Glomerular basement membrane (GBM) antigens used for immunological studies have until now been is... more Glomerular basement membrane (GBM) antigens used for immunological studies have until now been isolated from human and rat glomeruli but not from mice. However, given the growing awareness of extracellular matrix species-specificity, the need for a purification method of mouse GBM exists. We now report the purification of GBM from isolated mouse glomeruli. 10 ml 0.01 M phosphate buffered saline (PBS) containing 1.25% Fe3O4 was perfused through the aortae of (C57BL10 x DBA/2)F1 mice over 1 min, kidneys were decapsulated and passed through a 0.075 mm mesh metal screen and collected. After pelletting and washing, the tube was placed against one pole of a magnet and the pelleted glomeruli were washed. This procedure was repeated three times. The glomerular suspension was examined by light microscopy, sonicated, and lyophilized. In suspension no free fragments of tubuli or Bowman's capsule were present as observed by light microscopy. Mouse GBM was prepared from the isolated glomeruli using a modification of earlier described methods for human and rat GBM by enzymatic digestion. Elisa studies showed the presence of laminin, type IV collagen, and fibronectin. Monoclonal antibody directed against tubular epithelial antigen gp160 did not react to either mouse or rat GBM. Sera and glomerular eluates from (C57BL10 x DBA/2)F1 mice suffering from chronic graft-versus-host autoimmune disease gave a higher signal against mouse GBM than against rat GBM. Normal rabbit serum, normal mouse serum and normal mouse eluate did not show significant activity against mouse GBM. Immunoblotting showed the presence of both laminin B1 and B2 chains as well as type IV collagen alpha 1 and alpha 2 subunits. In summary, we describe a method by which it is possible to extract mouse GBM with a high purity.
Extraction of RNA has been described for rat and rabbit glomeruli but not for mouse glomeruli. Du... more Extraction of RNA has been described for rat and rabbit glomeruli but not for mouse glomeruli. Due to their small size, mouse glomeruli cannot be isolated by relatively simple sieving techniques. Based on recently reported methods for the isolation of mouse glomeruli, we developed an RNA isolation technique by performing comparative methodological studies. Two standard RNA extraction methods were compared. In addition in separate experiments the influence was studied of protease inhibitors and freezing and thawing of whole kidney prior to glomeruli isolation, on the yield and degradation of RNA. Therefore kidneys were perfused with 10 ml 0.01 M PBS containing 1.25% Fe 3 O 4 through the aorta. Kidneys were decapsulated and passed through a 75-um metal screen. After pelletting and washing, tubes were placed against a magnet and pelleted glomeruli were washed three times. In a second experiment protease inhibitors were added to the PBS. As a third method, kidneys were frozen before the isolation of glomeruli. From isolated glomeruli RNA was extracted using either caesium chloride or lithium chloride method. The yields of RNA (OD 260) were highest using the lithium chloride method. Hybridization of Northern blots of extracted RNA with cDNA probes showed the best results when RNA was extracted using the lithium chloride method, while the caesium chloride method led to considerable degradation of RNA. Freezing of kidney tissue prior to RNA extraction led to the virtual absence of any signal. We then applied this method succesfully in an in-vivo model of experimental lupus nephritis. This is the first description of an optimal protocol for the extraction of RNA from mouse glomeruli. From our studies we conclude that the lithium chloride method is superior for the extraction of RNA from mouse glomeruli. Adding of protease inhibitors during glomerular isolation is superfluous and freezing of kidney tissue prior to the isolation of glomeruli leads to total degradation of RNA.
The development of glomerulosclerosis was studied in murine chronic graft-versus-host disease (Gv... more The development of glomerulosclerosis was studied in murine chronic graft-versus-host disease (GvHD), which is a model for human systemic lupus erythematosus. The authors investigated the distribution patterns of six components of the extracellular matrix (ECM), i.e., laminin, fibronectin, collagen types I, III, IV, and VI during the course of the disease. All of these ECM components except collagen type I were found in the glomeruli of normal mice, where all of them were intrinsic constituents of the mesangium. Laminin, fibronectin, and collagen type IV were also found in the glomerular capillary walls. Starting 6 weeks after the induction of GvHD and continuing at week 8, the onset of an expansion of the mesangial matrix was observed. At the same time, the amounts of laminin, fibronectin, and collagen types IV and VI increased. Ten weeks after the onset of the disease, glomerulosclerosis developed. Traces of the interstitial collagen type I were found in sclerotic glomeruli. The l...
American Journal of Physiology - Endocrinology And Metabolism, 2015
A particular allele of the carnosinase gene (CNDP1) is associated with reduced plasma carnosinase... more A particular allele of the carnosinase gene (CNDP1) is associated with reduced plasma carnosinase activity and reduced risk for nephropathy in diabetic patients. On the one hand, animal and human data suggest that hyperglycemia increases plasma carnosinase activity. On the other hand, we recently reported lower carnosinase activity levels in elite athletes involved in high-intensity exercise, compared to untrained controls. Therefore, this study investigates whether exercise training and the consequent reduction in hyperglycemia can suppress carnosinase activity and content in adults with type 2 diabetes. Plasma samples were taken from 243 males and females with type 2 diabetes (mean age = 54.3 yr, SD = 7.1) without major microvascular complications before and after a 6-month exercise training program (4 groups: sedentary control (n=61), aerobic exercise (n=59), resistance exercise (n=63) and combined exercise training (n=60)). Plasma carnosinase content and activity, hemoglobin A1c, lipid profile and blood pressure were measured. A 6-month exercise training intervention, irrespective of training modality, did not decrease plasma carnosinase content or activity in type 2 diabetic patients. Plasma carnosinase content and activity showed a high inter-individual but very low intra-individual variability over the 6-month period. Age and sex, but not HbA1c, were significantly related to the activity or content of this enzyme. It can be concluded that the beneficial effects of exercise training on the incidence of diabetic complications is probably not related to a lowering effect on plasma carnosinase content or activity.
A polymorphism in the carnosine dipeptidase-1 gene (CNDP1), resulting in decreased plasma carnosi... more A polymorphism in the carnosine dipeptidase-1 gene (CNDP1), resulting in decreased plasma carnosinase activity, is associated with a reduced risk for diabetic nephropathy. Because carnosine, a natural scavenger/suppressor of ROS, advanced glycation end products, and reactive aldehydes, is readily degraded in blood by the highly active carnosinase enzyme, it has been postulated that low serum carnosinase activity might be advantageous to reduce diabetic complications. The aim of this study was to examine whether low carnosinase activity promotes circulating carnosine levels after carnosine supplementation in humans. Blood and urine were sampled in 25 healthy subjects after acute supplementation with 60 mg/kg body wt carnosine. Precooled EDTA-containing tubes were used for blood withdrawal, and plasma samples were immediately deproteinized and analyzed for carnosine and β-alanine by HPLC. CNDP1 genotype, baseline plasma carnosinase activity, and protein content were assessed. Upon car...
Clinical journal of the American Society of Nephrology : CJASN, Jan 7, 2014
Preeclampsia is characterized by hypertension and proteinuria, and increased shedding of podocyte... more Preeclampsia is characterized by hypertension and proteinuria, and increased shedding of podocytes into the urine is a common finding. This finding raises the question of whether preeclamptic nephropathy involves podocyte damage. This study examined podocyte-related changes in a unique sample of renal tissues obtained from women who died of preeclampsia. All patients with preeclampsia who died in The Netherlands since 1990 and had available autopsy tissue were identified using a nationwide database of the Dutch Pathology Registry (PALGA). This resulted in a cohort of 11 women who died from preeclampsia. Three control groups were also identified during the same time period, and consisted of normotensive women who died during pregnancy (n=25), and nonpregnant controls either with (n=14) or without (n=13) chronic hypertension. Glomerular lesions, including podocyte numbers, podocyte proliferation, and parietal cell activation, were measured. Patients with preeclampsia had prominent cha...
Pregnancy Hypertension: An International Journal of Women's Cardiovascular Health, 2013
Objectives: We hypothesized that women with PE and young women with chronic hypertension might sh... more Objectives: We hypothesized that women with PE and young women with chronic hypertension might show similarity in renal lesions. Furthermore, we investigated if the number of podocytes within the kidney is decreased under these different circumstances. Methods We performed a search for renal autopsy-tissues using a nationwide computerized database (PALGA) to collect a unique large cohort of preeclamptic patients (n = 11). Three control groups were included consisting of young women who died during pregnancy without hypertension (n = 25) and non-pregnant controls with (n = 14) and without (n = 13) chronic hypertension. WT-1 staining was used to quantify the number of podocytes. Results Women with PE had MPGN-like lesions without immune-deposits. Tram tracking and podocyte changes were exclusively observed in these patients. Endotheliosis was significantly more present in PE, but sporadically seen in pregnant-and hypertensive controls. Chronic ischaemic lesions were predominantly found in young women with chronic hypertension, and not in PE and other controls. No differences were found in glomerular podocyte number between the study groups.
Endothelial chimerism in transplanted organs is a fascinating phenomenon, indicative of a mechani... more Endothelial chimerism in transplanted organs is a fascinating phenomenon, indicative of a mechanism by which progenitor recipient cells replace the donor endothelium. It has been hypothesized that this replacement could lead to a decrease in alloreactivity and thus would positively influence graft outcome. However, recent studies have shown that the amount of recipient-derived endothelial cells found in donor organs is relatively small. What effect on graft survival can we expect from this low number of chimeric cells? There are several hypotheses that address this question, but distinguishing the true effect of donor endothelial replacement on outcome from other factors affecting graft survival is difficult. Furthermore, "contamination" of chimeric cells from sources other than the recipient would have to be excluded before the effect of donor endothelial replacement by recipient cells can be accurately assessed. Pregnancies and blood transfusions are the other sources that may induce chimerism. Most of the techniques currently used to detect chimeric cells in donor organs are not specific enough to distinguish chimeric cells that may have been present in the graft before transplantation and recipient-derived chimeric cells that replace the endothelium after transplantation. Also, the sensitivity of these techniques may be questioned: do we really detect all chimeric cells that are present? This review will elaborate on these questions and discuss future perspectives of research into chimerism.
Glomerular protein handling mechanisms have received much attention in studies of nephrotic syndr... more Glomerular protein handling mechanisms have received much attention in studies of nephrotic syndrome. Histopathological findings in renal biopsies from severely proteinuric patients support the likelihood of protein endocytosis by podocytes. ClC-5 is involved in the endocytosis of albumin in the proximal tubule.
Background The blood vessels of a transplanted organ are the interface between donor and recipien... more Background The blood vessels of a transplanted organ are the interface between donor and recipient. The endothelium in the blood vessels is thought to be the major target for graft rejection. Endothelial cells of a transplanted organ are believed to remain of donor origin after transplantation. We aimed to verify this concept.
Prevention of glomeruloscierosis by early cyclosporine treatment of experimental lupus nephritis.... more Prevention of glomeruloscierosis by early cyclosporine treatment of experimental lupus nephritis. The influence of cyclosporine A (CsA) treatment on the development of glomerulonephritis and glomeruloscierosis 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, vaiying 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
Endothelial cell chimerism occurs more often and earlier in female than in male recipients of kid... more Endothelial cell chimerism occurs more often and earlier in female than in male recipients of kidney transplants.
Chimerism indicates the presence of cells from one individual in another. Pregnancy and blood tra... more Chimerism indicates the presence of cells from one individual in another. Pregnancy and blood transfusions are considered the main sources for chimerism. Chimeric cells have been attributed a pathogenic role in various autoimmune diseases. However, data on the occurrence of chimeric cells in normal organs are scarce. In order to gain insight into the possible pathogenic potential of chimeric cells in autoimmune disease, it is necessary to determine the prevalence of chimeric cells in organs not affected by autoimmune disease. In situ hybridization for the Y-chromosome was performed on organs obtained at autopsy of 51 women. We investigated 44 thyroid, 38 lung, 21 skin and 7 lymph node samples. All women had sons, and data from their blood transfusion histories were retrieved for at least 10 years before death. Slides were scored semi-quantitatively for chimerism as low (1-3 Y-chromosome-positive cells per slide), moderate (4-10 positive cells per slide) or high (more than 10 positive cells per slide). Y-chromosome-positive cells were found in 8 thyroid, 10 lung, 3 skin and 1 lymph node samples of 18 women. There was no association between the presence of chimeric cells and blood transfusion history. Most organs in which chimerism was present contained a small to moderate level. Thus, chimerism can occur in normal organs of women without autoimmune disease. Our results indicate that chimerism is not necessarily associated with disease.
Mice with chronic graft-versus-host disease (GvHD) induced by injection of DBA/2 lymphocytes into... more Mice with chronic graft-versus-host disease (GvHD) induced by injection of DBA/2 lymphocytes into (DBA/2 x C57BL/10) F1 hybrids (DBA/2 GvHD) develop a lupus-like glomerulonephritis with global glomerulosclerosis 12 weeks after induction of the disease. In two other strain combinations with similar H-2 incompatibilities [BALB/c into BALB/c x BL10 (BALB/c GvHD) and BALB.D2 into BALB.D2 x BL10 (BALB.D2 GvHD)], GvHD induction leads to lupus nephritis without global glomerulosclerosis. This study investigated the identity of kidney-infiltrating leukocytes and their involvement in the development of glomerulosclerosis in these three strain combinations. In mice with DBA/2 GvHD, a significant increase in glomerular CD11a-positive cells was found 4 weeks after disease induction. Mice with BALB/c or BALB.D2 GvHD did not show an increase in glomerular CD11a-positive cells at any time point. In the interstitium, CD11a-positive cells were observed 4 weeks after disease induction only in mice with DBA/2 GvHD. In mice with BALB.D2 GvHD, no increase was found in interstitial CD11a-positive cells. In mice with BALB/c GvHD, interstitial CD11a-positive cells were found from week 4 onward. Further immunohistochemical analysis of the glomerular CD11a-positive cells in mice with DBA/2 GvHD showed that these cells were neither polymorphonuclear leukocytes (PMN), nor CD3-positive (T cells), B220-positive (B cells), or F4/80-positive (macrophages). They were all CD45-positive (leukocytes) and MHC class II-positive. In conclusion, we have shown in this model of chronic lupus nephritis that glomerular influx of as yet unidentified CD11a-positive leukocytes is associated with the development of glomerulosclerosis.
To determine whether CCL2 mRNA expression is beneficial or detrimental for cervical cancer patien... more To determine whether CCL2 mRNA expression is beneficial or detrimental for cervical cancer patients, the association between the expression of this molecule by cervical tumour cells, the number of tumour-associated macrophages, and clinicopathological parameters such as recurrence, relapse-free survival, and overall patient survival was investigated. In cervical cancer samples from 93 untreated cervical cancer patients, the CCL2 mRNA expression level was quantified using RNA in situ hybridization and verified using real-time quantitative RT-PCR. The number of tumour-associated macrophages was determined using immunohistochemistry. Furthermore, the study investigated whether lack of CCL2 expression was due to genetic alterations near the 17q11.2 (CCL2 genomic) region. CCL2 mRNA expression by cervical tumour cells was associated with the number of tumour-associated macrophages (p < 0.001). Lack of CCL2 mRNA expression (15 samples; 16%) was associated with increased cumulative relapse-free survival (log rank test, p = 0.030), increased cumulative overall survival (log rank test, p = 0.024), less post-operative surgery, reduced local and distant recurrence, reduced vascular invasion, and smaller tumour size (<40 mm). The absence of CCL2 mRNA expression corresponded with loss of heterozygosity (LOH) at 17q11.2 in five of six samples. The increased cumulative relapse-free survival and cumulative overall survival of cervical cancer patients lacking tumour cell-associated CCL2 mRNA suggest that the tumour-associated macrophages support tumour progression, presumably by promoting angiogenesis and production of growth factors.
Glomerular basement membrane (GBM) antigens used for immunological studies have until now been is... more Glomerular basement membrane (GBM) antigens used for immunological studies have until now been isolated from human and rat glomeruli but not from mice. However, given the growing awareness of extracellular matrix species-specificity, the need for a purification method of mouse GBM exists. We now report the purification of GBM from isolated mouse glomeruli. 10 ml 0.01 M phosphate buffered saline (PBS) containing 1.25% Fe3O4 was perfused through the aortae of (C57BL10 x DBA/2)F1 mice over 1 min, kidneys were decapsulated and passed through a 0.075 mm mesh metal screen and collected. After pelletting and washing, the tube was placed against one pole of a magnet and the pelleted glomeruli were washed. This procedure was repeated three times. The glomerular suspension was examined by light microscopy, sonicated, and lyophilized. In suspension no free fragments of tubuli or Bowman's capsule were present as observed by light microscopy. Mouse GBM was prepared from the isolated glomeruli using a modification of earlier described methods for human and rat GBM by enzymatic digestion. Elisa studies showed the presence of laminin, type IV collagen, and fibronectin. Monoclonal antibody directed against tubular epithelial antigen gp160 did not react to either mouse or rat GBM. Sera and glomerular eluates from (C57BL10 x DBA/2)F1 mice suffering from chronic graft-versus-host autoimmune disease gave a higher signal against mouse GBM than against rat GBM. Normal rabbit serum, normal mouse serum and normal mouse eluate did not show significant activity against mouse GBM. Immunoblotting showed the presence of both laminin B1 and B2 chains as well as type IV collagen alpha 1 and alpha 2 subunits. In summary, we describe a method by which it is possible to extract mouse GBM with a high purity.
Extraction of RNA has been described for rat and rabbit glomeruli but not for mouse glomeruli. Du... more Extraction of RNA has been described for rat and rabbit glomeruli but not for mouse glomeruli. Due to their small size, mouse glomeruli cannot be isolated by relatively simple sieving techniques. Based on recently reported methods for the isolation of mouse glomeruli, we developed an RNA isolation technique by performing comparative methodological studies. Two standard RNA extraction methods were compared. In addition in separate experiments the influence was studied of protease inhibitors and freezing and thawing of whole kidney prior to glomeruli isolation, on the yield and degradation of RNA. Therefore kidneys were perfused with 10 ml 0.01 M PBS containing 1.25% Fe 3 O 4 through the aorta. Kidneys were decapsulated and passed through a 75-um metal screen. After pelletting and washing, tubes were placed against a magnet and pelleted glomeruli were washed three times. In a second experiment protease inhibitors were added to the PBS. As a third method, kidneys were frozen before the isolation of glomeruli. From isolated glomeruli RNA was extracted using either caesium chloride or lithium chloride method. The yields of RNA (OD 260) were highest using the lithium chloride method. Hybridization of Northern blots of extracted RNA with cDNA probes showed the best results when RNA was extracted using the lithium chloride method, while the caesium chloride method led to considerable degradation of RNA. Freezing of kidney tissue prior to RNA extraction led to the virtual absence of any signal. We then applied this method succesfully in an in-vivo model of experimental lupus nephritis. This is the first description of an optimal protocol for the extraction of RNA from mouse glomeruli. From our studies we conclude that the lithium chloride method is superior for the extraction of RNA from mouse glomeruli. Adding of protease inhibitors during glomerular isolation is superfluous and freezing of kidney tissue prior to the isolation of glomeruli leads to total degradation of RNA.
The development of glomerulosclerosis was studied in murine chronic graft-versus-host disease (Gv... more The development of glomerulosclerosis was studied in murine chronic graft-versus-host disease (GvHD), which is a model for human systemic lupus erythematosus. The authors investigated the distribution patterns of six components of the extracellular matrix (ECM), i.e., laminin, fibronectin, collagen types I, III, IV, and VI during the course of the disease. All of these ECM components except collagen type I were found in the glomeruli of normal mice, where all of them were intrinsic constituents of the mesangium. Laminin, fibronectin, and collagen type IV were also found in the glomerular capillary walls. Starting 6 weeks after the induction of GvHD and continuing at week 8, the onset of an expansion of the mesangial matrix was observed. At the same time, the amounts of laminin, fibronectin, and collagen types IV and VI increased. Ten weeks after the onset of the disease, glomerulosclerosis developed. Traces of the interstitial collagen type I were found in sclerotic glomeruli. The l...
American Journal of Physiology - Endocrinology And Metabolism, 2015
A particular allele of the carnosinase gene (CNDP1) is associated with reduced plasma carnosinase... more A particular allele of the carnosinase gene (CNDP1) is associated with reduced plasma carnosinase activity and reduced risk for nephropathy in diabetic patients. On the one hand, animal and human data suggest that hyperglycemia increases plasma carnosinase activity. On the other hand, we recently reported lower carnosinase activity levels in elite athletes involved in high-intensity exercise, compared to untrained controls. Therefore, this study investigates whether exercise training and the consequent reduction in hyperglycemia can suppress carnosinase activity and content in adults with type 2 diabetes. Plasma samples were taken from 243 males and females with type 2 diabetes (mean age = 54.3 yr, SD = 7.1) without major microvascular complications before and after a 6-month exercise training program (4 groups: sedentary control (n=61), aerobic exercise (n=59), resistance exercise (n=63) and combined exercise training (n=60)). Plasma carnosinase content and activity, hemoglobin A1c, lipid profile and blood pressure were measured. A 6-month exercise training intervention, irrespective of training modality, did not decrease plasma carnosinase content or activity in type 2 diabetic patients. Plasma carnosinase content and activity showed a high inter-individual but very low intra-individual variability over the 6-month period. Age and sex, but not HbA1c, were significantly related to the activity or content of this enzyme. It can be concluded that the beneficial effects of exercise training on the incidence of diabetic complications is probably not related to a lowering effect on plasma carnosinase content or activity.
A polymorphism in the carnosine dipeptidase-1 gene (CNDP1), resulting in decreased plasma carnosi... more A polymorphism in the carnosine dipeptidase-1 gene (CNDP1), resulting in decreased plasma carnosinase activity, is associated with a reduced risk for diabetic nephropathy. Because carnosine, a natural scavenger/suppressor of ROS, advanced glycation end products, and reactive aldehydes, is readily degraded in blood by the highly active carnosinase enzyme, it has been postulated that low serum carnosinase activity might be advantageous to reduce diabetic complications. The aim of this study was to examine whether low carnosinase activity promotes circulating carnosine levels after carnosine supplementation in humans. Blood and urine were sampled in 25 healthy subjects after acute supplementation with 60 mg/kg body wt carnosine. Precooled EDTA-containing tubes were used for blood withdrawal, and plasma samples were immediately deproteinized and analyzed for carnosine and β-alanine by HPLC. CNDP1 genotype, baseline plasma carnosinase activity, and protein content were assessed. Upon car...
Clinical journal of the American Society of Nephrology : CJASN, Jan 7, 2014
Preeclampsia is characterized by hypertension and proteinuria, and increased shedding of podocyte... more Preeclampsia is characterized by hypertension and proteinuria, and increased shedding of podocytes into the urine is a common finding. This finding raises the question of whether preeclamptic nephropathy involves podocyte damage. This study examined podocyte-related changes in a unique sample of renal tissues obtained from women who died of preeclampsia. All patients with preeclampsia who died in The Netherlands since 1990 and had available autopsy tissue were identified using a nationwide database of the Dutch Pathology Registry (PALGA). This resulted in a cohort of 11 women who died from preeclampsia. Three control groups were also identified during the same time period, and consisted of normotensive women who died during pregnancy (n=25), and nonpregnant controls either with (n=14) or without (n=13) chronic hypertension. Glomerular lesions, including podocyte numbers, podocyte proliferation, and parietal cell activation, were measured. Patients with preeclampsia had prominent cha...
Pregnancy Hypertension: An International Journal of Women's Cardiovascular Health, 2013
Objectives: We hypothesized that women with PE and young women with chronic hypertension might sh... more Objectives: We hypothesized that women with PE and young women with chronic hypertension might show similarity in renal lesions. Furthermore, we investigated if the number of podocytes within the kidney is decreased under these different circumstances. Methods We performed a search for renal autopsy-tissues using a nationwide computerized database (PALGA) to collect a unique large cohort of preeclamptic patients (n = 11). Three control groups were included consisting of young women who died during pregnancy without hypertension (n = 25) and non-pregnant controls with (n = 14) and without (n = 13) chronic hypertension. WT-1 staining was used to quantify the number of podocytes. Results Women with PE had MPGN-like lesions without immune-deposits. Tram tracking and podocyte changes were exclusively observed in these patients. Endotheliosis was significantly more present in PE, but sporadically seen in pregnant-and hypertensive controls. Chronic ischaemic lesions were predominantly found in young women with chronic hypertension, and not in PE and other controls. No differences were found in glomerular podocyte number between the study groups.
Endothelial chimerism in transplanted organs is a fascinating phenomenon, indicative of a mechani... more Endothelial chimerism in transplanted organs is a fascinating phenomenon, indicative of a mechanism by which progenitor recipient cells replace the donor endothelium. It has been hypothesized that this replacement could lead to a decrease in alloreactivity and thus would positively influence graft outcome. However, recent studies have shown that the amount of recipient-derived endothelial cells found in donor organs is relatively small. What effect on graft survival can we expect from this low number of chimeric cells? There are several hypotheses that address this question, but distinguishing the true effect of donor endothelial replacement on outcome from other factors affecting graft survival is difficult. Furthermore, "contamination" of chimeric cells from sources other than the recipient would have to be excluded before the effect of donor endothelial replacement by recipient cells can be accurately assessed. Pregnancies and blood transfusions are the other sources that may induce chimerism. Most of the techniques currently used to detect chimeric cells in donor organs are not specific enough to distinguish chimeric cells that may have been present in the graft before transplantation and recipient-derived chimeric cells that replace the endothelium after transplantation. Also, the sensitivity of these techniques may be questioned: do we really detect all chimeric cells that are present? This review will elaborate on these questions and discuss future perspectives of research into chimerism.
Glomerular protein handling mechanisms have received much attention in studies of nephrotic syndr... more Glomerular protein handling mechanisms have received much attention in studies of nephrotic syndrome. Histopathological findings in renal biopsies from severely proteinuric patients support the likelihood of protein endocytosis by podocytes. ClC-5 is involved in the endocytosis of albumin in the proximal tubule.
Background The blood vessels of a transplanted organ are the interface between donor and recipien... more Background The blood vessels of a transplanted organ are the interface between donor and recipient. The endothelium in the blood vessels is thought to be the major target for graft rejection. Endothelial cells of a transplanted organ are believed to remain of donor origin after transplantation. We aimed to verify this concept.
Prevention of glomeruloscierosis by early cyclosporine treatment of experimental lupus nephritis.... more Prevention of glomeruloscierosis by early cyclosporine treatment of experimental lupus nephritis. The influence of cyclosporine A (CsA) treatment on the development of glomerulonephritis and glomeruloscierosis 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, vaiying 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
Endothelial cell chimerism occurs more often and earlier in female than in male recipients of kid... more Endothelial cell chimerism occurs more often and earlier in female than in male recipients of kidney transplants.
Chimerism indicates the presence of cells from one individual in another. Pregnancy and blood tra... more Chimerism indicates the presence of cells from one individual in another. Pregnancy and blood transfusions are considered the main sources for chimerism. Chimeric cells have been attributed a pathogenic role in various autoimmune diseases. However, data on the occurrence of chimeric cells in normal organs are scarce. In order to gain insight into the possible pathogenic potential of chimeric cells in autoimmune disease, it is necessary to determine the prevalence of chimeric cells in organs not affected by autoimmune disease. In situ hybridization for the Y-chromosome was performed on organs obtained at autopsy of 51 women. We investigated 44 thyroid, 38 lung, 21 skin and 7 lymph node samples. All women had sons, and data from their blood transfusion histories were retrieved for at least 10 years before death. Slides were scored semi-quantitatively for chimerism as low (1-3 Y-chromosome-positive cells per slide), moderate (4-10 positive cells per slide) or high (more than 10 positive cells per slide). Y-chromosome-positive cells were found in 8 thyroid, 10 lung, 3 skin and 1 lymph node samples of 18 women. There was no association between the presence of chimeric cells and blood transfusion history. Most organs in which chimerism was present contained a small to moderate level. Thus, chimerism can occur in normal organs of women without autoimmune disease. Our results indicate that chimerism is not necessarily associated with disease.
Mice with chronic graft-versus-host disease (GvHD) induced by injection of DBA/2 lymphocytes into... more Mice with chronic graft-versus-host disease (GvHD) induced by injection of DBA/2 lymphocytes into (DBA/2 x C57BL/10) F1 hybrids (DBA/2 GvHD) develop a lupus-like glomerulonephritis with global glomerulosclerosis 12 weeks after induction of the disease. In two other strain combinations with similar H-2 incompatibilities [BALB/c into BALB/c x BL10 (BALB/c GvHD) and BALB.D2 into BALB.D2 x BL10 (BALB.D2 GvHD)], GvHD induction leads to lupus nephritis without global glomerulosclerosis. This study investigated the identity of kidney-infiltrating leukocytes and their involvement in the development of glomerulosclerosis in these three strain combinations. In mice with DBA/2 GvHD, a significant increase in glomerular CD11a-positive cells was found 4 weeks after disease induction. Mice with BALB/c or BALB.D2 GvHD did not show an increase in glomerular CD11a-positive cells at any time point. In the interstitium, CD11a-positive cells were observed 4 weeks after disease induction only in mice with DBA/2 GvHD. In mice with BALB.D2 GvHD, no increase was found in interstitial CD11a-positive cells. In mice with BALB/c GvHD, interstitial CD11a-positive cells were found from week 4 onward. Further immunohistochemical analysis of the glomerular CD11a-positive cells in mice with DBA/2 GvHD showed that these cells were neither polymorphonuclear leukocytes (PMN), nor CD3-positive (T cells), B220-positive (B cells), or F4/80-positive (macrophages). They were all CD45-positive (leukocytes) and MHC class II-positive. In conclusion, we have shown in this model of chronic lupus nephritis that glomerular influx of as yet unidentified CD11a-positive leukocytes is associated with the development of glomerulosclerosis.
To determine whether CCL2 mRNA expression is beneficial or detrimental for cervical cancer patien... more To determine whether CCL2 mRNA expression is beneficial or detrimental for cervical cancer patients, the association between the expression of this molecule by cervical tumour cells, the number of tumour-associated macrophages, and clinicopathological parameters such as recurrence, relapse-free survival, and overall patient survival was investigated. In cervical cancer samples from 93 untreated cervical cancer patients, the CCL2 mRNA expression level was quantified using RNA in situ hybridization and verified using real-time quantitative RT-PCR. The number of tumour-associated macrophages was determined using immunohistochemistry. Furthermore, the study investigated whether lack of CCL2 expression was due to genetic alterations near the 17q11.2 (CCL2 genomic) region. CCL2 mRNA expression by cervical tumour cells was associated with the number of tumour-associated macrophages (p < 0.001). Lack of CCL2 mRNA expression (15 samples; 16%) was associated with increased cumulative relapse-free survival (log rank test, p = 0.030), increased cumulative overall survival (log rank test, p = 0.024), less post-operative surgery, reduced local and distant recurrence, reduced vascular invasion, and smaller tumour size (<40 mm). The absence of CCL2 mRNA expression corresponded with loss of heterozygosity (LOH) at 17q11.2 in five of six samples. The increased cumulative relapse-free survival and cumulative overall survival of cervical cancer patients lacking tumour cell-associated CCL2 mRNA suggest that the tumour-associated macrophages support tumour progression, presumably by promoting angiogenesis and production of growth factors.
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Papers by Hans Baelde