Antisense oligonucleotide-mediated terminal intron retention of endoglin: A potential strategy to inhibit renal interstitial fibrosis.

TGF-ß is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-ß co-receptor endoglin (ENG) tends to be upregulated in kidney fibrosis. ENG has two membrane bound isoforms generated via alternative splicing. Long-ENG was shown to enhance the extent of renal fibrosis in an unilateral ureteral obstruction mouse model, while short-ENG inhibited renal fibrosis. Here we aimed to achieve terminal intron retention of endoglin using antisense-oligo nucleotides (ASOs), thereby shifting the ratio towards short-ENG to inhibit the TGF-ß1-mediated pro-fibrotic response. We isolated mRNA from kidney biopsies of patients with chronic allograft disease (CAD) (n = 12) and measured total ENG and short-ENG mRNA levels. ENG mRNA was upregulated 2.3 fold (p < 0.05) in kidneys of CAD patients compared to controls, while the percentage short-ENG of the total ENG mRNA was significantly lower (1.8 fold; p < 0.05). Transfection of ASOs that target splicing regulatory sites of ENG into TK173 fibroblasts led to higher levels of short-ENG (2 fold; p < 0.05). In addition, we stimulated these cells with TGF-ß1 and measured a decrease in upregulation of ACTA2, COL1A1 and FN1 mRNA levels, and protein expression of αSMA, collagen type I, and fibronectin. These results show a potential for ENG ASOs as a therapy to reduce interstitial fibrosis in CKD.

Endoglin Is an Important Mediator in the Final Common Pathway of Chronic Kidney Disease to End-Stage Renal Disease.

Chronic kidney disease (CKD) is a slow-developing, progressive deterioration of renal function. The final common pathway in the pathophysiology of CKD involves glomerular sclerosis, tubular atrophy and interstitial fibrosis. Transforming growth factor-beta (TGF-ß) stimulates the differentiation of fibroblasts towards myofibroblasts and the production of extracellular matrix (ECM) molecules, and thereby interstitial fibrosis. It has been shown that endoglin (ENG, CD105), primarily expressed in endothelial cells and fibroblasts, can function as a co-receptor of TGF signaling. In several human organs, endoglin tends to be upregulated when chronic damage and fibrosis is present. We hypothesize that endoglin is upregulated in renal interstitial fibrosis and plays a role in the progression of CKD. We first measured renal endoglin expression in biopsy samples obtained from patients with different types of CKD, i.e., IgA nephropathy, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy (DN) and patients with chronic allograft dysfunction (CAD). We showed that endoglin is upregulated in CAD patients (p < 0.001) and patients with DN (p < 0.05), compared to control kidneys. Furthermore, the amount of interstitial endoglin expression correlated with eGFR (p < 0.001) and the amount of interstitial fibrosis (p < 0.001), independent of the diagnosis of the biopsies. Finally, we investigated in vitro the effect of endoglin overexpression in TGF-ß stimulated human kidney fibroblasts. Overexpression of endoglin resulted in an enhanced ACTA2, CCN2 and SERPINE1 mRNA response (p < 0.05). It also increased the mRNA and protein upregulation of the ECM components collagen type I (COL1A1) and fibronectin (FN1) (p < 0.05). Our results suggest that endoglin is an important mediator in the final common pathway of CKD and could be used as a possible new therapeutic target to counteract the progression towards end-stage renal disease (ESRD).

Endothelial Endothelin Receptor A Expression Is Associated With Podocyte Injury and Oxidative Stress in Patients With Focal Segmental Glomerulosclerosis.

INTRODUCTION: The podocyte is thought to be the mainly affected cell type in focal segmental glomerulosclerosis (FSGS). However, recent studies have also indicated a role for glomerular endothelial cells and podocyte-endothelial crosstalk in FSGS development. An experimental model for podocyte injury showed that increased endothelin-1 (ET-1) signaling between podocytes and endothelial cells induces endothelial oxidative stress and subsequent podocyte loss. In the current study, we investigated endothelial endothelin receptor A (ETAR) expression in patients with FSGS and its association with podocyte injury and glomerular oxidative stress. METHODS: We selected 39 biopsy samples of patients with FSGS and 8 healthy control subjects, and stained them for ETAR, nephrin and 8-oxo-guanine, a DNA lesion caused by oxidative damage. Glomeruli with ETAR-positive endothelium and with nephrin loss were scored, and the 8-oxo-guanine-positive glomerular area was measured. RESULTS: The mean percentage of glomeruli with ETAR-positive endothelial cells in patients with FSGS was higher compared to that in healthy control subjects (52% vs. 7%; P < 0.001). The presence of glomerular ETAR-positive endothelium was strongly associated with nephrin loss both on the biopsy level (rho = 0.47; P < 0.01), as on the level of individual glomeruli (odds ratio = 2.0; P < 0.001). Moreover, glomeruli with ETAR-positive endothelium showed more 8-oxo-guanine-positive staining (1.9% vs. 2.4%; P = 0.037). Finally, 8-oxo-guanine positivity in glomeruli was associated with increased levels of proteinuria. CONCLUSION: Taking together our findings, we show that ETAR is increased in glomerular endothelial cells of patients with FSGS and associated with podocyte damage and glomerular oxidative stress. These findings support the hypothesis that ET-1 signaling in glomerular endothelial cells contributes to disease development in patients with FSGS.

Endoglin Promotes Myofibroblast Differentiation and Extracellular Matrix Production in Diabetic Nephropathy.

Diabetic nephropathy (DN) is a complication of diabetes mellitus that can lead to proteinuria and a progressive decline in renal function. Endoglin, a co-receptor of TGF-ß, is known primarily for regulating endothelial cell function; however, endoglin is also associated with hepatic, cardiac, and intestinal fibrosis. This study investigates whether endoglin contributes to the development of interstitial fibrosis in DN. Kidney autopsy material from 80 diabetic patients was stained for endoglin and Sirius Red and scored semi-quantitatively. Interstitial endoglin expression was increased in samples with DN and was correlated with Sirius Red staining (p < 0.001). Endoglin expression was also correlated with reduced eGFR (p = 0.001), increased creatinine (p < 0.01), increased systolic blood pressure (p < 0.05), hypertension (p < 0.05), and higher IFTA scores (p < 0.001). Biopsy samples from DN patients were also co-immunostained for endoglin together with CD31, CD68, vimentin, or α-SMA Endoglin co-localized with both the endothelial marker CD31 and the myofibroblast marker α-SMA. Finally, we used shRNA to knockdown endoglin expression in a human kidney fibroblast cell line. We found that TGF-ß1 stimulation upregulated SERPINE1, CTGF, and ACTA2 mRNA and α-SMA protein, and that these effects were significantly reduced in fibroblasts after endoglin knockdown. Taken together, these data suggest that endoglin plays a role in the pathogenesis of interstitial fibrosis in DN.

Glomerular clusterin expression is increased in diabetic nephropathy and protects against oxidative stress-induced apoptosis in podocytes.

Clusterin, a glycoprotein encoded by the CLU gene, is expressed in many tissues, including the kidney, and clusterin expression is upregulated in the glomeruli of patients with various forms of kidney disease. Here, we investigated the role of clusterin in diabetic nephropathy (DN). In this study, we found that glomerular clusterin expression was increased in both patients with DN and streptozotocin-induced diabetic mice and that it co-localised with the podocyte marker WT1, indicating clusterin is expressed in podocytes. In our in vitro analysis, we found no significant change in CLU mRNA expression in podocytes following stimulation with high glucose and angiotensin II; in contrast, CLU mRNA expression was significantly upregulated following methylglyoxal stimulation. Methylglyoxal treatment also significantly decreased the mRNA expression of the slit diaphragm markers ZO-1 and NEPH1 and significantly increased the mRNA expression of the oxidative stress marker HO-1. Lastly, we showed that pre-incubating podocytes with recombinant human clusterin protein increased podocyte survival, prevented slit diaphragm damage, and reduced oxidative stress‒induced apoptosis following methylglyoxal stimulation. Taken together, our results indicate that glomerular clusterin is upregulated in DN, and this increase in clusterin expression may protect against oxidative stress-induced apoptosis in podocytes, providing a possible new therapeutic target for DN and other kidney diseases.

Reducing YAP expression in Pkd1 mutant mice does not improve the cystic phenotype.

The Hippo pathway is a highly conserved signalling route involved in organ size regulation. The final effectors of this pathway are two transcriptional coactivators, yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (WWTR1 or TAZ). Previously, we showed aberrant activation of the Hippo pathway in autosomal-dominant polycystic kidney disease (ADPKD), suggesting that YAP/TAZ might play a role in disease progression. Using antisense oligonucleotides (ASOs) in a mouse model for ADPKD, we efficiently down-regulated Yap levels in the kidneys. However, we did not see any effect on cyst formation or growth. Moreover, the expression of YAP/TAZ downstream targets was not changed, while WNT and TGF-ß pathways' downstream targets Myc, Acta2 and Vim were more expressed after Yap knockdown. Overall, our data indicate that reducing YAP levels is not a viable strategy to modulate PKD progression.

Glomerular C4d deposition can precede the development of focal segmental glomerulosclerosis.

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.

Four-jointed knock-out delays renal failure in an ADPKD model with kidney injury.

Autosomal Dominant Polycystic Kidney Disease is characterised by the development of fluid-filled cysts in the kidneys which lead to end-stage renal disease (ESRD). In the majority of cases, the disease is caused by a mutation in the Pkd1 gene. In a previous study, we demonstrated that renal injury can accelerate cyst formation in Pkd1 knock-out (KO) mice. In that study, we found that after injury four-jointed (Fjx1), an upstream regulator of planar cell polarity and the Hippo pathway, was aberrantly expressed in Pkd1 KO mice compared to WT. Therefore, we hypothesised a role for Fjx1 in injury/repair and cyst formation. We generated single and double deletion mice for Pkd1 and Fjx1, and we induced toxic renal injury using the nephrotoxic compound 1,2-dichlorovinyl-cysteine. We confirmed that nephrotoxic injury can accelerate cyst formation in Pkd1 mutant mice. This caused Pkd1 KO mice to reach ESRD significantly faster; unexpectedly, double KO mice survived significantly longer. Cyst formation was comparable in both models, but we found significantly less fibrosis and macrophage infiltration in double KO mice. Taken together, these data suggest that Fjx1 disruption protects the cystic kidneys against kidney failure by reducing inflammation and fibrosis. Moreover, we describe, for the first time, an interesting (yet unidentified) mechanism that partially discriminates cyst growth from fibrogenesis. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Analysis of the genomic architecture of a complex trait locus in hypertensive rat models links Tmem63c to kidney damage.

Unraveling the genetic susceptibility of complex diseases such as chronic kidney disease remains challenging. Here, we used inbred rat models of kidney damage associated with elevated blood pressure for the comprehensive analysis of a major albuminuria susceptibility locus detected in these models. We characterized its genomic architecture by congenic substitution mapping, targeted next-generation sequencing, and compartment-specific RNA sequencing analysis in isolated glomeruli. This led to prioritization of transmembrane protein Tmem63c as a novel potential target. Tmem63c is differentially expressed in glomeruli of allele-specific rat models during onset of albuminuria. Patients with focal segmental glomerulosclerosis exhibited specific TMEM63C loss in podocytes. Functional analysis in zebrafish revealed a role for tmem63c in mediating the glomerular filtration barrier function. Our data demonstrate that integrative analysis of the genomic architecture of a complex trait locus is a powerful tool for identification of new targets such as Tmem63c for further translational investigation.

The expression of somatostatin receptor 2 decreases during cyst growth in mice with polycystic kidney disease.

IMPACT STATEMENT: Somatostatin (SST) analogs have been shown to halt cyst growth and progression of autosomal dominant polycystic kidney disease by several clinical trials. However, two studies suggest that the effect of the SST analog octreotide on kidney growth during the first year of treatment is reduced in the subsequent follow-ups and the kidney enlargement resumes. This biphasic change in kidney growth during octreotide treatment may be partially explained by alterations in SSTR2 expression. Here, we found that SSTR2 is mainly expressed in distal tubules and collecting ducts in murine kidneys, and the expression of SSTR2 decreases during cyst growth in two PKD mouse models. Our data may thus provide possible explanations for the lack of efficacy in long-term treatment with SST analogs.

Endoglin Mediates Vascular Endothelial Growth Factor-A-Induced Endothelial Cell Activation by Regulating Akt Signaling.

In diabetic nephropathy, differential expression of growth factors leads to vascular changes, including endothelial cell activation, monocyte infiltration, and inflammation. Endoglin plays an important role in endothelial function and is also associated with inflammation. In the kidney, vascular endoglin expression is increased in animal models of renal injury, where it contributes to disease severity, possibly by promoting endothelial cell activation and inflammation. Herein, we investigated whether endoglin expression is associated with diabetic nephropathy. In addition, we examined whether reducing endothelial endoglin expression in vitro affects endothelial cell activation and monocyte adhesion and, if so, which intracellular pathways are involved. Finally, we analyzed whether glomerular endoglin expression is correlated with endothelial cell activation in patients with diabetic nephropathy. Endoglin levels were significantly increased in mice with type 1 diabetes compared with control mice. Reducing endoglin expression in cultured endothelial cells significantly impaired the vascular endothelial growth factor-A-induced up-regulation of activation markers and monocyte adhesion. This was mediated by increased phosphorylation of Akt, thereby inhibiting activating transcription factor 2 phosphorylation, which regulates vascular cell adhesion molecule-1 (VCAM1) gene transcription in these cells. Last, endoglin colocalized with VCAM-1 in the glomeruli of diabetic patients, glomerular VCAM-1 expression was significantly increased in these patients, and this increase in VCAM-1 expression was correlated with increased glomerular endoglin expression. Thus, targeting endoglin function may have therapeutic value in patients at risk for diabetic nephropathy.

Nephrin Loss Can Be Used to Predict Remission and Long-term Renal Outcome in Patients With Minimal Change Disease.

INTRODUCTION: Minimal change disease is a common cause of nephrotic syndrome. In general, patients with minimal change disease respond to corticosteroids and have excellent long-term renal survival. However, some patients have less favorable outcome. These patients are often thought to have progressed to focal segmental glomerulosclerosis. We previously reported that a segmental loss of podocyte markers is present before the development of focal segmental glomerulosclerosis in a rat model. Here, we investigated whether loss of podocyte marker nephrin can serve as a biomarker for predicting poor outcome in patients with minimal change disease. METHODS: We obtained 47 kidney biopsy samples from patients diagnosed with minimal change disease and stained sections with periodic acid-Schiff and for nephrin. Nephrin loss was scored by 2 independent researchers who were blinded to clinical outcome. Clinical data were collected retrospectively, and nephrin loss was correlated with clinical follow-up data. RESULTS: Nephrin loss was present in 34% of the biopsy samples. During follow-up, patients with nephrin loss achieved remission less frequently (61%) compared to patients without (96%) (P = 0.002). Moreover, 5-year eGFR was lower in the patients with renal nephrin loss. The risk of eGFR decreasing to < 60 ml/min per 1.73m2 increased with each percentage of glomeruli with nephrin loss (hazard ratio = 1.044, 95% confidence interval = 1.02-1.07). CONCLUSION: These results indicate that nephrin loss in patients with minimal change disease can help predict both remission and long-term renal outcome.

The VEGF-A inhibitor sFLT-1 improves renal function by reducing endothelial activation and inflammation in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Animal models of diabetic nephropathy show increased levels of glomerular vascular endothelial growth factor (VEGF)-A, and several studies have shown that inhibiting VEGF-A in animal models of diabetes can prevent albuminuria and glomerular hypertrophy. However, in those studies, treatment was initiated before the onset of kidney damage. Therefore, the aim of this study was to investigate whether transfecting mice with the VEGF-A inhibitor sFlt-1 (encoding soluble fms-related tyrosine kinase 1) can reverse pre-existing kidney damage in a mouse model of type 1 diabetes. In addition, we investigated whether transfection with sFlt-1 can reduce endothelial activation and inflammation in these mice. METHODS: Subgroups of untreated 8-week-old female C57BL/6J control (n = 5) and diabetic mice (n = 7) were euthanised 5 weeks after the start of the experiment in order to determine the degree of kidney damage prior to treatment with sFLT-1. Diabetes was induced with three i.p. injections of streptozotocin (75 mg/kg) administered at 2 day intervals. Diabetic nephropathy was then investigated in diabetic mice transfected with sFlt-1 (n = 6); non-diabetic, non-transfected control mice (n = 5); non-diabetic control mice transfected with sFlt-1(n = 10); and non-transfected diabetic mice (n = 6). These mice were euthanised at the end of week 15. Transfection with sFlt-1 was performed in week 6. RESULTS: We found that transfection with sFlt-1 significantly reduced kidney damage by normalising albuminuria, glomerular hypertrophy and mesangial matrix content (i.e. glomerular collagen type IV protein levels) (p < 0.001). We also found that transfection with sFlt-1 reduced endothelial activation (p < 0.001), glomerular macrophage infiltration (p < 0.001) and glomerular TNF-α protein levels (p < 0.001). Finally, sFLT-1 decreased VEGF-A-induced endothelial activation in vitro (p < 0.001). CONCLUSIONS/INTERPRETATION: These results suggest that sFLT-1 might be beneficial in treating diabetic nephropathy by inhibiting VEGF-A, thereby reducing endothelial activation and glomerular inflammation, and ultimately reversing kidney damage.

A Randomized Phase II Study Adding Axitinib to Pemetrexed-Cisplatin in Patients with Malignant Pleural Mesothelioma: A Single-Center Trial Combining Clinical and Translational Outcomes.

INTRODUCTION: Mesothelioma often presents with a high vessel count and increased vascular growth factors levels. Interference with angiogenesis may therefore improve outcome. This study reports on clinical and translational parameters in patients treated with the small molecule tyrosine kinase inhibitor axitinib and chemotherapy. METHODS: Chemonaive patients with mesothelioma were eligible. Patients received pemetrexed (500 mg/m(2) every 3 weeks) and cisplatin (75 mg/m(2) every 3 weeks) and were randomized to receive axitinib daily (two 5-mg tablets on days 2-19) or observation. Before treatment and after three cycles of chemotherapy, a thoracoscopy was performed to evaluate vascular changes. RESULTS: Twenty-five patients were randomized after a successful lead-in with six patients who received axitinib. Median follow-up was 45 months. In all but one patient, it was feasible to perform a second thoracoscopy. However, there was more grade 3 or 4 neutropenia leading to pneumonia in the axitinib group. The rates of partial response and stable disease in the axitinib arm were 36% and 43% compared with 18% and 73% in the chemotherapy-only arm. Median progression-free survival and overall survival (5.8 and 18.9 months versus 8.3 and 18.5 months) were not different between the two groups. Axitinib reduced vessel number and vessel immaturation. Yet, the mRNA levels of a number of vascular growth factors, their receptors, serum VEGF levels, and activation of tissue vascular endothelial growth factor receptor 2 were increased. Gene expression of platelet-derived growth factor receptor beta, fms-related tyrosine kinase 1, and fms-related tyrosine kinase 4 even correlated with outcome. CONCLUSIONS: Axitinib was well tolerated in combination with cisplatin and pemetrexed. Despite the lack of a clinical benefit, axitinib reduced angiogenesis. Whether changes in differentially expressed growth factors in tissue and serum may serve as a biomarker needs further investigation.

Blood and lymphatic microvessel damage in irradiated human skin: The role of TGF-ß, endoglin and macrophages.

BACKGROUND AND PURPOSE: Microvascular damage is an important component of late radiation-induced morbidity. In our pre-clinical models, we demonstrated that repair of vessel injury is dependent on proper endoglin-mediated transforming growth factor-beta (TGF-ß) signalling and that it can be affected by infiltrating macrophages. We now wanted to extend these findings in irradiated patients, using skin as a model system, and assess whether bisphosphonates could modulate the response. MATERIALS AND METHODS: Paired skin biopsies from irradiated and non-irradiated sites were obtained from 48 breast cancer patients. In 8 patients, biopsies were repeated after 4months of bisphosphonate treatment. Immunohistochemistry was used to assess vascular alterations and leucocyte infiltration. Western Blot and qPCR were used to assess expression of growth factors and their receptors. RESULTS: Decreased blood vessel numbers at early time points were followed by increased endoglin expression and restoration of vessel number. Loss of small lymphatic vessels was associated with increased TGF-ß levels, whereas dilation of lymphatic vessels correlated with increased macrophage infiltration. Bisphosphonate treatment reduced leucocyte infiltration, but also prevented restoration of blood vessel numbers after irradiation. CONCLUSION: Radiation injury of the microvasculature is mediated through TGF-ß, whereas repair is modulated by the co-receptor endoglin and promoted by macrophages.

Thalidomide ameliorates inflammation and vascular injury but aggravates tubular damage in the irradiated mouse kidney.

PURPOSE: The late side effects of kidney irradiation include vascular damage and fibrosis, which are promoted by an irradiation-induced inflammatory response. We therefore treated kidney-irradiated mice with the anti-inflammatory and angiogenesis-modulating drug thalidomide in an attempt to prevent the development of late normal tissue damage and radiation nephropathy in the mouse kidney. METHODS AND MATERIALS: Kidneys of C57Bl/6 mice were irradiated with a single dose of 14 Gy. Starting from week 16 after irradiation, the mice were fed with thalidomide-containing chow (100 mg/kg body weight/day). Gene expression and kidney histology were analyzed at 40 weeks and blood samples at 10, 20, 30, and 40 weeks after irradiation. RESULTS: Thalidomide improved the vascular structure and vessel perfusion after irradiation, associated with a normalization of pericyte coverage. The drug also reduced infiltration of inflammatory cells but could not suppress the development of fibrosis. Irradiation-induced changes in hematocrit and blood urea nitrogen levels were not rescued by thalidomide. Moreover, thalidomide worsened tubular damage after irradiation and also negatively affected basal tubular function. CONCLUSIONS: Thalidomide improved the inflammatory and vascular side effects of kidney irradiation but could not reverse tubular toxicity, which probably prevented preservation of kidney function.

Bone marrow-derived macrophages incorporate into the endothelium and influence vascular and renal function after irradiation.

PURPOSE: We recently demonstrated that endoglin, an ancillary transforming growth factor beta (TGF-ß) receptor, modulates vascular damage and fibrosis formation and influences renal function after kidney irradiation. We also suggested that this was partially accomplished by endoglin-mediated regulation of cytokine production in macrophages. Endoglin is expressed on both endothelial cells and on activated macrophages. Therefore, in the current study, we addressed the respective contribution of altered endoglin levels in the different cellular compartments to the development of kidney toxicity after irradiation. MATERIALS AND METHODS: Female endoglin wild-type (Eng(+/+) or WT) or heterozygous (Eng(+/-) or HET) mice were subjected to total body irradiation (2 × 6 Gy with a 6-hour interval) followed by kidney irradiation (1 × 3 Gy). Recipient mice were then transplanted with 4 × 10E(6) green fluorescent protein heterozygous (GFP(+/-)) bone marrow cells from either Eng(+/+) or Eng(+/-) male donor mice. Chimerism was determined 6 weeks thereafter. Blood samples were taken every 10 weeks after irradiation and at sacrifice at 35 weeks. One kidney was used to isolate macrophages; the other kidney was used for histology and to determine cytokine and chemokine concentrations. RESULTS: In all treatment groups, the majority of infiltrating macrophages were bone marrow-derived and this was not altered by endoglin. Bone marrow cells accumulated in damaged tissue areas in the interstitium, but also incorporated into the vasculature. Reducing endoglin levels in macrophages, but not in the endothelium, led to improved renal function (hematocrit, blood urea nitrogen) after irradiation. This was probably promoted by lowered production of pro-inflammatory cytokines and chemokines in macrophages. Other measurements of tissue toxicity (pericyte coverage, fibrosis, damage score) were not altered by differential endoglin expression. CONCLUSIONS: Endoglin regulates pro-inflammatory macrophage properties thereby influencing vascular and renal function after kidney irradiation.

Endoglin haploinsufficiency attenuates radiation-induced deterioration of kidney function in mice.

BACKGROUND AND PURPOSE: Endoglin is a transforming growth receptor beta (TGF-ß) co-receptor, which plays a crucial role in the development of late normal tissue damage. Mice with halved endoglin levels (Eng(+/-) mice) develop less inflammation, vascular damage and fibrosis after kidney irradiation compared to their wild type littermates (Eng(+/+) mice). This study was aimed at investigating whether reduced tissue damage in Eng(+/-) mice also results in superior kidney function. MATERIAL AND METHODS: Kidneys of Eng(+/+) and Eng(+/-) mice were irradiated with a single dose of 14 Gy. Functional kidney parameters and kidney histology were analysed at 20, 30 and 40 weeks after irradiation. RESULTS: Eng(+/-) mice displayed improved kidney parameters (haematocrit, BUN) compared to Eng(+/+) mice at 40 weeks after irradiation. Irradiation of Eng(+/+) kidneys damaged the vascular network and led to an increase in PDGFR-ß positive cells, indicative of fibrosis-promoting myofibroblasts. Compared to Eng(+/+) kidneys, vascular perfusion and number of PDGFR-ß positive cells were reduced in Eng(+/-) control mice; however, this did not further deteriorate after irradiation. CONCLUSIONS: Taken together, we show that not only kidney morphology, but also kidney function is improved after irradiation in Eng(+/-) compared to Eng(+/+) mice.

The TGF-ß co-receptor endoglin regulates macrophage infiltration and cytokine production in the irradiated mouse kidney.

BACKGROUND AND PURPOSE: We previously showed that mice with reduced levels of the transforming growth factor-beta (TGF-ß) co-receptor endoglin (Eng(+/-) mice) develop less fibrosis and vascular damage after kidney irradiation than their wild type (Eng(+/+) mice) littermates; however, the underlying mechanism was unclear. Results from current studies suggest that this occurs via modulation of the radiation-induced inflammatory response. MATERIALS AND METHODS: Kidneys of Eng(+/+) and Eng(+/-) mice were irradiated with 16Gy. Mice were sacrificed at 20weeks after irradiation and gene expression and protein levels were analyzed. RESULTS: Kidney irradiation triggered the infiltration of macrophages in both Eng(+/+) and Eng(+/-) mice, however, levels of macrophage-produced cytokines interleukin 1 beta (Il1b) and interleukin 6 (Il6) were reduced in irradiated Eng(+/-) compared to Eng(+/+) mice. Double immuno-stainings confirmed that IL-6 was produced by macrophages, whereas IL-1ß was mainly detected in other cell types. Accordingly, inflammatory cell precursors derived from the bone marrow of Eng(+/-) mice showed impaired ability to express Il1b and Il6 compared to wild type mice. CONCLUSIONS: Endoglin promotes kidney inflammation after irradiation by regulating macrophage infiltration and interleukin production, thereby promoting pathogenic changes after radiation exposure.

ALK1 heterozygosity delays development of late normal tissue damage in the irradiated mouse kidney.

BACKGROUND AND PURPOSE: Activin receptor-like kinase 1 (ALK1) is a transforming growth factor ß (TGF-ß) receptor, which is mainly expressed in endothelial cells regulating proliferation and migration in vitro and angiogenesis in vivo. Endothelial cells also express the co-receptor endoglin, which modulates ALK1 effects on endothelial cells. Our previous studies showed that mice with reduced endoglin levels develop less irradiation-induced vascular damage and fibrosis, caused by an impaired inflammatory response. This study was aimed at investigating the role of ALK1 in late radiation toxicity. MATERIAL AND METHODS: Kidneys of ALK(+/+) and ALK1(+/-) mice were irradiated with 14 Gy. Mice were sacrificed at 10, 20, and 30 weeks after irradiation and gene expression and protein levels were analyzed. RESULTS: Compared to wild type littermates, ALK1(+/-) mice developed less inflammation and fibrosis at 20 weeks after irradiation, but displayed an increase in pro-inflammatory and pro-fibrotic gene expression at 30 weeks. In addition, ALK1(+/-) mice showed superior vascular integrity at 10 and 20 weeks after irradiation which deteriorated at 30 weeks coinciding with changes in the VEGF pathway. CONCLUSIONS: ALK1(+/-) mice develop a delayed normal tissue response by modulating the inflammatory response and growth factor expression after irradiation.