E. de Heer

Genetic associations in diabetic nephropathy: a meta-analysis

Aims/hypothesisThis meta-analysis assessed the pooled effect of each genetic variant reproducibly associated with diabetic nephropathy.MethodsPubMed, EMBASE and Web of Science were searched for articles assessing the association between genes and diabetic nephropathy. All genetic variants statistically associated with diabetic nephropathy in an initial study, then independently reproduced in at least one additional study, were selected. Subsequently, all studies assessing these variants were included. The association between these variants and diabetic nephropathy (defined as macroalbuminuria/proteinuria or end-stage renal disease [ESRD]) was calculated at the allele level and the main measure of effect was a pooled odds ratio. Pre-specified subgroup analyses were performed, stratifying for type 1/type 2 diabetes mellitus, proteinuria/ESRD and ethnic group.ResultsThe literature search yielded 3,455 citations, of which 671 were genetic association studies investigating diabetic nephropathy. We identified 34 replicated genetic variants. Of these, 21 remained significantly associated with diabetic nephropathy in a random-effects meta-analysis. These variants were in or near the following genes: ACE, AKR1B1 (two variants), APOC1, APOE, EPO, NOS3 (two variants), HSPG2, VEGFA, FRMD3 (two variants), CARS (two variants), UNC13B, CPVL and CHN2, and GREM1, plus four variants not near genes. The odds ratios of associated genetic variants ranged from 0.48 to 1.70. Additional variants were detected in subgroup analyses: ELMO1 (Asians), CCR5 (Asians) and CNDP1 (type 2 diabetes).Conclusions/interpretationThis meta-analysis found 24 genetic variants associated with diabetic nephropathy. The relative contribution and relevance of the identified genes in the pathogenesis of diabetic nephropathy should be the focus of future studies.

Therapeutic potential of vasopressin V2 receptor antagonist in a mouse model for autosomal dominant polycystic kidney disease: optimal timing and dosing of the drug

BACKGROUND The renoprotective effect of vasopressin V2 receptor antagonist (V2RA) is currently being tested in a clinical trial in early autosomal dominant polycystic kidney disease (ADPKD). If efficacious, this warrants life-long treatment with V2RA, however, with associated side effects as polydipsia and polyuria. We questioned whether we could reduce the side effects without influencing the renoprotective effect by starting the treatment later in the disease or by lowering drug dosage. METHODS To investigate this, we administered V2RA OPC-31260 at a high (0.1%) and low (0.05%) dose to a tamoxifen-inducible kidney epithelium-specific Pkd1-deletion mouse model starting treatment at Day 21 (early) or 42 (advanced). After 3 and 6 weeks of treatment, we monitored physiologic and potential renoprotective effects. RESULTS Initiation of V2RA treatment at advanced stage of the disease lacked renoprotective effects and had less pronounced physiologic effects than early initiation. After 3 weeks on a high dose, cyst ratio and kidney weight were reduced versus untreated controls (18 versus 25%, P = 0.05, and 0.33 versus 0.45 g, P = 0.03, respectively). After 6 weeks of treatment, however, this did not reach significance anymore, even at a high dose (cyst ratio 24 versus 27%, P = 0.12, and kidney weight 0.55 versus 0.66 g, P = 0.38). CONCLUSIONS Our results suggest that intervention with V2RA should be instituted early in ADPKD and that it might be necessary to further increase the dosage of this drug later in the disease to decrease cyst growth.

Early Renal Ischemia‐Reperfusion Injury in Humans Is Dominated by IL‐6 Release from the Allograft

The pathophysiology of ischemia/reperfusion (I/R) injury is complex, and current knowledge of I/R injury in humans is incomplete. In the present study, human living‐donor kidney transplantation was used as a highly reproducible model to systematically study various processes potentially involved in early I/R injury. Unique, direct measurements of arteriovenous concentration differences over the kidney revealed massive release of interleukin (IL)‐6 in the first 30 minutes of graft reperfusion and a modest release of IL‐8. Among the assessed markers of oxidative and nitrosative stress, only 15(S)‐8‐iso‐PGF2α was released. When assessing cell activation, release of prothrombin factor 1 + 2 indicated thrombocyte activation, whereas there was no release of markers for endothelial activation or neutrophil activation. Common complement activation complex sC5b‐9 was not released into the bloodstream, but was released into urine rapidly after reperfusion. To investigate whether IL‐6 plays a modulating role in I/R injury, a mouse experiment of renal I/R injury was performed. Neutralizing anti‐IL‐6 antibody treatment considerably worsened kidney function. In conclusion, this study shows that renal I/R in humans is dominated by local IL‐6 release. Neutralization of IL‐6 in mice resulted in a significant aggravation of renal I/R injury.

ECM homeostasis in renal diseases: a genomic approach

Chronic renal disease is in general histologically accompanied by a vast amount of scar tissue, ie glomerulosclerosis and interstitial fibrosis. Scarring results from excessive accumulation of extracellular matrix (ECM) components, a process driven by a plethora of cytokines and growth factors. Studies in experimental renal disease which target these regulators using gene therapy limit or prevent the development of scarring. This review focuses specifically on the role of transforming growth factor‐beta, platelet‐derived growth factor, connective tissue growth factor, hepatocyte growth factor, and epidermal growth factor. The results obtained in animal models hold promise for molecular intervention strategies in human renal disease. Microarray technology allows large‐scale gene expression profiling in kidney tissue to identify common molecular pathways in a step towards discovery of new drug targets. Molecular techniques are expected to be used for diagnostic and prognostic purposes in nephrological practice to supplement renal biopsy. Several studies already show that molecular techniques might be of use in routine diagnostic practice. Improvement of diagnosis and prediction of outcome in renal patients might lead to more efficient and earlier therapeutic intervention. Copyright

Anti-C1q autoantibodies in murine lupus nephritis

Autoantibodies against C1q can be found in the circulation of patients with several autoimmune diseases including systemic lupus erythematosus (SLE). In SLE there is an association between the occurrence of these antibodies and renal involvement. How anti‐C1q autoantibodies contribute to renal disease is currently unknown. Cohorts of MRL‐lpr mice, which are known to develop age‐dependent SLE‐like disease, were used to study the relationship between levels of anti‐C1q autoantibodies and renal disease. We collected serum, urine and renal tissue and analysed autoantibodies, complement levels and renal deposition as well as renal function. At 2 months of age all mice already had elevated levels of anti‐C1q autoantibodies, and elution of kidneys revealed the presence of these antibodies in renal immune deposits in MRL‐lpr mice and not in control MRL+/+ mice. In conclusion, anti‐C1q antibodies are already present in serum and immune deposits of the kidney early in life and therefore can play a role in nephritis during experimental SLE‐like disease in mice.

GMP-17-positive T-lymphocytes in renal tubules predict progression in early stages of IgA nephropathy

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.

Lower frequency of the 5/5 homozygous CNDP1 genotype in South Asian Surinamese

We investigated the frequency of the 5/5 homozygous CNDP1 (carnosinase) genotype, which was found to be associated with a reduced risk of developing diabetic nephropathy, in three ethnic groups in The Netherlands. Particularly interesting were the South Asian Surinamese, who have a high prevalence of diabetic nephropathy. Furthermore, we investigated the association between this gene and carnosinase activity in South Asian Surinamese and whether carnosinase was expressed in the kidney. We genotyped 290 South Asian Surinamese, 532 African Surinamese, and 472 White Dutch in a cross-sectional population study. Furthermore, an independent cohort of South Asian Surinamese was genotyped. In this population, carnosinase activity was measured in serum. Immunostaining and in situ hybridization for CNDP1 were performed on kidney tissue. Both South Asian populations had lower frequencies of the 5/5 homozygous genotype than African Surinamese and White Dutch (23.0%, 27.2%, 38.2%, and 41.3%, respectively; chi-square, p<0.001). This genotype showed a lower carnosinase activity in South Asian Surinamese (Wilcoxon rank-sum, p=0.03). CNDP1 was expressed in the kidney. South Asian Surinamese have a lower frequency of the 5/5 homozygous genotype, which was associated with lower carnosinase activity. Our study provides an indication that South Asian Surinamese are genetically at risk for developing diabetic nephropathy.

Image analysis and quantification in lung tissue

On 9–10 September 1999, an international workshop on image analysis and quantification in lung tissue was held at the Leiden University Medical Center, Leiden, The Netherlands. Participants with expertise in pulmonary and/or pathology research discussed the validity and applicability of techniques used for quantitative examination of inflammatory cell patterns and gene expression in bronchial or parenchymal tissue in studies focusing on asthma and chronic obstructive pulmonary disease (COPD). Differences in techniques for tissue sampling and processing, immunohistochemistry, cell counting and densitometry are hampering the comparison of data between various laboratories. The main goals of the workshop were to make an inventory of the techniques that are currently available for each of these aspects, and in particular to address the validity and unresolved problems of using digital image analysis (DIA) as opposed to manual scoring methods for cell counting and assessment of gene and protein expression. Obviously, tissue sampling and handling, fixation and (immunohistochemical) staining, and microscope settings, are having a large impact on any quantitative analysis. In addition, careful choices will have to be made of the commercially available optical and recording systems as well as the application software in order to optimize quantitative DIA. Finally, it appears to be of equal importance to reach consensus on which histological areas are to be analysed. The current proceedings highlight recent advances and state of the art knowledge on digital image analysis for lung tissue, and summarize the established issues and remaining questions raised during the course of the workshop.

Stable transplantation results of magnetically retracted islets: a novel method

Aims/hypothesisLarge quantities of pure viable donor islets are necessary for clinical transplantation. At present, low yields and low viability of pancreatic islets after transplantation necessitate the use of multiple donors for a single recipient. In this study an improved method for obtaining large quantities of pure viable islets of Langerhans for transplantation was developed in the rat.MethodsIslets of Langerhans were isolated from Albino Oxford rats. The donor pancreata were perfused in situ with iron oxide, which resulted in entrapment of iron particles in the capillaries of the islets. Subsequently, the islets were isolated by magnetic retraction. Islets obtained with this method were compared with islets obtained by density gradient-isolated islets with respect to yields, purity, and insulin production capacity. Islets isolated with the magnetic retraction method were transplanted under the renal capsule of streptozotocin-induced diabetic recipients. Blood-glucose levels in the recipients were monitored for 2 months after transplantation.ResultsThis method yielded more pure and viable islets than the conventional protocol. No contamination of exocrine tissue was observed after isolation. Furthermore, the islets isolated by magnetic retraction stained strongly positive for insulin during the entire observation period in vitro, and produced high amounts of insulin upon a challenge with glucose. The islets that were obtained by this new protocol were suitable for safe and effective transplantation.Conclusions/interpretationWe have shown that both the quantity and quality of islets obtained with this method were sufficient to induce insulin independence in a diabetic recipient using islets from only one donor.

Reestablishment of self tolerance by suppressor T-cells after active Heymann's nephritis.

Adoptive transfer experiments were performed to obtain evidence that the down-regulation of the autoimmune response in rats with active Heymanns nephritis (HN) is due to suppressor T cells. Late in the course of HN antigen-specific OX8+ suppressor T cells were found in the spleen, but never in the draining lymph nodes. These cells were shown to suppress the autoimmune response when transferred to naive recipients that were subsequently challenged. By mixing B cells or helper T cells from rats with HN with suppressor T cells from high-dose tolerant rats we showed that OX8+ suppressor T cells appeared to have a direct suppressive effect on autoreactive B cells. A profound suppressive effect on helper T cells appeared after 10 weeks. Possible mechanisms to account for the failure of Lewis rats to maintain self tolerance are discussed.