of renal biopsy specimens: correlation between labelling indices and tubular damage.

AIMS: To examine the correlation between bromodeoxyuridine (BrdU) labelling indices (LI) and tubular damage in renal biopsy specimens; to evaluate the diagnostic and prognostic potential of measuring cell proliferation in a variety of renal lesions. METHODS: In vitro BrdU labelling of renal biopsy specimens was undertaken and labelled cells were detected in routinely fixed, paraffin wax embedded sections by immunohistochemistry. The BrdU LI were calculated as percentages for the three types of tubular cells--proximal and distal convoluted tubules and medulla (LI/PCT, LI/DCT, LI/Med)--and a total tubular BrdU LI (LI/Tub) was also calculated for each biopsy specimen. Histological features indicative of tubular damage were also scored and a total tubular damage score obtained for each biopsy specimen. RESULTS: The one hour labelling process did not affect tissue morphology or impede subsequent diagnosis. Four biopsy specimens were obtained from three renal transplant recipients. Diagnosis of 19 non-transplant biopsy specimens revealed a variety of renal lesions. Total tubular damage scores ranged from 0 to 25 and the LI/Tub ranged from 0 to 3.68% in all 23 biopsy specimens. Analyses of variance showed highly significant correlations between the total tubular damage score and both LI/Tub (p = 0.004) and LI/PCT (p = 0.004); a weaker correlation was found between the total tubular damage score and LI/DCT (p = 0.013). CONCLUSIONS: A correlation was found between tubular damage and BrdU LI. This was most clearly seen in the proximal tubules. However, as the study was limited to a few examples of specific forms of glomerular or interstitial disease, firm conclusions about the value of BrdU labelling in routine diagnosis and prognosis could not be drawn.

Renal allograft rejection: expression and function of VCAM-1 on tubular epithelial cells.

The interaction between vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4) is known to play an important role in stabilizing the adhesion of lymphocytes to endothelial cells. Such cellular adhesion is crucial to many immunological processes including lymphocyte-mediated cell lysis. In this study the expression of VCAM-1 on renal tubular epithelial cells is demonstrated on biopsy sections recovered during acute renal allograft rejection. Experiments performed using epithelial cells cultured from renal tubules show that VCAM-1 is up-regulated by addition of the inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Combination of TNF-alpha and IFN-gamma synergized to induce high levels of VCAM-1 expression. Further experiments demonstrated that the cytokines produced by activated lymphocytes in mixed leucocyte culture also up-regulate expression of VCAM-1. Assays of the adhesion of lymphoid cells to cultured renal epithelial cells showed that cytokine pretreatment of the renal cells enhanced the binding of lymphoid cells. The proportion of bound lymphoid cells was significantly reduced by addition of an antibody capable of blocking the interaction of VCAM-1 with VLA-4. This result indicated that the VCAM-1 induced on renal epithelial cells by inflammatory cytokines is functionally capable of binding VLA-4, thereby enhancing the adhesion of potentially graft-damaging lymphoid cells.

Tissue distribution of amyloid P component as defined by a monoclonal antibody produced by immunization with human glomerular basement membranes.

A monoclonal antibody reactive against amyloid P component (NCL-AMP) has been developed following immunization of mice with partially-purified human glomerular basement membranes (GBM) and standard hybridization and cloning techniques. The antibody reactivity was evaluated by enzyme-linked immunosorbent assay (ELISA) and by the indirect immunoperoxidase technique on sections of frozen and fixed human kidney and other tissues. The distribution of amyloid P component in various normal tissues is described and the possible co-localization with the Goodpasture antigen is discussed. In addition, the suitability of the antibody for detection of amyloid deposits in renal amyloidosis is demonstrated and its potential for use in other pathological conditions is considered.

Anti-glomerular basement membrane glomerulonephritis (anti-GBM GN) in the mouse: BrdU-labelling indices and histological damage.

In-vivo BrdU incorporation and visualization by immunohistochemistry, previously reported in normal mouse kidney, were applied to a mouse model of anti-GBM GN, induced by immunization with rabbit anti-mouse GBM antiserum, to assess the contribution of capsular cell proliferation in the development of crescents. A significant increase (P = 0.003) in the BrdU-labelling index (LI) for capsular cells was observed, as compared to normal mice (5.76 +/- 1.1 vs 0.70% +/- 0.12%). Elevated LI were also observed for tuft and tubular cells but these increases were not statistically significant. It was concluded that, in this model, capsular cell proliferation is a major contributory factor to the formation of cellular crescents. In addition, other pathological features, indicative of glomerular damage, were assessed semi-quantitatively alongside numbers of labelled capsular cells per glomerulus. It was found that podocyte vacuolation is strongly associated with, and may precede, proliferation, suggesting some common causative factor. Fibrin, when present, was confined within the tuft capillary loops and was only weakly associated with either podocyte vacuolation or capsular cell proliferation. It was concluded that this protein does not play a major role in the initiation of pathological damage. Finally, glomerular lesions were found to be randomly distributed. Thus, the idea of intraglomerular signalling, resulting in 'clustering' of damaged glomeruli, is not supported.

Remnant kidney pathology after five-sixth nephrectomy in rat. II. Electron microscopy study.

In Wistar male rats, hypertension was induced by 5/6 nephrectomy (5/6N). Electron microscopy revealed an increase in the number of epithelial cells on the Bowman's capsule during the early stages (4 weeks). After 8 and 12 weeks, frequent adhesion was observed between the glomerular tuft and Bowman's capsule. The abnormal podocytes showed nuclear irregularities and distortions. Characteristic foot process fusion formed cytoplasmic plates. There was a considerable increase in mesangial matrix and cells. No immune deposits or breaks in the glomerular basement membrane were observed. In the endothelial cells, the fenestration disappeared in sclerosed glomeruli. Some capillary loops were obliterated by fibrin, macrophages and foam cells. These findings combined with our previous light microscopy and immunofluorescent observations suggest a non-immunogenic glomerular sclerosis.

Remnant kidney pathology after five-sixth nephrectomy in rat. I. A biochemical and morphological study.

In Wistar male rats, hypertension was induced by 5/6 nephrectomy (5/6N). Body weight, blood pressure measurements, morphological and biochemical changes were followed (at four weekly intervals) for 12 weeks after 5/6N. Renal function was assessed by daily total urinary protein (TUP), plasma creatinine concentration [(Cr)p] and creatinine clearance rate. Plasma renin concentration (PRC), aldosterone concentration and erythrocyte content of sodium [Na]E and potassium [K]E were also investigated. Significant increases in systolic blood pressure (SBP), TUP, [(Cr)p] and [Na]E occurred after 4, 8, and 12 weeks of 5/6N. Progressive glomerulosclerosis (GSC), tubular atrophy and interstitial fibrosis were observed. Positive correlations were found between GSC and SBP and TUP. Positive correlations were also found between SBP and [Na]E and [(Cr)P]. PRC was not increased and showed no correlation with SBP. It is concluded that 5/6N produced hypertension associated with a series of morphological and biochemical alterations in kidney structure and function. In this model, mechanisms other than the renin-angiotensin system may be involved.

Tissue artefacts caused by sponges.

Artefacts which occur during the processing of small biopsy specimens can cause sufficient tissue distortion to impair interpretation and can be a considerable source of nuisance. Triangular artefacts were noted in renal and liver biopsy specimens which were caused by foam sponges in embedding cassettes. Scanning electron microscopic examination of the sponges showed they comprised a mesh of scimitar-shaped rigid spikes which closely match the artefacts seen in the tissues.

Immunoperoxidase techniques and histology in the diagnosis of rhabdomyolysis related acute renal failure.

A case of rhabdomyolysis associated acute renal failure (RM-ARF) occurring as a result of strenuous exercise is presented. Diagnostic renal biopsy was performed. The histological appearances, combined with immunoperoxidase staining for myoglobin, allowed a positive diagnosis of RM-ARF to be made and excluded the possibility of glomerulonephritis. The patient recovered completely after a stormy clinical course.

Three-dimensional reconstruction of human juxtaglomerular apparatus (JGA).

A three-dimensional and morphological study of the human JGA was undertaken to establish a background for understanding the changes in this vital apparatus during various physiological and pathological conditions. Three-dimensional reconstruction was carried out using a computer program "GLOM". Serial sections of normal human kidneys were used after staining with specific human renin antiserum. Three-dimensional reconstruction revealed renin-positive cells in the afferent and efferent arterioles and interlobular arteries away from the JGA area. A close contact was demonstrated between renin-positive cells and the macula densa. The frequency of positively stained JGAs was significantly higher in the superficial glomeruli compared to the deep glomeruli. The high renin content of the superficial glomeruli suggests higher generation of angiotensin, which may contribute to the regulation of the GFR as proposed by other workers. This preliminary study on normal human JGA is to be extended to hypertensive and renal failure patients.