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Recent studies suggest that complement plays a role in the pathogenesis of focal segmental glomerulosclerosis (FSGS). Moreover, co-localization of IgM and C3 deposits with FSGS lesions has frequently been reported. Here, we investigated whether glomerular complement deposition precedes the development of FSGS and whether it represents local complement activation. Renal biopsies from 40 patients with primary FSGS, 84 patients with minimal change disease, and 10 healthy individuals were stained for C4d, C1q, and mannose-binding lectin. C4d deposits were also measured in renal allograft biopsies from 34 patients with native primary FSGS, 18 of whom subsequently developed recurrent FSGS. Lastly, we measured C4d deposits in the Munich Wistar Frömter rat model of FSGS. The prevalence of C4d-positive glomeruli was significantly higher among patients with FSGS (73%) compared to patients with minimal change disease (21%) and healthy individuals (10%). Moreover, segmental sclerosis was absent in 42% of C4d-positive glomeruli. Glomerular C1q was significantly more prevalent in FSGS compared to minimal change disease or healthy individuals, while mannose-binding lectin was infrequently observed. C4d deposition was significantly more prevalent in recurrent FSGS (72%) before the development of sclerotic lesions compared to control transplant samples (27%). Finally, at the onset of albuminuria but before the development of FSGS lesions, Munich Wistar Frömter rats had a significantly higher percentage of C4d-positive glomeruli (31%) compared to control rats (4%). Thus, glomerular C4d deposition can precede the development of FSGS, suggesting that complement activation may play a pathogenic role in the development of FSGS., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

We present a consensus report pertaining to the improved clarity of definitions and classification of glomerular lesions in lupus nephritis that derived from a meeting of 18 members of an international nephropathology working group in Leiden, Netherlands, in 2016. Here we report detailed recommendations on issues for which we can propose adjustments based on existing evidence and current consensus opinion (phase 1). New definitions are provided for mesangial hypercellularity and for cellular, fibrocellular, and fibrous crescents. The term "endocapillary proliferation" is eliminated and the definition of endocapillary hypercellularity considered in some detail. We also eliminate the class IV-S and IV-G subdivisions of class IV lupus nephritis. The active and chronic designations for class III/IV lesions are replaced by a proposal for activity and chronicity indices that should be applied to all classes. In the activity index, we include fibrinoid necrosis as a specific descriptor. We also make recommendations on issues for which there are limited data at present and that can best be addressed in future studies (phase 2). We propose to proceed to these investigations, with clinicopathologic studies and tests of interobserver reproducibility to evaluate the applications of the proposed definitions and to classify lupus nephritis lesions., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

In 2012, the Systemic Lupus International Collaborating Clinics (SLICC) presented a new classification for systemic lupus erythematosus (SLE). In this classification, biopsy-confirmed lupus nephritis with positive antinuclear or anti-double-stranded DNA antibodies became a stand-alone criterion. Because of the unknown diagnostic performance among patients from nephrology clinics, we aimed to test the validity of the SLICC classification, compared with the American College of Rheumatology classification, in a cohort of patients whose renal biopsies would raise the clinicopathologic suspicion of lupus nephritis. All patients with a renal biopsy showing full house glomerular deposits and clinical follow-up in our center were included and reevaluated, after which clinicians and a pathologist reached a consensus on the reference-standard clinical diagnosis of SLE. The diagnostic performance and net reclassification improvement were assessed in 149 patients, 117 of whom had clinical SLE. Compared with the American College of Rheumatology classification, the SLICC classification had better sensitivity (100 vs. 94%); although, this was at the expense of specificity (91 vs. 100%; net reclassification improvement -0.03). Excluding the stand-alone renal criterion, the specificity of the SLICC classification reached 100%, with a significant net reclassification improvement of 0.06 compared with the American College of Rheumatology classification. The SLICC classification performed well in terms of diagnostic sensitivity among patients with full house glomerular deposits; whereas, the stand-alone renal criterion had no additional value and compromised the specificity. Thus, presumed patients with lupus nephritis in nephrology clinics reflect a distinct SLE disease spectrum warranting caution when applying SLE classification criteria., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The reported prevalence of diabetic nephropathy (DN) among patients with diabetes varies widely. Most studies use the presence of microalbuminuria for clinical onset of DN in the absence of a histopathologic evaluation. In this autopsy study, we collected and analyzed data from a cohort of patients with type 1 or 2 diabetes and determined the prevalence of histologically proven DN in patients with or without clinical manifestations of renal disease. We also examined the distribution among histopathologic classes with respect to clinical parameters. Renal tissue specimens from autopsies and clinical data were collected retrospectively from 168 patients with diabetes. The histopathologic classification for DN was scored as were interstitial and vascular parameters. In this cohort, 106 of 168 patients had histopathologic changes in the kidney characteristic of DN. Twenty of the 106 histologically proven DN cases did not present with DN-associated clinical manifestations within their lifetime. Glomerular and interstitial lesions were associated with renal function but not with proteinuria. We also found that underdiagnosed DN may encompass all histopathologic classes except the sclerotic class. Thus, the prevalence of histologically proven DN was higher than previously appreciated, and we found a relatively high proportion of DN that was clinically underdiagnosed yet histologically proven, suggesting that DN lesions may develop before the onset of clinical findings., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The introduction of C4d in daily clinical practice in the late nineties aroused an ever-increasing interest in the role of antibody-mediated mechanisms in allograft rejection. As a marker of classical complement activation, C4d made it possible to visualize the direct link between anti-donor antibodies and tissue injury at sites of antibody binding in a graft. With the expanding use of C4d worldwide several limitations of C4d were identified. For instance, in ABO-incompatible transplantations C4d is present in the majority of grafts but this seems to point at 'graft accommodation' rather than antibody-mediated rejection. C4d is now increasingly recognized as a potential biomarker in other fields where antibodies can cause tissue damage, such as systemic autoimmune diseases and pregnancy. In all these fields, C4d holds promise to detect patients at risk for the consequences of antibody-mediated disease. Moreover, the emergence of new therapeutics that block complement activation makes C4d a marker with potential to identify patients who may possibly benefit from these drugs. This review provides an overview of the past, present, and future perspectives of C4d as a biomarker, focusing on its use in solid organ transplantation and discussing its possible new roles in autoimmunity and pregnancy.

To study the predictive value of biopsy lesions in IgA nephropathy in a range of patient ages we retrospectively analyzed the cohort that was used to derive a new classification system for IgA nephropathy. A total of 206 adults and 59 children with proteinuria over 0.5 g/24 h/1.73 m(2) and an eGFR of stage-3 or better were followed for a median of 69 months. At the time of biopsy, compared with adults children had a more frequent history of macroscopic hematuria, lower adjusted blood pressure, and higher eGFR but similar proteinuria. Although their outcome was similar to that of adults, children had received more immunosuppressants and achieved a lower follow-up proteinuria. Renal biopsies were scored for variables identified by an iterative process as reproducible and independent of other lesions. Compared with adults, children had significantly more mesangial and endocapillary hypercellularity, and less segmental glomerulosclerosis and tubulointerstitial damage, the four variables previously identified to predict outcome independent of clinical assessment. Despite these differences, our study found that the cross-sectional correlation between pathology and proteinuria was similar in adults and children. The predictive value of each specific lesion on the rate of decline of renal function or renal survival in IgA nephropathy was not different between children and adults.

IgA nephropathy is the most common glomerular disease worldwide, yet there is no international consensus for its pathological or clinical classification. Here a new classification for IgA nephropathy is presented by an international consensus working group. The goal of this new system was to identify specific pathological features that more accurately predict risk of progression of renal disease in IgA nephropathy, thus enabling both clinicians and pathologists to improve individual patient prognostication. In a retrospective analysis, sequential clinical data were obtained on 265 adults and children with IgA nephropathy who were followed for a median of 5 years. Renal biopsies from all patients were scored by pathologists blinded to the clinical data for pathological variables identified as reproducible by an iterative process. Four of these variables: (1) the mesangial hypercellularity score, (2) segmental glomerulosclerosis, (3) endocapillary hypercellularity, and (4) tubular atrophy/interstitial fibrosis were subsequently shown to have independent value in predicting renal outcome. These specific pathological features withstood rigorous statistical analysis even after taking into account all clinical indicators available at the time of biopsy as well as during follow-up. The features have prognostic significance and we recommended they be taken into account for predicting outcome independent of the clinical features both at the time of presentation and during follow-up. The value of crescents was not addressed due to their low prevalence in the enrolled cohort.

Pathological classifications in current use for the assessment of glomerular disease have been typically opinion-based and built on the expert assumptions of renal pathologists about lesions historically thought to be relevant to prognosis. Here we develop a unique approach for the pathological classification of a glomerular disease, IgA nephropathy, in which renal pathologists first undertook extensive iterative work to define pathologic variables with acceptable inter-observer reproducibility. Where groups of such features closely correlated, variables were further selected on the basis of least susceptibility to sampling error and ease of scoring in routine practice. This process identified six pathologic variables that could then be used to interrogate prognostic significance independent of the clinical data in IgA nephropathy (described in the accompanying article). These variables were (1) mesangial cellularity score; percentage of glomeruli showing (2) segmental sclerosis, (3) endocapillary hypercellularity, or (4) cellular/fibrocellular crescents; (5) percentage of interstitial fibrosis/tubular atrophy; and finally (6) arteriosclerosis score. Results for interobserver reproducibility of individual pathological features are likely applicable to other glomerulonephritides, but it is not known if the correlations between variables depend on the specific type of glomerular pathobiology. Variables identified in this study withstood rigorous pathology review and statistical testing and we recommend that they become a necessary part of pathology reports for IgA nephropathy. Our methodology, translating a strong evidence-based dataset into a working format, is a model for developing classifications of other types of renal disease.

Treatment of patients with IgA nephropathy (IgAN) depends on a reliable assessment of disease progression based on measurements of glomerular filtration rate (GFR), proteinuria, hypertension, and tubulointerstitial changes. We sought to determine whether progression could be predicted from analysis of glomerular and tubulointerstitial inflammation in biopsies taken at an early stage of IgAN. We retrospectively analyzed biopsies from 50 patients, relating the subsequent clinical course to infiltration with B- and T-lymphocytes, granule membrane protein of 17 kDa (GMP-17) positive cytotoxic T cells, macrophages, fibroblasts, and tubulointerstitial expression of human leukocyte antigen-D related (HLA-DR). At biopsy, 19 patients had decreased GFR while 13 of 31 patients with normal GFR and progressive IgAN differed significantly from 18 non-progressors in the level of proteinuria and in the severity of scores for mesangial proliferation, tubular atrophy, interstitial fibrosis, and interstitial infiltrates. On multivariate regression analysis these differences disappeared; however, associations with GMP-17-positive cytotoxic T-lymphocytes in intact renal tubules and of B-lymphocytes in the interstitium remained significant. Our study may have identified a marker of disease progression in early stages of IgAN.

Micro-vascular and renal complications in diabetic patients are a considerable clinical challenge. In a previous study, we found a significant decrease in vascular endothelial growth factor A (VEGF-A) mRNA levels in glomeruli from patients with diabetic nephropathy (DN). We now set out to investigate the relationship between reduced VEGF-A and connective tissue growth factor (CTGF) expression levels, the number of podocytes, and the extent of interstitial fibrosis. Laser capture microdissection was applied to obtain glomerular RNA from 28 patients with DN and 22 controls. mRNA levels of VEGF-A, CTGF, nephrin, podocin, and Wilms tumor1 (WT1) were measured using real-time polymerase chain reaction. Protein expression was evaluated using immuno-stainings for VEGF-A and CTGF, as well as markers for podocytes (WT1) and endothelial cells (CD31). We found a significant decrease in glomerular mRNA levels for VEGF-A (2.5 times), CTGF (1.6), nephrin (2.8), podocin (3.3), and WT1 (1.7) in patients with DN. There was a significant correlation between expression of podocyte markers and VEGF-A mRNA levels, and an inverse correlation between podocin message and the extent of interstitial fibrosis. CD31-positive area was significantly decreased (3.2 times) in patients with DN. Reduction of angiogenic factors correlated with the extent of interstitial fibrosis. This downregulation was related to a reduction of podocytes in DN. The results may suggest that downregulation of VEGF-A and CTGF in DN is a result of podocyte loss.

Although it is clear that genetic predispositions play a role in progressive glomerulosclerosis, identification of specific genes is difficult because of natural genetic heterogeneity among individuals. We have reported a differential susceptibility to progressive glomerulosclerosis after induction of experimental glomerulonephritis anti-Thy-1 nephritis in Lewis rat substrains. Glomerular lesions in Lewis/Møllegard rats resolve spontaneously, whereas Lewis/Maastricht (Lew/Maa) rats develop progressive glomerulosclerosis. This predisposition for progressive glomerulosclerosis is governed by unknown genes that are expressed by renal cells. Here, differential gene expression analysis using a rat complementary DNA micro array revealed neuronal activity-regulated pentraxin (Narp) as a candidate gene involved in the remodeling or progression of damaged glomeruli. Glomerular Narp mRNA expression was monitored during disease in both Lewis sub strains. Immunohistochemistry revealed that Narp protein is exclusively expressed in Lew/Maa glomeruli 7 and 14 days after induction of anti-Thy-1 nephritis. Double-immunofluorescent staining showed that proliferating mesangial cells and parietal epithelial cells (PECs) at sites of adhesion to podocytes are partially Narp-positive, whereas podocytes fail to express Narp. Immunohistochemistry in nephritic Wistar, unilaterally nephrectomized Wistar and Sprague-Dawley rats showed that Narp protein is present only in strains that develop progressive glomerulosclerosis but never in strains that show remodeling. We conclude that Narp is a predictor for anti-Thy-1 nephritis-induced glomerulosclerosis and its expression by PECs may be involved in the progression to glomerulosclerosis.

Until recently, intravenous cyclophosphamide pulses with oral corticosteroids were regarded standard therapy for proliferative lupus nephritis (LN). Azathioprine, a less toxic alternative, was never proven to be inferior. In the first Dutch lupus nephritis study (enrollment between 1995 and 2001), we randomized 87 proliferative LN patients to either cyclophosphamide pulses (750 mg/m(2), 13 pulses in 2 years) combined with oral prednisone (CY) or to azathioprine (2 mg/kg/day in 2 years) combined with intravenous pulses of methylprednisolone (3 x 3 pulses of 1000 mg) and oral prednisone (AZA). After a median follow-up of 5.7 years (interquartile range 4.1-7.2 years), doubling of serum creatinine was more frequent in the AZA group, although not statistically significant (relative risk (RR): 4.1, with 95% confidence interval (95% CI): 0.8-20.4). Relapses occurred more often in the AZA group (RR: 8.8, 95% CI: 1.5-31.8). Creatinine and proteinuria at last visit did not differ between the two treatment arms. Moreover, 88.4% of the patients in the AZA arm were still free of cyclophosphamide treatment. During the first 2 years, the frequency of remission was not different, but infections, especially herpes zoster virus infections (HZV) were more frequent in the AZA group. Parameters for ovarian function did not differ between the two groups. In conclusion, in this open-label randomized controlled trial, cyclophosphamide was superior to azathioprine with regard to renal relapses and HZV. At last follow-up, there were no differences in serum creatinine or proteinuria between the two groups. However, since our study lacked sufficient power, longer follow-up is needed to reveal putative differences.

Background: Endothelial chimerism occurs in renal transplants, but factors involved in its development and its impact on outcome, are unknown. Most studies on chimerism are restricted to gender-mismatched combinations of female donor organs into male recipients. By using blood group antigen mismatches to detect chimeric cells, we circumvented this restriction. We determined which factors predispose for the development of endothelial chimerism, and how it influences graft survival., Methods: We studied 85 renal transplant biopsies of 24 patients with either blood group A or B, who received a blood group O kidney. Biopsies were scored according to BANFF '97. Blood group antigens were stained by immunohistochemistry. Semiquantitative scoring was performed by four independent observers., Results: Endothelial chimerism was found in 27/85 biopsies from 16/24 patients. All female recipients, but only half of the male recipients, had endothelial chimerism in their grafts (P < 0.025). In female recipients, endothelial chimerism occurred significantly earlier than in male recipients (P < 0.02). The presence of endothelial chimerism was not associated with: rejection, outcome, original renal disease, previous transplantations, age, warm/cold ischemia time, pretransplantation blood urea levels, or erythropoietin therapy., Conclusion: We are the first to report that endothelial chimerism occurs significantly more often in female than in male recipients of renal transplants. Endothelial chimerism had no influence on graft outcome. We hypothesize that hormonal factors may influence the development of endothelial chimerism, in parallel with differences in endothelial function between males and females in cardiovascular disease.

Background: In kidney transplantation, chronic allograft nephropathy (CAN) is the major cause of graft loss. Causes of CAN include chronic rejection and chronic cyclosporine A (CsA) nephrotoxicity. It is necessary to differentiate between these two entities in order to apply the appropriate therapeutic regimen for the individual patient, but this is hampered by the lack of discriminating functional and morphologic parameters. We investigated whether renal cortical mRNA levels for several matrix proteins can serve as discriminating parameters., Methods: Patients with chronic rejection (N= 19) and chronic CsA toxicity (N= 17) were selected by clinical and histologic criteria. Protocol biopsies without histologic abnormalities, taken at 6 months after transplantation from patients receiving CsA, were used as controls (N= 6). Total RNA was extracted from the renal biopsy tissue, and mRNA levels of transforming growth factor-beta (TGF-beta) and the extracellular matrix (ECM) molecules collagen Ialpha1, IIIalpha1, IValpha3, decorin, fibronectin, and laminin beta2 were measured by real-time polymerase chain reaction (PCR)., Results: In both patient groups, the mean collagen IValpha3 and fibronectin mRNA levels were significantly elevated compared to those in controls, whereas only in CsA toxicity were the laminin beta2 and TGF-beta mRNA levels significantly increased. The increase of laminin beta2 and TGF-beta mRNA levels was significantly higher in the CsA toxicity group than in the chronic rejection group (P < 0.001 and P= 0.004, respectively). Receiver-operating characteristic (ROC) curve analysis showed that with a 15.6-fold increase in laminin beta2 mRNA expression as cut-off point, the presence of CsA toxicity could be predicted with an 87% sensitivity and an 88% specificity., Conclusion: Renal laminin beta2 and TGF-beta mRNA levels can be used to differentiate between chronic rejection and chronic CsA toxicity in renal transplants. The method of mRNA quantification might be applicable as an additional diagnostic tool in clinical practice.

Background: Chronic transplant glomerulopathy is an uncommon cause of chronic transplant dysfunction of unknown pathogenesis. We evaluated the epidemiologic, clinical, and histologic features of chronic transplant glomerulopathy. To determine the possible contribution of humoral immune responses, we assessed glomerular deposition of C4d., Methods: From a cohort of 1111 kidney transplants (1983 to 2001) with at least 6 months of graft function, we identified 18 cases with chronic transplant glomerulopathy (1.6%) showing double contours of the glomerular basement membrane (GBM) on light microscopy. To assess the risk factors, this group was compared with 739 patients with stable function using multivariate Cox regression analysis. Paraffin sections of 11/18 biopsies were stained with polyclonal C4d antibodies. Sera of 13/18 patients could be tested for antidonor human leukocyte antigen (HLA) antibodies by enzyme-linked immunosorbent assay (ELISA). Patients with chronic allograft nephropathy without chronic transplant glomerulopathy or predominant cyclosporine nephrotoxicity were used as controls., Results: Chronic transplant glomerulopathy was diagnosed at a median of 8.3 (range 2.6-12.5) years posttransplantation. Panel reactive antibodies at time of transplantation, RR 1.23 (1.05-1.45) per 10% increase, and late acute rejection episodes, RR 7.6 (1.8-31.7), were independently associated with chronic transplant glomerulopathy. We found glomerular C4d deposits in 10/11 biopsies showing chronic transplant glomerulopathy and in only 2/13 controls. Peritubular capillary C4d deposits and donor-specific anti-HLA antibodies were demonstrated in 4 and 3 of the 10 patients with glomerular C4d deposits, respectively., Conclusion: Presensitization and late acute rejection episodes were the risk factors identified. Glomerular C4d deposits suggest that chronic transplant glomerulopathy emerges from in situ humoral rejection. Chronic transplant glomerulopathy should be considered as a manifestation of immune-mediated injury.

The currently used classification reflects our understanding of the pathogenesis of the various forms of lupus nephritis, but clinicopathologic studies have revealed the need for improved categorization and terminology. Based on the 1982 classification published under the auspices of the World Health Organization (WHO) and subsequent clinicopathologic data, we propose that class I and II be used for purely mesangial involvement (I, mesangial immune deposits without mesangial hypercellularity; II, mesangial immune deposits with mesangial hypercellularity); class III for focal glomerulonephritis (involving <50% of total number of glomeruli) with subdivisions for active and sclerotic lesions; class IV for diffuse glomerulonephritis (involving > or =50% of total number of glomeruli) either with segmental (class IV-S) or global (class IV-G) involvement, and also with subdivisions for active and sclerotic lesions; class V for membranous lupus nephritis; and class VI for advanced sclerosing lesions. Combinations of membranous and proliferative glomerulonephritis (i.e., class III and V or class IV and V) should be reported individually in the diagnostic line. The diagnosis should also include entries for any concomitant vascular or tubulointerstitial lesions. One of the main advantages of the current revised classification is that it provides a clear and unequivocal description of the various lesions and classes of lupus nephritis, allowing a better standardization and lending a basis for further clinicopathologic studies. We hope that this revision, which evolved under the auspices of the International Society of Nephrology and the Renal Pathology Society, will contribute to further advancement of the WHO classification.

Background: Assessment of mRNA levels has the potential to predict renal outcome. The objective of this study was to optimize several steps in the protocol for obtaining cDNA from routine clinical kidney biopsy material., Methods: RNA integrity was compared between different methods for extraction of RNA, synthesis of cDNA, and storage of renal tissue. Hereby, RNAlater, an RNA-preserving compound, was tested for implementation in a protocol for renal biopsies that combines routine histology and RNA expression studies. Gel electrophoresis and real-time polymerase chain reactions (PCR) were outcome parameters for assessment of RNA integrity., Results: The Trizol method rendered higher RNA yields from fresh renal tissue than the NP40 method and RNeasy spin columns. RNA yields were not affected when renal tissue was stored at -70 degrees C for up to 2 months in phosphate-buffered saline (PBS). cDNA levels obtained using avian myeloblastosis virus (AMV) reverse transcriptase (RT) were at least twice as high as those obtained with Sensiscript and Superscript RT. RNAlater maintained RNA integrity in whole renal cortex stored at 4 degrees C for up to 3 months. Dissection of small biopsies in RNAlater rendered similar RNA yields in comparison with dissection in PBS, but the yield of glomeruli from the cortices was 50% lower (P < 0.005). Integrity of RNAlater-treated tissue, evaluated by light microscopy and immunofluorescence, was diminished., Conclusion: This study shows optimization of several steps in the protocol for extraction and handling of RNA in renal cortical tissue. RNA extraction and cDNA synthesis can be optimized by the use of the Trizol method and AMV RT, respectively. RNAlater is beneficial for preserving RNA integrity in whole renal cortex during storage and processing, but is not suitable for implementation in routine diagnostic histologic stainings combined with RNA expression studies in dissected biopsy material.

Background: Conversion from cyclosporine to azathioprine after renal transplantation has been shown to be beneficial in terms of allograft function, cardiovascular risk factor profile, and the incidence of gout. A higher incidence of acute rejection, however, has also been reported and uncertainty still exists about the long-term outcome after conversion. We report on the extended follow-up of an open-label, randomized trial that examined conversion to azathioprine as early as three months after transplantation., Methods: One hundred twenty-eight patients were enrolled in this single-center study. Three months after transplantation they were randomly assigned to continue cyclosporine treatment (N = 68), or they were converted to azathioprine (N = 60). The steroid dose was temporarily increased in the patients who were converted., Results: Patient survival was not different in the two groups. Graft survival tended to be lower (64.7% vs. 76.5% at 15 years) in the cyclosporine continuation group (P = 0.14) when data were analyzed on an intention to treat basis. The graft survival of the patients that stayed on their assigned treatment was significantly higher in the azathioprine arm, starting at two years' post-transplantation. The glomerular filtration rate was significantly higher in the patients who were converted to azathioprine. More allograft biopsies were taken from patients remaining on cyclosporine for suspicion of cyclosporine-related nephrotoxicity and prompted a high rate of late conversions (19%). The relative risk of chronic allograft nephropathy was significantly higher in the group that continued cyclosporine [relative risk, 4.3 (95% CI, 1.4 to 12.9); P = 0.009]. Conversion to azathioprine reduced the need of blood pressure and lipid-lowering drugs., Conclusion: Conversion to a calcineurin inhibitor-free immunosuppressive regiment three months after renal transplantation improved allograft function, reduced the need of cardiovascular risk factor-controlling medication, and reduced the incidence of chronic allograft nephropathy.

Background: The predictive value of clinical and renal histological features for renal outcome in patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis was investigated in a prospective analysis of 96 patients with ANCA-associated vasculitis, and moderate renal involvement (creatinine <500 micromol/L)., Methods: The extent of 39 histological features in 96 biopsies (performed at entry in a clinical trial) was scored by two independent observers, according to a standardized protocol. Age, gender, diagnosis, glomerular filtration rate at entry (GFR0), ANCA-specificity, proteinuria, and treatment of these 96 patients were also taken into account. Treatment was standardized and started after the biopsy was performed. End-points included renal function at 18 months (GFR18), GFR18 corrected for GFR0 (CORGFR18), and the occurrence of relapse or death., Results: Parameters that most strongly correlated with GFR18 were GFR0 (r = 0.67), interstitial fibrosis (r = -0.45), glomerulosclerosis (r = -0.37), and tubular atrophy (r = -0.36). Parameters that most strongly correlated with CORGFR18 were segmental (r = 0.45) and cellular (r = 0.30) crescents, and fibrinoid necrosis (r = 0.46). None of the clinical and histological features predicted the occurrence of relapse or death. By applying a stepwise linear multiple regression analysis, we designed a formula for the estimation of renal function at 18 months: GFR18 (mL/min) = 17 + 0.71 x GFR0 (mL/min) + 0.34 x fibrinoid necrosis (%) + 0.33 x segmental crescents (%), (r2 = 0.60; standard deviation = 19 mL/min). Our results were independent of diagnosis, ANCA-specificity, and treatment limb., Conclusions: These data suggest that in ANCA-associated glomerulonephritis, GFR0 and predominantly chronic renal lesions are potent predictors of GFR18. Active lesions are associated with renal function recovery and may be reversible. The formula for the estimation of GFR18 shows that a combination of GFR0 and renal histology is a better predictor for GFR18 than GFR0 only.

Damage to the kidney generally elicits tissue repair mechanisms, but these processes themselves conversely may result in the progression of chronic renal disease. In a majority of patients chronic renal insufficiency progresses to a common histological end point, marked by the presence of a vast amount of scar tissue, that is, glomerulosclerosis and interstitial fibrosis. These lesions are the result of an excessive production of extracellular matrix (ECM) components. Studies on RNA expression in experimental kidney disease have shown that renal mRNA levels for ECM components and cytokines can function as prognostic tools. This suggests that mRNA levels potentially predict outcome and reaction to therapy in patients with renal diseases. Timely detection of molecular alterations could allow early therapeutic intervention that slows down or even prevents the development of sclerotic and fibrotic lesions. This review first provides a short introduction on mechanisms of initiation and progression of renal disease. Molecular techniques are available to identify renal RNA sequences potentially involved in disease progression. We discuss several molecular techniques that are being used in kidney research for quantitation and detection of mRNA. This is followed by a brief overview of investigation in experimental renal diseases, which reveal that alterations in tissue ECM mRNA levels precede histological damage and can function as predictors of clinical outcome. In particular, studies in human kidney biopsies that evaluate the prognostic value of mRNA levels with respect to renal function are examined, paying special attention to the pitfalls that potentially are encountered when interpreting the results of such studies. Then, we elaborate on ways of optimal exploitation of mRNA quantification as a prognostic tool. The potential and limitations of microarray technology in the search for genes specifically involved in progression of renal disease are reviewed, including RNA expression profiling and large-scale DNA mutation screening. Finally, the future utilities of microarray in nephrology and renal pathology are discussed.

Background: Glomerulosclerosis is a severe complication of many immunologically-mediated kidney diseases, eventually resulting in loss of renal function. In chronic graft-versus-host disease (GvHD) in mice, a model for human lupus nephritis, the end-stage sclerotic lesions were previously shown to contain large amounts of fibronectin (FN). This study investigated a domain-specific accumulation process of circulating plasma FN (pFN) in sclerotic lesions., Methods: GvHD mice were injected with FITC-conjugated pFN or pFN-fragments, with or without heparin pre-incubation. pFN fragments were generated by digestion of pFN by cathepsin D, after which the fragments were separated on a heparin affinity column. Thus, two batches of fragments were obtained with either low or high affinity for heparin., Results: FN accumulation was accompanied by an up-regulated expression of integrin alpha5beta1, the FN receptor, in the periphery of sclerotic lesions. pFN-FITC injected into GvHD mice was trapped in sclerotic glomeruli within 24 hours. Both heparin and non-anti-coagulant heparin blocked the accumulation of pFN-FITC, indicating that the protective effect of heparin in the trapping of FN is independent of its anticoagulant properties, and probably results from preventing direct binding of FN in the sclerotic lesions. To investigate whether FN binds in the glomerulus via the heparin-binding regions, pFN fragments were generated and injected into GvHD mice. Whereas the fraction with high affinity for heparin did not accumulate in the sclerotic glomeruli, the fraction with low affinity for heparin did. Partial sequencing of the isolated peptides showed that in the glomerulus fibronectin does not bind via the heparin II binding region., Conclusions: We hypothesize that the protective effect of heparin treatment may be the result of steric hindrance of the specific binding sites, that is, the I1-5 and/or III1 self-assembly sites of FN.

Background: Differences in renal histopathology between microscopic polyangiitis (MPA) and Wegener's granulomatosis (WG), and between anti-neutrophil cytoplasm autoantibody (ANCA) test results in patients with ANCA-associated vasculitis may provide insight into the differences in pathogenesis and raise the opportunity of classifying the vasculitides more accurately. The possible differences in histopathology are investigated in this study., Methods: We report an analysis of 173 patients with renal disease in microscopic polyangiitis or Wegener's granulomatosis. A total of 173 renal biopsies, performed at diagnosis, were scored by two observers separately, using a previously standardized protocol. Consensus on each biopsy was achieved during a central review., Results: Normal glomeruli were more common in WG than in MPA (P < 0.001). Glomerulosclerosis was more prominent in MPA than in WG (P=0.003). Interstitial fibrosis (P < 0.001), tubular atrophy (P < 0.001), and tubular casts (P=0.005) were more frequently present and more severe in MPA than in WG. Presence of glomerulosclerosis was more extensive in patients with myeloperoxidase (MPO)-ANCA than with proteinase 3 (PR3)-ANCA (P=0.022). Interstitial fibrosis (P=0.008), tubular necrosis (P=0.030), tubular atrophy (P=0.013), and intra-epithelial infiltrates (P=0.006) were more frequently present and more severe in MPO-ANCA than in PR3-ANCA., Conclusions: Glomerulonephritis in relation to MPA has more characteristics of chronic injury at the time of presentation than glomerulonephritis in relation to WG. This difference may be due to a delayed establishment of diagnosis in patients with MPA compared to patients with WG. Both active and chronic lesions are more abundantly present in MPO-ANCA-positive patients than in patients with PR3-ANCA-positivity, which suggests that the pathogenesis of renal disease in these ANCA subsets could be different. Our results also suggest that ANCA test results may be useful in classifying ANCA-associated vasculitides.

A variety of antineutrophil cytoplasmic auto-antibodies (ANCAs) are known to be associated with small vessel vasculitides such as Wegener's granulomatosis and microscopic polyangiitis. To visualize colocalization patterns of the fibrinoid necrotic lesions and ANCA-antigens more accurately, we have developed a double staining technique in which an immunohistochemical staining is followed by a histological staining. Instead of using sequential biopsy slides of histologically and immunohistochemically stained sections, which may lead to an underestimation of the number and size of the lesions, our technique permits the visualization of the colocalized patterns of fibrinoid necrosis with an ANCA-antigen in a single slide. The double staining procedure is presented in this Technical Note.

Background: Investigation of the prognostic value of the expression of mRNA for extracellular matrix (ECM) components with respect to deterioration of kidney function in patients with renal disease requires an evaluation of the basal expression of ECM mRNA in healthy individuals and of the reliability of ECM mRNA measurements. In the current study, the collagen alpha 1(IV)/GAPDH (C4:G) and collagen alpha 1(I)/GAPDH (C1:G) mRNA ratios and the accumulation of collagen IV and collagen I protein were investigated in renal cortices of individuals of various age. Furthermore, we examined whether the C4:G mRNA ratio measured in a renal biopsy is representative of that in the rest of the kidney., Methods: To investigate the effect of age on collagen expression, kidneys obtained at autopsy from patients with a normal renal function (N = 18; age 19 to 92) were used. C4:G and C1:G mRNA ratios were measured by real-time polymerase chain reaction (PCR) analysis. Accumulation of collagen IV and collagen I protein was measured by quantitative image analysis on immunohistochemically stained sections. To determine whether the site at which a biopsy is taken affects the C4:G mRNA ratio, this ratio was measured in cortical biopsies taken from different locations from each of four kidneys: one without renal disease, one with diabetes mellitus type I, and two with diabetes mellitus type II. C4:G mRNA ratios were measured by using real-time PCR., Results: The C4:G mRNA ratio, but not the C1:G mRNA ratio or collagen IV protein accumulation, increased significantly with age (r = 0.55, P < 0.03). Collagen I protein accumulation increased with age (r = 0.85, P < 0.001) and correlated with the extent of interstitial fibrosis (r = 0.50, P < 0.05). The C4:G mRNA ratio did not differ significantly within a kidney., Conclusions: This report shows, to our knowledge for the first time, that in the aging, normally functioning human kidney, there is a dissociation between the levels of mRNA for collagen IV and collagen I and the accumulation of these proteins. The levels of mRNA for collagen IV in a single renal biopsy can be regarded as representative of those in the rest of the kidney. These observations should be taken into account when ECM mRNA levels are used for diagnostic purposes.

Background: Renal transplant loss from chronic rejection remains substantial. To increase our understanding of this syndrome, we identified risk factors predicting late graft loss, with a special emphasis on the impact of human lymphocyte antigen (HLA) matching., Methods: We studied all 654 cadaveric kidney transplants performed in our center between 1983 and 1996 that had survived for more than six months. Eighty-two transplants, lost because of chronic rejection, were used as the outcome variable. The influence of HLA mismatches and shares on long-term graft survival was evaluated at the level of private antigens and cross-reactive groups (CREG) of multiple histocompatibility complex (MHC) class I. HLA and other recipient, donors and transplant parameters were studied using univariate and multivariate Cox regression analysis., Results: The cohort had a mean number of 1.9 HLA mismatches. Because of the homozygosity of HLA antigens, HLA mismatches were not reciprocal to shares. CREG and HLA-A-B mismatches had a relative risk for graft loss of 1.19 (95% CI, 0.97 to 1.45) and 1.05 (0.84 to 1.32) per mismatch. In contrast, the relative risk per shared CREG and broad HLA-A-B antigen was 0.76 (0.63 to 0.92) and 0.79 (0.61 to 1.03). Multivariate analysis revealed that individuals sharing less than four CREGs had a relative risk of 2.13 (1.29 to 3.75) for late graft loss. Other independent predictors were a recipient age of less than 50 years, relative risk 1.95 (1.02 to 3.71); a donor age of more than 50 years, relative risk 1.68 (1.01 to 2.80); acute rejection (vascular vs. no rejection), relative risk 3.52 (1.72 to 7.18); proteinuria (dipstick > 1+ vs. negative), relative risk 2.86 (1.29 to 6.35); and a serum creatinine concentration of more than 150 micromol/liter at six months, relative risk 3.41 (1.96 to 5.94)., Conclusion: We identified several coexisting recipient-, donor-, and transplant-related risk factors for graft loss from chronic rejection. In this well-matched group of renal transplants, HLA mismatches and shares had a nonreciprocal relationship. Sharing of HLA antigens, especially CREG of MHC class I, was associated with improved long-term survival.

Background: In kidney biopsies of patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, a variety of histopathological lesions occur, and their relationship to renal outcome is virtually unknown. This multicenter European study reports a clinicopathological analysis of biopsies from 157 patients with systemic vasculitis., Methods: The biopsies were evaluated according to a previously standardized scoring protocol. Serum creatinine values were measured at the time of biopsy and one year later. In addition, the lowest creatinine level during follow-up was taken into account as the optimum level of renal function recovery. The clinical prognostic value of the histopathological parameters was analyzed with the Kruskal-Wallis one-way analysis of variance and the Mann-Whitney U-test., Results: The percentage of normal glomeruli correlated most significantly with renal outcome at all points of measurement (all P < 0.001). Other lesions predicting for renal function were glomerular sclerosis (P < 0.0005 at one year after the biopsy), diffuse interstitial infiltrates (P < 0.0001 at entry, P < 0.0003 at one year), tubular necrosis (P < 0.0025 at entry), and tubular atrophy (P < 0.002 at entry, P < 0.0002 at one year)., Conclusion: Traditionally, attention is focused on the extent of active lesions in the renal biopsy in order to determine the severity of renal disease and its implication for renal outcome. Because of their significant impact on renal function, combined with their easy recognition, we recommend the use of the percentage of normal glomeruli in an adequate biopsy in predicting renal function of patients with systemic vasculitis.

Changes in heparan sulfate metabolism may be important in the pathogenesis of diabetic nephropathy. Recent studies performed on renal biopsies from patients with diabetic nephropathy revealed a decrease in heparan sulfate glycosaminoglycan staining in the glomerular basement membrane without changes in staining for heparan sulfate proteoglycan-core protein. To understand this phenomenon at the cellular level, we investigated the effect of high glucose conditions on the synthesis of heparan sulfate proteoglycan by glomerular cells in vitro. Human adult mesangial and glomerular visceral epithelial cells were cultured under normal (5 mM) and high glucose (25 mM) conditions. Immunofluorescence performed on cells cultured in 25 mM glucose confirmed and extended the in vivo histological observations. Using metabolic labeling we observed an altered proteoglycan production under high glucose conditions, with predominantly a decrease in heparan sulfate compared to dermatan sulfate or chondroitin sulfate proteoglycan. N-sulfation analysis of heparan sulfate proteoglycan produced under high glucose conditions revealed less di- and tetrasaccharides compared to larger oligosaccharides, indicating an altered sulfation pattern. Furthermore, with quantification of glomerular basement membrane heparan sulfate by ELISA, a significant decrease was observed when mesangial and visceral epithelial cells were cultured in high glucose conditions. We conclude that high glucose concentration induces a significant alteration of heparan sulfate production by mesangial cells and visceral epithelial cells. Changes in sulfation and changes in absolute quantities are both observed and may explain the earlier in vivo observations. These changes may be of importance for the altered integrity of the glomerular charge-dependent filtration barrier and growth-factor matrix interactions in diabetic nephropathy.

Impairment of renal function in various types of glomerular disease is associated with tubulointerstitial changes. The mechanism of mononuclear cell infiltration in the interstitium is not fully understood. Recently, monocyte chemoattractant protein-1 (MCP-1) has been identified as a monocyte-specific chemotactic factor. We analyzed the presence of MCP-1 in renal biopsies from patients with various forms of glomerular disease and demonstrated that MCP-1 expression is increased in renal tubular epithelial cells during disease. Further analysis showed that various cell lines of human proximal tubular epithelial cells (PTEC) produce MCP-1 in culture under serum-free conditions and that the production is inhibited by cycloheximide. IL-1 alpha and TNF-alpha enhanced the production by each cell line in a dose- and time-dependent manner as measured by radioimmunoassay. Northern blot analysis demonstrated that IL-1 alpha and TNF-alpha markedly enhanced the expression of MCP-1 mRNA. Taken together these observations support the notion that MCP-1 is synthesized de novo by PTEC. MCP-1 produced by PTEC is found to be 13 kD by gel filtration chromatography. It is chemotactically active for monocytes. We conclude that in various types of glomerular disease, MCP-1 expression in tubular epithelial cells is associated with up-regulation of MCP-1 production by PTEC. These findings raise the possibility that macrophages may accumulate in renal interstitium as a consequence of MCP-1 production by PTEC.

The influence of cyclosporine A (CsA) treatment on the development of glomerulonephritis and glomerulosclerosis was investigated in chronic graft-versus-host disease (GvHD), a murine model for lupus nephritis. The renal disease is characterized by the formation of IgG-containing electron-dense deposits along the glomerular basement membrane (GBM) and in the mesangium, followed by the onset of proteinuria which starts, varying per individual mouse, about six weeks after the induction of the disease. Glomerular mRNA levels for matrix molecules were increased from week 4, preceding mesangial matrix expansion and GBM thickening which occurred from week 6. These initial events finally led to development of glomerulosclerosis, and end-stage renal failure. Groups of mice received three intraperitoneal (i.p.) injections per week with different doses of CsA, and treatment was started 2, 4, or 6 weeks after induction of the disease. Treatment with 10 or 50 mg CsA/kg/week did not influence the development of glomerulonephritis or glomerulosclerosis. Injection of 100 mg CsA/kg/week delayed the onset of proteinuria only when treatment was started in week 2. In week 6 some mice had already developed proteinuria whereas others had not. Treatment with 250 mg CsA/kg/week starting in week 6 abrogated glomerulonephritis and glomerulosclerosis only in those animals which were not yet proteinuric at that time. This, despite comparable increased autoantibody levels against DNA, GBM, and renal tubular epithelium (RTE) in both treated and untreated GvHD mice. Further increase in proteinuria and development of glomerulosclerosis could not be prevented if the mice already had developed proteinuria when CsA treatment was started. Dot blot analysis and in situ hybridization showed significantly decreased mRNA levels for alpha 1(I) and alpha 1(IV) collagen in kidneys of CsA-treated mice as compared to those of untreated mice 12 weeks after induction of the disease, if the highest dose of CsA was administered before the onset of proteinuria. No effect on these whole-kidney mRNA levels was observed in mice which had already developed proteinuria before CsA injections were started. Increased mRNA expression for matrix molecules in this group and in untreated GvHD mice was observed mainly in the interstitium. The kidneys of the treated GvHD mice and those of mice injected with 250 mg CsA/kg/week without induction of GvHD showed no morphological signs of CsA nephrotoxicity. We conclude that treatment with 250 mg CsA/kg/week prevents the development of glomerulonephritis and glomerulosclerosis in this model of lupus nephritis, if started before the onset of proteinuria.(ABSTRACT TRUNCATED AT 400 WORDS)

The antigenic specificity of the mesangial IgA in IgA nephropathy (IgAN) remains unknown. Because shared antigenic specificities may be reflected in the usage of shared idiotypes, we prepared five monoclonal anti-idiotypic antibodies (MoAbs) specific for the mesangial IgA eluted from the kidney of an IgAN patient. All five MoAbs reacted with the same idiotype, which proved to be of a public nature. Although the idiotype could be identified in the mesangial deposits of the majority of IgAN patients studied, it was not specific for the disease because it was also found in the glomerular deposits of other types of glomerulonephritis. The idiotype was also expressed in polyethylene glycol precipitates of sera and in pokeweed mitogen-induced plasma cells from both IgAN patients and healthy controls. The conclusion that no disease-specific idiotypes are present in the renal eluate was further supported by the failure to produce polyclonal anti-idiotypic antibodies by immunizing a rabbit with the eluted mesangial IgA. Our results support the concept that mesangial IgA deposits in IgAN are of a polyclonal nature.

BALB/c mice infected with Trypanosoma brucei and treated seven days after inoculation with Diminazene aceturate develop polyclonal B-cell stimulation, including production of antibodies to known nephritogenic autoantigens and glomerular disease associated with severe albuminuria. To investigate if the susceptibility for glomerular disease in this model is linked to MHC or non-MHC genes, we studied this disease in six mouse strains that were partly congenic for their MHC and partly congenic for their non-MHC genes. The course of the infection was measured by parasitemia and related to (auto)antibody production, proteinuria and glomerular deposition of immunoglobulins. The mouse strains could be divided into two groups. The first group consisted of the C57BL/6 (H-2b), C57BL/10 (H-2b) and B10.D2 (H-2d) strains, which proved to be relatively resistant to infection with Trypanosoma brucei (that is, spontaneous survival > 25 days). In sera of these mice antibodies to a broad range of antigens could be found 14 days after inoculation; no proteinuria was observed. The second group consisted of the BALB/c (H-2d), BALB.B (H-2b) and DBA/2 (H-2d) strains, which were relatively susceptible to the infection. In these animals proteinuria occurred and a broad polyclonal B-cell stimulation was seen 42 days after inoculation. No correlation was found between the specificity of circulating antibodies and the occurrence of proteinuria or a glomerular fluorescence pattern. These results indicate that in this model non-MHC genes govern the outcome of the infection as well as the development of polyclonal B-cell stimulation and proteinuria.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor necrosis factor alpha (TNF alpha) production by proximal tubuli was studied by immunoperoxidase staining of 20 renal biopsies from transplant patients. A positive staining for TNF alpha on proximal tubuli was seen in nine out of 15 patients with interstitial infiltrate, five without clinical significant rejection and four with moderate to severe interstitial rejection. TNF alpha was only expressed on tubuli within areas of interstitial infiltrate. Expression of TNF alpha in the mononuclear cell infiltrate was seen only in three patients with interstitial rejection. Absence of TNF alpha could be seen in biopsies with no renal abnormalities. To obtain more information on the regulation of TNF alpha production, proximal tubular epithelial cells (PTEC) cultures were established and assessed for production of TNF alpha. Heterogenicity in production of TNF alpha was found in 14 tested PTEC lines cultured under serum free conditions. The presence of IL-1 alpha in the cultures induced a time- and dose-dependent enhancement of TNF alpha production by PTEC. Enhanced production of TNF alpha was not seen after stimulation with other cytokines such as IL-2 or IFN gamma. Inhibition studies with cycloheximide indicated de novo synthesis of TNF alpha. Western blot analysis of supernatants of unstimulated and IL-1 alpha stimulated PTEC indicated a 17 kd product, a size similar to that of recombinant TNF alpha. Northern blot analysis revealed the presence of a 2.0 kb hybridization signal in total RNA of PTEC cultures and up regulation upon treatment of PTEC with 1 ng/ml of IL-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

BALB/c mice neonatally injected with 1 x 10(8) (A/J x BALB/c)F1 hybrid spleen cells develop polyclonal B cell activation and autoimmune features as a consequence of a host-versus-graft (HVG) reaction. In this study, we first analyzed the time-course development of the renal lesions in HVG mice. From week 2 to week 6, linear deposits of IgG were observed by immunofluorescence along the glomerular capillary walls. From week 8 to week 12, the immunofluorescence pattern of IgG changed from linear to granular, and by immunoelectron microscopy, the IgG deposits were located on the epithelial side of the glomerular basement membrane (GBM). In addition, focal glomerulosclerosis complicated this membranous glomerulopathy in about 50% of the 12-week-old HVG mice and albuminuria was increased in most of them. Circulating antibodies to antigens of the GBM (laminin, type IV collagen) and of the renal tubular epithelial (RTE) cells (dipeptidyl peptidase IV, gp330) were already detected at week 2 and were still present at week 12. Immunoglobulins eluted from isolated glomeruli contained antibodies directed against type IV collagen, laminin, and to a lesser degree against gp330. F1 donor B cells were involved in the production of nephritogenic antibodies as indicated by (a) the presence of A/J allotypic determinants on serum anti-laminin antibodies and (b) the abrogation of the in vitro production of anti-GBM, anti-laminin and anti-RTE antibodies when spleen cells from HVG mice were depleted of F1 donor B cells. Finally, mixed lymphocyte culture experiments established that T cells from HVG mice stimulate normal B cells from F1 donor hybrids to produce anti-GBM, anti-laminin, anti-type IV collagen, anti-RTE, anti-gp330 and anti-dipeptidyl peptidase IV antibodies. We conclude that mice neonatally injected with semi-allogeneic spleen cells develop a glomerulonephritis characterized by the transition from a linear to a granular IF pattern, and that the production of nephritogenic antibodies results from the activation of donor B cells by host helper T cells.