Correlation of enhanced thrombospondin-1 expression, TGF-beta signalling and proteinuria in human type-2 diabetic nephropathy.

BACKGROUND: Activation of the thrombospondin-1 (TSP-1)-TGF-beta pathway by glucose and the relevance of TSP-1-dependent activation of TGF-beta for renal matrix expansion, renal fibrosis and sclerosis have previously been demonstrated by our group in in vivo and in vitro studies. Design and methods. We investigated renal biopsies (n = 40) and clinical data (n = 30) of patients with diabetic nephropathy. Ten kidneys without evidence of renal disease served as controls. Glomerular and cortical expression of TSP-1, p-smad2/3, fibrosis and glomerular sclerosis (PAS) were assessed by immunhistochemical staining and related with clinical data. RESULTS: Glomerular (g) and cortical (c) TSP-1 were increased during diabetic nephropathy (g: 2.62 +/- 2.65; c: 4.5 +/- 4.2) compared to controls (g: 0.67 +/- 0.7; c: 1.5 +/- 1.2). P-smad2/3 was significantly increased (g: 16.7 +/- 12.9; c: 148.7 +/- 92.8) compared to controls (g: 7.1 +/- 3.6; c: 55 +/- 25; P < 0.05). TSP-1 was coexpressed with p-smad2/3 as an indicator of TGF-beta activation. TSP-1 correlated with enhanced tubulointerstitial p-smad2/3 positivity (r = 0.39 and r = 0.4, P < 0.05) and glomerular p-smad2/3 correlated with proteinuria (r = 0.35, P < 0.05). CONCLUSIONS: In summary, the present study suggests a functional activity of the TSP-1/TGF-beta axis, especially in the tubulointerstitium of patients with diabetic nephropathy. The positive correlation of glomerular p-smad2/3 positivity with proteinuria further supports the importance of the TSP-1/TGF-beta system as a relevant mechanism for progression of human type-2 diabetic nephropathy.

Analysis of NO-synthase expression and clinical risk factors in human diabetic nephropathy.

BACKGROUND: Changes of renal nitric oxide (NO) production have been associated with glomerular hyperfiltration, vascular permeability, albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. Several studies demonstrated an up- as well as downregulated expression of NO-synthases (NOS) in experimental diabetic nephropathy. It is still not yet specified whether the regulation and activity of NOS is changed in human diabetic nephropathy. METHODS: Renal biopsies and clinical data of 45 patients with diabetic nephropathy and of 10 control subjects were investigated. Glomerular and cortical endothelial NOS (eNOS) and inducible NOS (iNOS) expression were assessed by immunohistochemical staining and related to clinical data such as the duration of diabetes, insulin therapy and arterial hypertension, albuminuria/proteinuria, eGFR according to the formula modification of diet in renal disease (MDRD), presence of vascular complications or diabetic retinopathy. RESULTS: The mean age of patients at biopsy was 60.3 years and the mean duration of diabetes 12.9 years. Expression of cortical and glomerular eNOS was increased in type 2 diabetes (P < 0.05). Increased expression of glomerular and cortical eNOS correlated with more severe vascular complications (r = 0.44; P < 0.05). Glomerular eNOS was strongly increased among different degrees of proteinuria (P < 0.01). In contrast to expression levels of eNOS, the glomerular expression pattern of iNOS changed from an endothelial pattern in glomeruli with preserved morphology towards expression predominantly by inflammatory cells. CONCLUSIONS: Thus, increased eNOS expression by the renal endothelium could be demonstrated in type 2 diabetic nephropathy, whereas iNOS was unchanged but spatially differentially expressed. The eNOS expression was related to vascular lesions and the degree of proteinuria.

Histologic analysis of renal leukocyte infiltration in antineutrophil cytoplasmic antibody-associated vasculitis: importance of monocyte and neutrophil infiltration in tissue damage.

OBJECTIVE: The histopathologic lesions in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) have been studied extensively, but the exact composition of the cellular infiltrate is unclear. We undertook this study to analyze renal leukocyte infiltration and the cellular distribution within glomeruli and interstitium in 65 renal biopsy samples obtained from patients newly diagnosed as having AAV. METHODS: Renal cellular tissue infiltration was assessed with an immunoperoxidase method. Furthermore, the infiltrating cell types were correlated with clinical and histopathologic data. RESULTS: The predominant interstitial infiltrating cells were T lymphocytes, while monocytes and, to a lesser extent, granulocytes constituted the dominant infiltrating cell types in glomeruli. Interestingly, lymphocyte infiltration was predominantly periglomerular, especially around glomeruli with sclerosis or heavy crescent formation, while interstitial monocyte and neutrophil infiltration was diffusely distributed over the interstitial tissue. A significant correlation was found for the glomerular infiltration of CD68-positive macrophages with the presence of glomerular necrosis as well as with the number of glomeruli with crescents (P < 0.0001 and P = 0.005, respectively). No correlation was found for interstitial fibrosis with the infiltration of any leukocyte subset. Furthermore, a significant correlation was found for the interstitial as well as for the glomerular infiltration of CD68-positive macrophages with serum creatinine concentration at the time of biopsy (P = 0.001 and P = 0.006, respectively). CONCLUSION: These data underscore a major role of monocytes in addition to neutrophils in the tissue damage of AAV.

Expression of cyclooxygenase-2 in biopsies obtained from human transplanted kidneys undergoing rejection.

BACKGROUND: The inducible cyclooxygenase (COX)-2 is a target of immunosuppressive drugs routinely administered to patients after transplantation. This study investigates a potential involvement of COX-2 in transplant rejection. Therefore, we examined the expression of COX-2 in biopsies obtained for diagnostic purposes. METHODS: COX-2 was detected by immunohistochemistry and in situ hybridization. Congruent staining was obtained by both methods: in specimens of a kidney explanted as the result of vascular rejection, tubular epithelial cells and endothelial cells stained positively for COX-2. Furthermore, in appendiceal specimens obtained at surgery, epithelial cells of the crypts, interstitial cells, and mesothelial cells were positive by both methods, affirming the specificity of the antibody. RESULTS: Compared with healthy control subjects, intensive staining of COX-2 was observed in most of the 28 biopsies obtained from patients diagnosed with vascular rejection combined with cellular interstitial rejection and tubulitis. Glomeruli and the macula densa area were essentially negative compared with prominent staining in cortical and medullary epithelial cells of the tubuli. Staining was distinct with individual positive cells in the tubular cross sections. Few arteries expressed COX-2 in intimal cells. Less prominent expression of COX-2 was detected in the biopsies of six kidneys obtained from patients diagnosed with acute tubular necrosis. CONCLUSION: This is the first report to show the up-regulation of COX-2 in human transplanted kidneys, despite ongoing immunosuppressive treatment. It remains to be established whether the up-regulation of COX-2 is part of the rejection process or has to be considered implicated in renal preservative mechanisms.

An increase in myeloid-related protein serum levels precedes acute renal allograft rejection.

Upon interaction with activated endothelium, monocytes and neutrophils form complexes of myeloid-related protein 8 (MRP8) (S100A8) and MRP14 (S100A9), two members of the calcium-binding S100 family that are secreted during transendothelial migration. In a pilot study of 20 renal transplant recipients and a validation study of 36 renal transplant recipients, MRP8/14 serum levels were measured with an enzyme-linked immunosorbent assay for 28 d, associated with C-reactive protein and creatinine serum levels, and grouped according to biopsy-proven acute rejection. Serum levels of MRP8/14 but not C-reactive protein were significantly increased for several days during the first 2 wk for the acute rejection groups in both studies (P < 0.005, on day 6 after transplantation). As determined by using receiver operating characteristic curves, the optimal cutoff for 100% specificity and high sensitivity (67%) for acute rejection on day 6 after transplantation was calculated to be 4.2 microg/ml for MRP8/14 in the pilot study; this value was confirmed in the validation study. Positive MRP8/14 serum levels preceded acute rejection episodes by a median of 5 d. A 3-d course of intravenous methylprednisolone therapy reduced prerejection MRP8/14 serum levels from 5.7 microg/ml to 3.3 microg/ml (P < 0.05). All MRP8/14 serum levels were below the cutoff during urinary tract infections, delayed graft function, or cytomegalovirus infections, and these values did not differ significantly from control values. It is concluded that the MRP8/14 complex is a very early serum marker suitable for monitoring of acute rejection with high sensitivity and specificity.