Hans J. Baelde

Expression of Podocyte-Associated Molecules in Acquired Human Kidney Diseases

Proteinuria is a poorly understood feature of many acquired renal diseases. Recent studies concerning congenital nephrotic syndromes and findings in genetically modified mice have demonstrated that podocyte molecules make a pivotal contribution to the maintenance of the selective filtration barrier of the normal glomerulus. However, it is unclear what role podocyte molecules play in proteinuria of acquired renal diseases. This study investigated the mRNA and protein expression of several podocyte-associated molecules in acquired renal diseases. Forty-eight patients with various renal diseases were studied, including minimal change nephropathy, focal segmental glomerulosclerosis, IgA nephropathy, lupus nephritis, and diabetic nephropathy, together with 13 kidneys with normal glomerular function. Protein levels of nephrin, podocin, CD2-associated protein, and podocalyxin were investigated using quantitative immunohistochemical assays. Real-time PCR was used to determine the mRNA levels of nephrin, podocin, and podoplanin in microdissected glomeruli. The obtained molecular data were related to electron microscopic ultrastructural changes, in particular foot process width, and to clinical parameters. In most acquired renal diseases, except in IgA nephropathy, a marked reduction was observed at the protein levels of nephrin, podocin, and podocalyxin, whereas an increase of the glomerular mRNA levels of nephrin, podocin, and podoplanin was found, compared with controls. The mean width of the podocyte foot processes was inversely correlated with the protein levels of nephrin (r = -0.443, P < 0.05), whereas it was positively correlated with podoplanin mRNA levels (r = 0.468, P < 0.05) and proteinuria (r = 0.585, P = 0.001). In the diseases studied, the decrease of slit diaphragm proteins was related to the effacement of foot processes and coincided with a rise of the levels of the corresponding mRNA transcripts. This suggests that the alterations in the expression of podocyte-associated molecules represent a compensatory reaction of the podocyte that results from damage associated with proteinuria.

Role of the VEGF-A Signaling Pathway in the Glomerulus: Evidence for Crosstalk between Components of the Glomerular Filtration Barrier

Background/Aims: Vascular endothelial growth factor is a major regulator of angiogenesis and vascular permeability [Carmeliet et al.: Nature 1996;380:435–439]. The podocyte, the outermost layer of the glomerular filtration barrier, produces large amounts of VEGF-A. The observation that levels of VEGF-A are altered in glomerular diseases, the identification of a link between pre-eclampsia and elevated levels of a circulating soluble VEGF receptor, and the entry of anti-VEGF therapies into the clinical arena have generated intense interest in the functional role of VEGF-A in the glomerulus. Methods: A variety of studies have been performed to address the role of VEGF-A signaling in the glomerulus. These include descriptions of expression patterns in human renal biopsies, cell culture studies to dissect paracrine versus autocrine signaling roles, and manipulation of VEGF-A expression in animal models using pharmacologic, biologic or genetic approaches. Results: Exquisite dosage sensitivity to VEGF-A exists in the developing glomerulus as small reductions in the expression of VEGF-A lead to profound changes in glomerular structure and function in mice. The use of VEGF inhibitors is associated with damage to the glomerular endothelium in animal models and proteinuria in patients, suggesting that local VEGF-A production is also required for maintenance of this specialized vascular bed. Conclusions: Tight regulation of VEGF-A signaling is required for development and maintenance of the glomerular filtration barrier (GFB) and emphasizes the role of podocyte-endothelial crosstalk in the glomerulus. The relative contributions of various VEGF-A isoforms, the role of autocrine signaling in vivo and identification of factors and mechanisms that regulate constitutive expression, storage and delivery of VEGF-A in the glomerulus are still under investigation.

Glomerular Structure and Function Require Paracrine, Not Autocrine, VEGF–VEGFR-2 Signaling

VEGF is a potent vascular growth factor produced by podocytes in the developing and mature glomerulus. Specific deletion of VEGF from podocytes causes glomerular abnormalities including profound endothelial cell injury, suggesting that paracrine signaling is critical for maintaining the glomerular filtration barrier (GFB). However, it is not clear whether normal GFB function also requires autocrine VEGF signaling in podocytes. In this study, we sought to determine whether an autocrine VEGF-VEGFR-2 loop in podocytes contributes to the maintenance of the GFB in vivo. We found that induced, whole-body deletion of VEGFR-2 caused marked abnormalities in the kidney and also other tissues, including the heart and liver. By contrast, podocyte-specific deletion of the VEGFR-2 receptor had no effect on glomerular development or function even up to 6 months old. Unlike cell culture models, enhanced expression of VEGF by podocytes in vivo caused foot process fusion and alterations in slit diaphragm-associated proteins; however, inhibition of VEGFR-2 could not rescue this defect. Although VEGFR-2 was dispensable in the podocyte, glomerular endothelial cells depended on VEGFR-2 expression: postnatal deletion of the receptor resulted in global defects in the glomerular microvasculature. Taken together, our results provide strong evidence for dominant actions of a paracrine VEGF-VEGFR-2 signaling loop both in the developing and in the filtering glomerulus. VEGF produced by the podocyte regulates the structure and function of the adjacent endothelial cell.

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.

Pro-inflammatory chemokine-chemokine receptor interactions within the Ewing sarcoma microenvironment determine CD8(+) T-lymphocyte infiltration and affect tumour progression.

Ewing sarcoma is an aggressive round cell sarcoma with poor patient prognosis, particularly in cases of advanced‐stage disease. Dynamic tumor‐host immune interations within the tumor microenvironment may polarize in situ immune responses and shape tumor development and/or progression. To gain insight into the nature of tumour–host immune interactions within the Ewing sarcoma microenvironment, the presence and spatial distribution of infiltrating CD8+/CD4+ T‐lymphocytes were evaluated in therapy‐naive Ewing sarcoma. Expression profiling of 40 different chemokines and several chemokine receptors was performed in therapy‐naive tumours and cell lines by qPCR, immunohistochemistry, and flow cytometry. Considerable inter‐tumour variation was observed regarding density, type, and distribution of infiltrating T‐lymphocytes. Tumour‐infiltrating T‐cells contained significantly higher percentages of CD8+ T‐lymphocytes as compared to stroma‐infiltrating cells, suggesting preferential migration of this T‐cell type into tumour areas. Gene expression levels of several type 1‐associated, pro‐inflammatory chemokines (CXCR3‐ and CCR5‐ligands CXCL9, CXCL10, and CCL5) correlated positively with infiltrating (CD8+) T‐lymphocyte numbers expressing corresponding chemokine receptors. Survival analyses demonstrated an impact of tumour‐infiltrating, and not stroma‐infiltrating, CD8+ T‐lymphocytes on tumour progression. At protein level, both tumour and stromal cells expressed the IFNγ‐inducible chemokines CXCL9 and CXCL10. CCR5‐ligand CCL5 was exclusively expressed by non‐tumoural stromal/infiltrating cells. Together, our results indicate that an inflammatory immune microenvironment with high expression of type 1‐associated chemokines may be critical for the recruitment of (CD8+) T‐lymphocytes expressing corresponding chemokine receptors. The observed impact of tumour‐infiltrating (CD8+) T‐lymphocytes is consistent with a role for adaptive anti‐tumour immunity in the prevention of Ewing sarcoma progression. Recognition of the merits and exploitation/induction of an inflammatory microenvironment may improve the efficacy of natural immune responses against, and (adoptive) immunotherapeutic approaches for, Ewing sarcoma. Copyright

Preeclampsia Is Characterized by Placental Complement Dysregulation

Increasing evidence suggests that preeclampsia is associated with complement dysregulation. The origin of complement dysregulation in preeclampsia is unknown, and further unraveling this mechanism could provide both diagnostic tools and therapeutic targets. Because the placenta is believed to play a crucial role in the pathogenesis of preeclampsia, we investigated placentas from preeclamptic women (n=28) and controls (n=44) for the presence of complement activation products. Immunohistochemistry was performed for C1q, mannose-binding lectin, properdin, and C4d. Staining patterns were related to pregnancy outcome. Possible causes of complement activation were investigated, including the presence of immune deposits at the syncytiotrophoblast and changes in the placental mRNA expression of complement regulatory proteins. C4d was rarely present in placentas from healthy controls (3%), whereas it was observed in 50% of placentas obtained from preeclamptic women (P=0.001). In these placentas, C4d was observed in a focal (9/14) or diffuse (5/14) staining pattern at the syncytiotrophoblast. With respect to C1q, mannose-binding lectin, and properdin, no differences were observed between cases and controls. In preeclamptic women, diffuse placental C4d was associated with a significantly lower gestational age at delivery. Furthermore, the mRNA expression of the complement regulatory proteins CD55 and CD59 was significantly upregulated in preeclampsia. In conclusion, there is evidence for increased classical pathway activation and altered complement regulation in preeclampsia. The relation between C4d and lower gestational age at birth suggests that the extent of complement dysregulation is associated with the severity of preeclampsia. Inhibiting excessive complement activation may be a promising therapeutic approach in the management of preeclampsia.

Vegfa Protects the Glomerular Microvasculature in Diabetes

Vascular endothelial growth factor A (VEGFA) expression is increased in glomeruli in the context of diabetes. Here, we tested the hypothesis that this upregulation of VEGFA protects the glomerular microvasculature in diabetes and that therefore inhibition of VEGFA will accelerate nephropathy. To determine the role of glomerular Vegfa in the development and progression of diabetic nephropathy, we used an inducible Cre-loxP gene-targeting system that enabled genetic deletion of Vegfa selectively from glomerular podocytes of wild-type or diabetic mice. Type 1 diabetes was induced in mice using streptozotocin (STZ). We then assessed the extent of glomerular dysfunction by measuring proteinuria, glomerular pathology, and glomerular cell apoptosis. Vegfa expression increased in podocytes in the STZ model of diabetes. After 7 weeks of diabetes, diabetic mice lacking Vegfa in podocytes exhibited significantly greater proteinuria with profound glomerular scarring and increased apoptosis compared with control mice with diabetes or Vegfa deletion without diabetes. Reduced local production of glomerular Vegfa in a mouse model of type 1 diabetes promotes endothelial injury accelerating the progression of glomerular injury. These results suggest that upregulation of VEGFA in diabetic kidneys protects the microvasculature from injury and that reduction of VEGFA in diabetes may be harmful.

Genetic variants in pre-eclampsia: a meta-analysis

BACKGROUND Pre-eclampsia has a clear familial component, suggesting that the condition may be partly attributable to genetic susceptibility. The search for susceptibility genes has led to a drastic increase in the number of published studies associating genetic factors with pre-eclampsia. However, attempts to replicate these findings have yielded inconsistent results. This meta-analysis assessed the pooled effect of each genetic variant that is reproducibly associated with pre-eclampsia. METHODS Studies that assessed the association between genes and pre-eclampsia were searched in PubMed, Embase and Web of Science. We selected all genetic variants that were significantly associated with pre-eclampsia in an initial study and were subsequently independently reproduced in at least one additional study. All studies that assessed these reproduced variants were then included. The association between genetic variants and pre-eclampsia was calculated at the allele level, and the main measure of effect was a pooled odds ratio in a random-effects model. RESULTS The literature search yielded 2965 articles, of which 542 investigated genetic associations in pre-eclampsia. We identified 22 replicated genetic variants, of which 7 remained significantly associated with pre-eclampsia following meta-analysis. These variants were in or near the following genes: ACE, CTLA4, F2, FV, LPL and SERPINE1. CONCLUSIONS This meta-analysis identified seven genetic variants associated with pre-eclampsia. Importantly, many of these variants are also risk factors for developing cardiovascular disease, revealing that pre-eclampsia and cardiovascular disease have shared genetic risk factors. The contribution of the identified genetic variants in the pathogenesis of pre-eclampsia should be the focus of future studies.

Vegetarianism, female gender and increasing age, but not CNDP1 genotype, are associated with reduced muscle carnosine levels in humans

Carnosine is found in high concentrations in skeletal muscles, where it is involved in several physiological functions. The muscle carnosine content measured within a population can vary by a factor 4. The aim of this study was to further characterize suggested determinants of the muscle carnosine content (diet, gender and age) and to identify new determinants (plasma carnosinase activity and testosterone). We investigated a group of 149 healthy subjects, which consisted of 94 men (12 vegetarians) and 55 women. Muscle carnosine was quantified in M. soleus, gastrocnemius and tibialis anterior using magnetic resonance proton spectroscopy and blood samples were collected to determine CNDP1 genotype, plasma carnosinase activity and testosterone concentrations. Compared to women, men have 36, 28 and 82% higher carnosine concentrations in M. soleus, gastrocnemius and tibialis anterior muscle, respectively, whereas circulating testosterone concentrations were unrelated to muscle carnosine levels in healthy men. The carnosine content of the M. soleus is negatively related to the subjects’ age. Vegetarians have a lower carnosine content of 26% in gastrocnemius compared to omnivores. In contrast, there is no difference in muscle carnosine content between omnivores with a high or low ingestion of β-alanine. Muscle carnosine levels are not related to the polymorphism of the CNDP1 gene or to the enzymatic activity of the plasma carnosinase. In conclusion, neither CNDP1 genotype nor the normal variation in circulating testosterone levels affects the muscular carnosine content, whereas vegetarianism, female gender and increasing age are the factors associated with reduced muscle carnosine stores.

Chimerism in Kidneys, Livers and Hearts of Normal Women: Implications for Transplantation Studies

Tissue chimerism was recently described in transplanted organs from female donors into male recipients, by demonstration of the Y‐chromosome in tissue‐derived cells. It was claimed that these Y‐chromosome positive cells were recipient derived. To find out whether the chimeric cells, derived from pregnancies of sons or blood transfusions, could have been present in the solid organs before transplantation, we performed the following study. In situ hybridization for the Y‐chromosome was performed on the normal organs (51 kidneys, 51 livers, 69 hearts) from 75 women of the normal population, whose child and blood transfusion status were known. Chimeric cells were found in 13 kidneys, 10 livers and 4 hearts, of 23 women. There was no relation between the child status or the blood transfusion history with the presence of Y‐chromosome positive cells. We have for the first time demonstrated that male cells are present in normal kidneys, livers and hearts. Theoretically, these organs could have been used for the transplantation. Therefore, our findings demonstrate that the chimeric cells thus far described in transplantation studies, are not necessarily donor derived, and could have been present in the organs before the transplantation.