The importance of glomerular deposits of von Willebrand factor in human renal allografts

OBJECTIVE: The aim of this study was to evaluate the importance of glomerular expression of von Willebrand factor (vWF) in human renal allografts. METHODS: We investigated graft biopsies from 72 renal transplant recipients, 40 with acute rejection (AR) and 32 with chronic allograft nephropathy (CAN). All biopsy specimens were immunostained with vWF and CD68 and graded using 3-tiered scales. The follow-up biopsies of patients with AR were reevaluated for development of glomerular sclerosis. RESULTS: A significant difference was found between type 1 and type 2 AR with regard to glomerular vWF expression (P < 0.01). None of the patients with type 1 AR showed mesangial vWF expression, but 36.4% of patients with type 2 AR showed segmental mesangial vWF expression. In follow-up biopsies, 18 of 40 patients developed significant glomerular sclerosis, and patients with mesangial vWF expression (grade 3 GvWF) showed glomerular sclerosis earlier than did others (P < 0.01). In addition, the outcome for grafts that showed grade 3 glomerular vWF was significantly worse than was the outcome noted for grafts that showed grade 1 or grade 2 glomerular vWF (P < 0.001). Half of the biopsy specimens in the CAN group showed global mesangial vWF expression. Glomerular macrophage infiltration was correlated with degree of glomerular vWF expression both in the AR and in the CAN groups (P < 0.05, P= 0.001, respectively). CONCLUSION: We hypothesized that the increasing amount of glomerular vWF may be used as a marker of acute vascular rejection and may help for the evaluation of renal allograft biopsies without sufficient arteries. In addition, it can also be a marker for development of early glomerular sclerosis and may help us determine which patients are in need of further treatment.     

Relationship of intraglomerular coagulation and platelet aggregation to glomerular sclerosis

In order to investigate the relationship between intraglomerular coagulation and glomerular sclerosis, the distribution of fibrin-related antigen (FRA) in glomeruli without extracapillary lesions was examined by immunoperoxidase microscopy in 80 patients with IgA nephropathy (IgA-N). A total of 302 glomeruli were examined, including 20 with global sclerosis, 31 with segmental sclerosis (SS glomeruli), and 251 nonsclerosed glomeruli. In the nonsclerotic areas of SS glomeruli, the deposition of FRA was significantly greater than in the nonsclerosed glomeruli. In the nonsclerosed glomeruli FRA was mainly found in the mesangium, while in the nonsclerotic areas of SS glomeruli FRA was not only present in the mesangium but also in the endothelium of the glomerular capillary loops. FRA-positive microclots were also often observed attached to the endothelium of the capillaries of the nonsclerotic areas of SS glomeruli. Cross-linked FRA was also observed in the endothelium of the same capillaries using the monoclonal antibody DD3B6/22. Deposition of von Willebrand factor (vWF) was greater in the endothelium than in the mesangium in the same areas. Aggregated platelets adhering to the glomerular capillary walls in these areas were frequently detected using the monoclonal antibody P2. Such distribution of platelets and vWF showed that the endothelium of the nonsclerotic areas of SS glomeruli was more severely damaged than that of nonsclerosed glomeruli. These findings suggest that endothelial cell damage might activate the intraglomerular coagulation, which might be one of the factors in the development of global glomerular sclerosis.     

CD40 expression on graft infiltrates and parenchymal CD154 (CD40L) induction in human chronic renal allograft rejection

BACKGROUND: CD40-CD154 (CD40L) costimulatory signaling plays a pivotal role in the effector mechanisms of transplant graft rejection. In animal models, CD40-CD154 blockade induces long-term graft acceptance concurrent with an absence of chronic rejection (CR) lesions. Given the critical importance of CD40-CD154 interactions in the development of chronic transplant allograft rejection, the relevance of in situ CD40 and CD154 expression was assessed in human chronic renal allograft rejection. METHODS: The expression of CD40, CD154, CD68, and T-cell receptor (TCR)alpha/beta was analyzed by immunohistochemistry. Serial cryostat sections of snap-frozen core renal allograft biopsies were obtained from 30 renal transplant patients. Biopsy specimens received diagnoses of CR (N = 23) according to the Banff classification and were compared with controls (N = 7) consisting of stable allografts and normal kidney tissue. RESULTS: Striking CD40 staining of graft cellular infiltrates (P = 0.016) was observed in renal allografts with CR compared with controls. The CD40+ cellular infiltrates in CR were predominantly TCR alpha/beta + T cells and some CD68+ macrophages. These findings were contrasted by the low-level CD40 expression detected in glomeruli and tubules of CR and controls. However, glomerular induction of CD154 was observed in CR allografts (P = 0.028) as compared with controls. CD154 immunoreactivity was demonstrated on glomerular endothelial, epithelial, and mesangial cells. Moderate CD154 expression was detected on tubular epithelial cells, and only weak CD154 immunoreactivity was observed on the infiltrates in isolated CR cases. CONCLUSION: In human chronic renal allograft rejection, CD40 is expressed on graft-infiltrating cells of the T cell and macrophage compartments. CD154 expression is induced on glomerular and tubular epithelial cells during CR, demonstrating another novel source of CD154 expression. The data substantiate the potential contributory role of an interaction between CD40+ graft-destructive effector T cells and macrophages with CD154+ renal allograft parenchymal cells in the development of chronic renal allograft rejection.     

C4d complement split product expression in chronic rejection of renal allograft

Chronic allograft rejection remains the major cause of late renal graft loss. Its pathogenesis is complex, depending on both immunological and nonimmunological factors. An important role in development of chronic rejection is ascribed to an ongoing immunological reaction mainly of the humoral type. C4d complement split product, as a stable fragment of complement degradation activated by antigen-antibody complexes, is considered to be an indicator of humoral activity in allografts. The aim of the present study was to establish a correlation between C4d expression and morphological findings specific for chronic rejection among biopsy specimens from patients with deteriorating graft function versus protocol biopsy specimens versus biopsy specimens of native kidneys with glomerular diseases. C4d deposits in peritubular capillaries and glomeruli were observed in 83% of patients with morphological changes of chronic rejection. No C4d expression was found in the protocol biopsy group. C4d deposits in glomeruli localizations were found in kidneys from patients with glomerulopathies; the pattern of distribution was similar to that for antibodies characteristic for glomerulonephritis. There was a positive correlation between C4d expression and morphological features of chronic rejection. In our opinion, only peritubular capillary localization is specific for a rejection process; glomerular localization is nonspecific and probably secondary to antigen-antibody complex deposition in course of some types of glomerulopathies.     

Expression of fractalkine, CX3CR1, and vascular endothelial growth factor in human chronic renal allograft rejection

AIM: Fractalkine/CX3CR1 system may contribute to the pathogenesis of renal allograft chronic rejection (CR). Vascular endothelial growth factor (VEGF) is an endothelial mitogen, which shows increased expression in inflammation and vasculopathy. This study sought describe the expression and distribution of Fractalkine/CX3CR1 and VEGF, and their relationship to human renal allograft CR. METHODS: Renal tissue from 10 patients with CR was examined for Fractalkine/CX3CR1 and VEGF protein by immunohistochemistry for comparison with patients displaying hyperacute rejection (n = 10), acute rejection (n = 10), and normal kidneys (n = 10). All patients were selected based upon histologically proven diagnoses between 1992 and 2003. RESULTS: Immunohistochemistry revealed that Fractalkine/CX3CR1 were mostly expressed in the tubulointerstitium and tubular epithelial cell basolateral membrane. Some vessels showed positive staining for Fractalkine/CX3CR1 as well as occasionally glomerular parietal wall cells. Among the CR group, VEGF was mostly expressed in tubular epithelium and the tubulointerstitium. A proportion of glomeruli and vessels had positive staining for VEGF, which was up-regulated most strikingly in the interstitial compartment in CR. There was markedly increased expression of Fractalkine/CX3CR1 and VEGF protein in the interstitium of the CR compared with other groups (P < .05). VEGF colocalized with the expression of Fractalkine/CX3CR1. CONCLUSION: Fractalkine/CX3CR1 and VEGF may play an important role in the development of interstitial fibrosis via mononuclear cell-induced cytokine production and myofibroblast stimulation in CR. Further studies are necessary to identify the role in the pathogenesis of CR.     

Differential expression of tissue factor (TF) in calcineurin inhibitor-induced nephrotoxicity and rejection--implications for development of a possible diagnostic marker

Deposition of fibrin in the form of fibrinoid necrosis is a common feature of severe acute renal allograft rejection. The role of the coagulation system and its initiator tissue factor (TF) during this process is, however, still poorly understood. In this study, we analyzed the expression of TF in 88 renal transplants afflicted with different forms of rejection and calcineurin inhibitor-induced nephrotoxicity, to see whether there was differential expression of this protein. TF immunoreactivity was evaluated semiquantitatively in six different renal structures: the podocytes, Bowman epithelium, the endothelium of the glomeruli, the brush border of tubular cells, the thin ascending loop of Henle, and small arteries/arterioles. The TF expression of normal renal tissue (n=6) was restricted to the glomerular podocytes and Bowman epithelium, and to some extent the ascending loop of Henle. Renal allografts undergoing acute rejection (AR) of grades I-III, (n=13, n=17 and n=12, respectively) did not show any altered TF expression in the glomeruli or vascular endothelium. In the ascending loop of Henle, a reduced expression could be seen (ARI, p=0.015; and ARII, p=0.043). TF staining of the brush border of renal transplants undergoing acute cyclosporin A (CsA) nephrotoxicity (n=18) was significantly higher than in normal kidneys (p=0.0003), as well as in transplants undergoing various degrees of acute rejection (ARI, p=0.027; ARII, p=0.0012; and ARIII, p=0.0001). Tubular brush border-expressed TF was also evident in 10 of 15 allografts suffering from chronic CsA nephrotoxicity, compared to 4 out of 13 cases with chronic allograft vasculopathy (CAV), but the increase was not statistically significant relative to normal kidneys. The majority of the grafts afflicted with either of the two chronic conditions displayed a TF-positive arterial endothelium (CAV, p=0.0034; and chronic CsA nephrotoxicity, p=0.0026) relative to controls. In conclusion, these results indicate that vascular TF expression is not altered during acute rejection, but may be of importance in chronic allograft nephropathy. Furthermore, TF immunoreactivity in the tubular brush border may be specific to acute CsA nephrotoxicity and might be used as a biomarker for this condition. Further studies are required to evaluate the possible role of brush border-expressed TF in the pathogenesis of CsA nephrotoxicity.     

Localization of decay accelerating factor in normal and diseased kidneys

Decay accelerating factor (DAF) is a cell membrane associated glycoprotein that inhibits C3 activation. In the present study we evaluated the presence of DAF in normal (N = 15) and diseased human kidneys (N = 76). Sections of frozen tissue were stained for DAF by immunoperoxidase, utilizing three mouse monoclonal anti-DAF anti-bodies. In normal kidneys, DAF was localized in the glomerular vascular pole, apparently in the juxtaglomerular apparatus (JGA). All other structures were negative for DAF. By contrast, in diseased kidneys, two types of abnormalities were detected. First, JGA-DAF was significantly decreased and this abnormality correlated with the pathologic diagnosis and with the presence of C3, IgM and/or fibrinogen in the glomeruli. Second, DAF was present in the glomerular mesangium (67%), renal interstitium (68%) and/or blood vessels (38%). The presence of DAF in the mesangium and interstitium of the kidney correlated with each other and correlated with C1q and C3 deposition in the glomerulus. Finally, vascular DAF was significantly more common in patients with electron dense deposits in the glomeruli. In summary, DAF is present in the normal kidney and is located exclusively in the glomerular vascular pole. In diseased kidneys, DAF tends to be lost from the JGA but is often present in glomerular mesangium, interstitium and blood vessels. This pattern is specially prominent in patients demonstrating complement deposition in the glomerulus. We speculate that kidney DAF may play a role in protecting the kidney against the products of complement activation.     

The Importance of Glomerular Deposits of Von Willebrand Factor in Human Renal Allografts

Objective. The aim of this study was to evaluate the importance of glomerular expression of von Willebrand factor (vWF) in human renal allografts. Methods. We investigated graft biopsies from 72 renal transplant recipients, 40 with acute rejection (AR) and 32 with chronic allograft nephropathy (CAN). All biopsy specimens were immunostained with vWF and CD68 and graded using 3-tiered scales. The follow-up biopsies of patients with AR were reevaluated for development of glomerular sclerosis. Results. A significant difference was found between type 1 and type 2 AR with regard to glomerular vWF expression (P < 0.01). None of the patients with type 1 AR showed mesangial vWF expression, but 36.4% of patients with type 2 AR showed segmental mesangial vWF expression. In follow-up biopsies, 18 of 40 patients developed significant glomerular sclerosis, and patients with mesangial vWF expression (grade 3 GvWF) showed glomerular sclerosis earlier than did others (P < 0.01). In addition, the outcome for grafts that showed grade 3 glomerular vWF was significantly worse than was the outcome noted for grafts that showed grade 1 or grade 2 glomerular vWF (P < 0.001). Half of the biopsy specimens in the CAN group showed global mesangial vWF expression. Glomerular macrophage infiltration was correlated with degree of glomerular vWF expression both in the AR and in the CAN groups (P < 0.05, P = 0.001, respectively). Conclusion. We hypothesized that the increasing amount of glomerular vWF may be used as a marker of acute vascular rejection and may help for the evaluation of renal allograft biopsies without sufficient arteries. In addition, it can also be a marker for development of early glomerular sclerosis and may help us determine which patients are in need of further treatment.     

Chronic renal allograft rejection. Selective involvement of the glomerular endothelium in humoral immune reactivity and intravascular coagulation

To study immune reactive and thrombotic mechanisms involved in chronic renal allograft rejection, Lewis rat kidneys were transplanted into bilaterally nephrectomized Brown Norway recipients tolerant of LEW erythrocyte antigens. Such BN rats fail to produce anti class I MHC alloantibodies after insertion of a LEW kidney. The LEW renal allografts experience a transient rejection episode without proteinuria followed by the development of chronic rejection, clinically characterized by glomerular proteinuria in the presence of stable renal function. Immunohistological studies of such chronically rejected LEW renal allografts showed the occurrence of glomerular and interstitial infiltration of predominantly monocytes and T cells. CD4-positive T cells dominated over CD8-positive T cells in the chronically rejected LEW renal grafts. IgG deposition was found deposited throughout the renal vasculature--this in contrast to IgM, which was observed only in the glomerular vasculature. Glomerular antibodies were not directed to endothelial class II MHC antigens, and showed only weak complement fixation as demonstrated by C3 staining. Selective glomerular IgM deposition was associated with vascular (platelet-containing) thrombi, and focal and segmental fibrinoid necrosis. In contrast, acutely rejected LEW renal grafts in unmodified BN recipients showed IgM deposition as well as thrombus formation throughout the entire renal vasculature. The results demonstrate that the antibody response to endothelial--and, in particular, glomerular endothelial non-MHC antigens--may bring about chronic vascular renal allograft rejection. How the formation of glomerular thrombotic lesions may be assisted by endothelial reactivity to cytokines from local immune reactive cells is discussed.     

猪肾小球内皮细胞与系膜细胞的发育过程及相互关系

目的:明确中国实验用小型猪肾小球内皮细胞与系膜细胞的发育过程及相互关系。方法:采集不同时间点(胚胎28～112d及出生后1d、7d、14d、21d)中国实验用小型猪肾组织,应用免疫荧光技术检测胚肾发育不同阶段(帽状间充质、肾小囊体、逗号形体、"S"形体、毛细血管袢期肾小球及成熟肾小球)内皮细胞标志物CD31与系膜细胞标志物α-SMA的表达情况。结果:内皮细胞标志物CD31在胚肾早期呈散在性分布,继而围绕肾小囊体和逗号形体呈"环抱"状分布,然后进入"S"形体下端的血管裂隙内聚集形成无管腔的"前毛细血管束",最后表达于成熟肾小球毛细血管内皮细胞。系膜细胞标志物α-SMA在早期胚肾、肾小囊体和逗号形体阶段均无表达;"S"形体早期分布于"S"形体周围,后期进入"S"形体下端的血管裂隙;毛细血管袢期聚集在肾小球血管极根部;随着肾小球发育逐渐向肾小球内延伸,最终表达于成熟肾小球系膜区。CD31与α-SMA双重染色的结果显示,在毛细血管袢期CD31阳性的内皮细胞聚集形成无管腔的前毛细血管束,而α-SMA阳性的系膜细胞聚集在肾小球血管极的根部;随着肾小球发育,α-SMA阳性的系膜细胞逐渐由血管极根部向肾小球内迁移,同时CD31阳性的内皮细胞逐渐形成带有管腔的毛细血管丛。结论:中国实验用小型猪肾小球内皮细胞的发育开始于后肾间充质阶段,系膜细胞的发育开始于"S"形体阶段,即肾小球系膜细胞发育在内皮细胞之后;在肾小球血管丛形成过程中,内皮细胞与系膜细胞间可能存在重要的相互作用。     

The presence and prognostic importance of glomerular macrophage infiltration in renal allografts

AIM: The purpose of this study was to analyze the role of intraglomerular macrophage infiltration in human renal allografts by examining biopsies from kidney grafts that were dysfunctional after transplantation. METHODS: Eighty-three patients (58 men, 25 women) of a mean age of 30.2 +/- 1.4 years were evaluated. In all cases, biopsy specimens were examined for the presence of macrophage infiltration in the glomeruli. The infiltration of these cells was evaluated immunohistochemically using monoclonal antibody CD68, which labels macrophage cytoplasm. 10 renal allograft biopsies with normal histopathology were used as control group. The CD68-positive macrophages in all glomeruli were counted and the glomerular macrophage index (GMI) was calculated. RESULTS: Of the 83 patients, 40 showed acute rejection (AR), 33 showed chronic rejection (CR) and 10 showed cyclosporin A (CsA) toxicity. Only the biopsies of 28 patients stained positive for CD68 in the glomeruli. Neither patients with CsA toxicity nor controls showed intraglomerular macrophages. The CD68-positive group consisted of 7/33 CR and 21/40 AR patients. We observed intraglomerular macrophages in only 6 of the 20 AR cases that responded to steroid therapy (mean GMI 0.3 +/- 0.1) and in 15 of the 20 steroid-resistant AR cases (mean GMI 1.7 +/- 1.2; p < 0.01). The outcome of grafts that contained intraglomerular macrophages was significantly worse than the outcomes of other grafts noticed during the follow-up. CONCLUSION: We conclude that the presence of glomerular macrophages can be considered a marker for rejection and is a valuable additional criterion of rejection in the histological examination of renal allograft biopsies. The presence of intraglomerular macrophages indicates that the outcome of the graft will be significantly worse than that of grafts without intraglomerular macrophage infiltration.     

Expression of the chemokine receptor CCR1 in human renal allografts.

BACKGROUND: Chemokines are involved in the recruitment of leukocytes to vascularized allografts. CCR1 is a receptor for various proinflammatory chemokines and CCR1 blockade reduces renal allograft injury in rabbits. The purpose of the study was to characterize CCR1-positive cells in human renal allografts. METHODS: Formalin-fixed, paraffin-embedded allograft nephrectomies (n = 9) and non-involved parts of tumour nephrectomies (n = 10) were studied. Immunohistochemistry for CCR1, CD3 and CD68 was performed on consecutive sections. Double immunofluorescence for CCR1 and CD3, CD20, CD68, DC-SIGN and S100 was used on selected cases. Expression of CCR1 mRNA and the ligands CCL3 and CCL5 was studied in renal allograft biopsies with acute rejection (n = 10), with chronic allograft nephropathy (n = 8) and controls (n = 8). RESULTS: CCR1 protein was expressed by circulating cells in glomerular and peritubular capillaries, colocalizing with CD68. In renal allografts CCR1-positive cells were present within glomerular tufts, but only scattered CCR1-positive cells were found in tubulointerstitial infiltrates. CCR1 did not colocalize with the majority of CD68-positive cells in the interstitium. The small number of CCR1-positive interstitial cells were identified as CD20- or DC-SIGN-positive by double immunofluorescence. CCR1 mRNA was significantly increased in renal biopsies with acute allograft rejection (P < 0.001), and with chronic allograft nephropathy (P < 0.05), it correlated with the expression of CCL3 and CCL5, and with serum-creatinine. CONCLUSIONS: CCR1 mRNA expression was associated with renal function in allografts. CCR1 protein expression was restricted to monocytes, CD20-positive B cells and DC-SIGN-positive dendritic cells. Thus most interstitial macrophages were CCR1 negative, which may relate to down-regulation after migration into the interstitium in human renal allografts.     

Monocytes and peritubular capillary C4d deposition in acute renal allograft rejection

BACKGROUND: Peritubular capillary (PTC) deposition of complement split factor C4d in renal allografts has been shown to be closely associated with circulating antidonor antibodies and a marker for relatively poor graft survival. Monocyte/macrophage (MO) infiltration of renal allografts has been shown to adversely affect graft survival. The purpose of this study was to assess whether the two phenomena are related. METHODS: Twenty-three biopsies (from 15 patients) demonstrated diffuse strong staining of PTC for C4d (C4d+ group) and acute tubular injury with or without significant cellular rejection, while 28 biopsies (with acute rejection) but negative for PTC C4d served as controls (C4d- group). RESULTS: The C4d+ group demonstrated significantly greater glomerular and interstitial MO infiltration than did the C4d- group [3.4 +/- 2.0 vs. 0.2 +/- 0.3 MO/glomerulus, P < 0.0001; 12.9 +/- 9.2 vs. 6.5 +/- 5.0 MO/high power field (hpf), P = 0.0030]. Neutrophilic (PMN) infiltration of glomeruli and PTC was also significantly greater in the C4d+ group than in the C4d- one (0.8 +/- 0.6 vs. 0.3 +/- 0.3 PMN/glomerulus, P = 0.0003; 0.9 +/- 0.8 vs. 0.4 +/- 0.3 PTC PMN/hpf, P = 0.0035). CONCLUSION: The results indicate a close association between PTC C4d deposition and MO infiltration, particularly glomerular, and confirm previous observations regarding the correlation of PTC C4d staining and PMN infiltration.     

Vascular endothelial growth factor (VEGF) ligand and receptor induction in rat renal allograft rejection

BACKGROUND: Acute rejection is the single most important risk factor for the subsequent development of chronic allograft nephropathy (CAN), which is still the primary reason for late allograft loss in kidney transplantation. Vascular endothelial growth factor (VEGF) is a proangiogenic factor that has an important role in the development and maintenance of physiological endothelium. While its role has been characterized in the pathology of diabetic nephropathy and preeclampsia, its role in the development of acute and chronic allograft rejection remains unclear. METHODS: Kidney transplantations were performed from DA to WF rats and syngeneic control transplantations were performed between DA rats. Normal kidneys were used as controls to evaluate physiological VEGF and VEGFR-1 expression. Allografted rats were immunosuppressed with cyclosporine (CsA) (1.5 mg/kg/d subcutaneously); and no immunosuppression was given to syngeneic grafts. Grafts were harvested at 5 and 90 days after transplantation for histology and immunohistochemistry (VEGF, VEGFR-1). RESULTS: In normal kidneys VEGF ligand and receptor expression was almost nonexistent. Only mild glomerular, arterial, and tubular VEGF expression was seen. In syngeneic grafts, no histological signs of acute or chronic rejection were seen, whereas characteristics of both acute and chronic rejection were seen in CsA-treated allografts. Altough VEGF expression was increased in syngenic grafts when compared to controls it still remained mild in both the early and the late posttransplant period. In CsA-treated allografts moderate VEGF expression was seen already 5 days after transplantation; the expression increased at 90 days after transplantation. The same pattern was also discovered for VEGFR-1 expression although the difference was not as remarkable after 5 days. CONCLUSIONS: Our results demonstrated that VEGF ligand and receptor expression was increased in both acute and chronic rejection. Our data suggested that VEGF may have an important role in the pathology of chronic rejection. Based on our findings VEGF inhibition could be a potential intervention to prevent CAN in clinical kidney transplantation.     

Renal distribution of collagen types III, IV and V in various glomerular diseases

The purpose of this study is to examine the immunochemical changes of the glomerular basement membrane (GBM) and the mesangium, in pretreated paraffin-embedded sections with trypsin by utilizing monoclonal antibodies to type III (anti-III), type IV (anti-IV) and type V (anti-V) collagens. We observed 6 normal kidneys and 44 kidneys with various renal diseases. In normal human kidney the staining with anti-IV demonstrated GBM, mesangium, Bowman's BM, tubular BM and capillary BM. Anti-V was also seen in the interstitium. On the other hand, anti-III stained only interstitium. Thickened GBM in membranoproliferative glomerulonephritis (MPGN) and diabetic nephropathy, and irregular GBM in Membranous Nephropathy and Alport's syndrome were also evident in anti-IV stain, while widened mesangial area was seen in anti-V rather than anti-IV stain. In severely proliferative GN, anti-III as well as anti-IV and anti-V was detected in the mesangium in spite of existence of neither adhesion nor Bowman's gap. In MPGN type II, anti-III was observed along the GBM. In obsolescent glomeruli, anti-IV was not always detected although anti-V was constantly seen. On the other hand, anti-III was markedly positive in the crescents and obsolescent glomeruli. These results suggest that it is possible for mesangial, endothelial and epithelial cell to produce several types of collagens and type III collagen is closely related to the process of the glomerular obsolescence.     

Thy-1.1 in glomeruli of rat kidneys

The antigenic composition of the rat kidney was studied by indirect immunofluorescence and immunoelectron microscopy using monoclonal antibodies against Thy-1.1 (OX7 and ER Ly 1), Thy-1.2 (30-H12), and W3/13 as well as rabbit antisera against rat brain or thymic blasts. The anti-Thy-1.1 reagents caused pronounced staining of the glomerular mesangium as well as a weak stain around some of the medullary collecting ducts and/or vessels. The rabbit antisera contained additional antibodies that bound to renal basement membranes and smooth muscle cells. This was confirmed by blocking and absorption experiments. The presence of Thy-1.1 in glomeruli was further demonstrated by in vivo perfusion studies with OX7 and rabbit anti-thymic blast serum. Immunoelectron microscopy of isolated glomeruli showed binding of OX7 antibodies to mesangial cells and the mesangial matrix. It is concluded that in rat kidneys the Thy-1.1 antigen is expressed on glomerular mesangial cells.     

Ultrastructural examination of glomerular and tubular changes in renal allografts with acute rejection

Acute rejection is the most important threat to transplanted kidneys in the early phase after transplantation. With the advances in renal transplant surgery and immunosuppressive therapies, one-year graft survival rates reached 90%, but long-term graft survival did not improve to a similar degree. To prevent acute rejection more effectively and decrease the risk of chronic nephropathy development, the pathogenesis and effects of acute rejection on renal grafts should be further explored. This study aimed to examine the glomerular and tubular changes ultrastructurally. Tissues were obtained from 11 renal allografts with acute rejection, fixed in 1% Osmium tetra oxide embedded in Epon. The changes in glomerular basement membrane, podocyte, mesangium, and proximal tubules were examined by electron microscope. Tubular changes such as tubular basement membrane multi-lamellation, MN and PMN cells in peritubular capillaries, tubular vacuolization, mitochondrial changes (increase in number, alterations in cristae organization, or cristae effacement), and infiltration of tubular epithelium by MN cells (mainly lymphocytes) were found statistically significant (p < 0.01) when compared to those of control group. Some forms of endothelial injury (swelling of endothelial cells or fenestrae loss) were also statistically significant (p < 0.01). Acute rejection is an important predictor of long-term graft survival, and there may be no clinical clue to make diagnosis easier. Therefore ultrastructural changes may help solve this problem together with molecular studies.     

Comprehensive immunohistological analysis of the endothelin system in human kidney grafts.

BACKGROUND: In experimental models of renal transplantation, upregulation of the endothelin (ET) system and amelioration of renal injury by ET-receptor blockers have been documented. In contrast, little information is available on the expression of the ET system in human kidney allografts. It was the purpose of the present study to analyse by immunohistology the expression of ET-1 as well as of the two ET receptors (ET-RA and ET-RB) in the different cells and compartments of kidney grafts and control kidneys. METHODS: Fifty-five graft biopsies were taken from 55 kidney allograft recipients (mean age: 32+/-2.8 years) who were all on a calcineurin inhibitor. The indication for biopsies was delayed graft function or suspected rejection. The underlying diagnoses were acute allograft rejection (n = 14), chronic allograft nephropathy (n = 14), cyclosporin A (CSA) toxicity (n = 10), post-operative acute tubular necrosis (ATN) (n = 11) and recurrent primary disease (n = 6). As control, tissues of non-grafted kidneys with ATN (mean age: 35+/-24 years), of primary glomerulonephritis (mean age: 69+/-10 years) and of non-tumour-bearing parts of eight tumour nephrectomy specimens (mean age: 67+/-5 years) were assessed. The biopsies were scored using the 1997 Banff criteria. Expression of ET-1, ET-RA and ET-RB as well as of vascular endothelial growth factor was evaluated by immunohistochemistry and a semi-quantitative scoring system. Interstitial infiltrating cells were characterized using antibodies against T cells, B cells and macrophages (CD3, CD20 and CD68). RESULTS: Control cases showed only faint expression of ET-1 in glomeruli (in podocytes and endothelial cells), whereas marked expression was seen in distal, but less in proximal tubular cells. The interstitium was completely negative. ET-1 expression was seen in vascular endothelial cells (VEC) and vascular smooth muscle cells (VSMC). Only faint expression of ET-RA and ET-RB was found in glomeruli and tubuli (distal more than proximal). Marked ET-RA and ET-RB expression was seen in VEC and VSMC. In all transplanted kidneys, irrespective of the underlying diagnosis, expression of ET-1, ET-RA and ET-RB was markedly higher compared with control kidneys. ET-1 was strikingly upregulated in glomeruli and tubuli, but surprisingly not in the vasculature of grafts with CSA toxicity. Expression of ET-RB was markedly increased in CSA toxicity in glomeruli, tubuli and vessels. In grafts with ATN and acute rejection, pronounced expression of ET-RA was noted. There was a strong correlation between proteinuria and expression of ET-1 in glomeruli and proximal tubuli and of ET-RB in proximal tubuli. CONCLUSIONS: The above data in human kidney allograft biopsies are consistent with an important role of the ET system in different types of renal allograft damage. This finding extends and clarifies the somewhat contradictory results in animal models.     

Increased angiotensin-converting enzyme and type 1 angiotensin receptors in cortical vasculature and tubulointerstitium of chronically rejected human kidney allografts

SUMMARY: Chronic rejection of human renal allografts after transplantation is characterized by interstitial infiltration, arteriosclerosis and glomerulosclerosis in the grafts. Apart from tissue HLA compatibility, angiotensin II (AII) may be implicated in accelerating the processes of chronic rejection. In the present study, the cellular distribution of angiotensin‐converting enzyme (ACE) and type 1 AII (AT₁) receptors was mapped and compared in non‐rejected human kidneys (n = 8) and chronically rejected renal allografts (n = 9) using complementary immunohistochemistry and quantitative in vitro autoradiography. Chronically rejected allografts showed typical histopathological characteristics of tissue rejection, including concentric intimal thickening of intrarenal arteries, extensive or focal glomerulosclerosis, tubular atrophy and severe tubulointerstitial fibrosis. In rejected allografts, total ACE binding in the cortex was decreased to 46% of that in non‐rejected kidneys (P < 0.01), whereas AT₁ receptor binding in the glomeruli and the inner stripe of the outer medulla was maintained. However, ACE and AT₁ receptor binding were increased in the cortical tubulointerstitium of chronically rejected allografts. In non‐rejected kidneys, strong ACE immunostaining occurred in proximal tubules and vascular endothelium, whereas AT₁ receptors occurred in vascular smooth muscle cells, as expected. In rejected allografts, intense ACE and AT₁ receptor immunostaining were detected not only in the same sites as those non‐rejected kidneys, but also in cortical tubulointerstitium between atrophied tubules and surrounding the glomeruli. AT₁ receptors were markedly up‐regulated in vascular smooth muscle cells of thickened atherosclerotic vessels. These results provide novel morphological evidence that increased expression and/or altered distribution of ACE and AT₁ receptors in cortical tubulointerstitium and in the neointima of intrarenal arteries may play an important role in the progression of chronic rejection of human renal allografts after transplantation.