CCN2 reduction mediates protective effects of BMP7 treatment in obstructive nephropathy

Treatment with rhBMP7 exerts profound protective effects in a wide variety of experimental models of renal disease. However, little is known about how these protective effects are mediated, and which cells in the kidney are targeted by exogenous rhBMP7 treatment. To determine if rhBMP7 increases glomerular and tubulointerstitial canonical BMP signaling, we performed Unilateral Ureteral Obstruction w(UUO, a widely used obstructive nephropathy model) in mice reporting transcriptional activity downstream of canonical BMP signaling by the expression of GFP under the BMP Responsive Element of the Id1 promoter (BRE:gfp mice). We also analysed the impact of rhBMP7 treatment on severity of the UUO phenotype, on TGFβ signaling, and on expression of CCN2 (CTGF). Despite profound protective effects with respect to morphological damage, macrophage infiltration, and fibrosis, no significant difference in GFP-expression was observed upon rhBMP7 administration. Also TGFβ signalling was similar in rhBMP7 and vehicle treated mice, but CCN2 expression in obstructed kidneys was significantly reduced by rhBMP7 treatment. Of note, in heterozygous CCN2 mice (CCN2+/−) treatment with rhBMP7 did not (further) reduce the severity of kidney damage in the UUO-model. These data suggest that protection against obstructive nephropathy by exogenous rhBMP7 treatment relies primarily on non-canonical BMP signaling, and may be mediated in large part by downregulation of CCN2 expression

Targeting podocyte-associated diseases

Injury to the podocytes is the initiating cause of many renal diseases, leading to proteinuria with possible progression to end-stage renal disease. Podocytes are highly specialized cells, with an important role in maintaining the glomerular filtration barrier and producing growth factors for both mesangial cells and endothelial cells. With their foot processes they cover the glomerular basement membrane, and form slit diaphragms with neighboring podocytes. Human podocytopathies include focal and segmental glomerulosclerosis, minimal change disease, membranous nephropathy, collapsing glomerulopathy and diabetic nephropathy. Research in the last two decades has demonstrated great progress in understanding the molecular mechanisms leading to podocytopathies. These include single gene defects in slit diaphragm proteins, but also discovery of apoptotic, enzymatic and other pathways involved in podocyte injury. With this progress, a great number of animal models is now available to study either specific podocytopathies, e.g. in mouse models with single gene mutations, or more general podocyte injury patterns, such as the lipopolysaccharide or protamine sulfate model of foot process effacement. In this review, the morphology of the glomerulus will be discussed, with a focus on the podocyte, its interactions with surrounding cells, and the highly differentiated slit diaphragm separating the apical from the basal membrane. We also provide an overview of human podocytopathies and animal models to study these diseases. In the last part we discuss targeted therapies addressing pathways and proteins affected in podocyte injury

BMP Signaling and Podocyte Markers Are Decreased in Human Diabetic Nephropathy in Association With CTGF Overexpression

Diabetic nephropathy is characterized by decreased expression of bone morphogenetic protein-7 (BMP-7) and decreased podocyte number and differentiation. Extracellular antagonists such as connective tissue growth factor (CTGF; CCN-2) and sclerostin domain-containing-1 (SOSTDC1; USAG-1) are important determinants of BMP signaling activity in glomeruli. We studied BMP signaling activity in glomeruli from diabetic patients and non-diabetic individuals and from control and diabetic CTGF+/+ and CTGF+/− mice. BMP signaling activity was visualized by phosphorylated Smad1, -5, and -8 (pSmad1/5/8) immunostaining, and related to expression of CTGF, SOSTDC1, and the podocyte differentiation markers WT1, synaptopodin, and nephrin. In control and diabetic glomeruli, pSmad1/5/8 was mainly localized in podocytes, but both number of positive cells and staining intensity were decreased in diabetes. Nephrin and synaptopodin were decreased in diabetic glomeruli. Decrease of pSmad1/5/8 was only partially explained by decrease in podocyte number. SOSTDC1 and CTGF were expressed exclusively in podocytes. In diabetic glomeruli, SOSTDC1 decreased in parallel with podocyte number, whereas CTGF was strongly increased. In diabetic CTGF+/− mice, pSmad1/5/8 was preserved, compared with diabetic CTGF+/+ mice. In conclusion, in human diabetic nephropathy, BMP signaling activity is diminished, together with reduction of podocyte markers. This might relate to concomitant overexpression of CTGF but not SOSTDC1. (J Histochem Cytochem 57:623–631, 2009

Elevated Urinary Connective Tissue Growth Factor in Diabetic Nephropathy Is Caused by Local Production and Tubular Dysfunction

Connective tissue growth factor (CTGF; CCN2) plays a role in the development of diabetic nephropathy (DN). Urinary CTGF (uCTGF) is elevated in DN patients and has been proposed as a biomarker for disease progression, but it is unknown which pathophysiological factors contribute to elevated uCTGF. We studied renal handling of CTGF by infusion of recombinant CTGF in diabetic mice. In addition, uCTGF was measured in type 1 DN patients and compared with glomerular and tubular dysfunction and damage markers. In diabetic mice, uCTGF was increased and fractional excretion (FE) of recombinant CTGF was substantially elevated indicating reduced tubular reabsorption. FE of recombinant CTGF correlated with excretion of endogenous CTGF. CTGF mRNA was mainly localized in glomeruli and medullary tubules. Comparison of FE of endogenous and recombinant CTGF indicated that 60% of uCTGF had a direct renal source, while 40% originated from plasma CTGF. In DN patients, uCTGF was independently associated with markers of proximal and distal tubular dysfunction and damage. In conclusion, uCTGF in DN is elevated as a result of both increased local production and reduced reabsorption due to tubular dysfunction. We submit that uCTGF is a biomarker reflecting both glomerular and tubulointerstitial hallmarks of diabetic kidney disease

Hemizygous deletion of CTGF/CCN2 does not suffice to prevent fibrosis of the severely injured kidney

Background: Connective Tissue Growth Factor (CTGF/CCN2) is an important mediator of kidney fibrosis. Previous observations indicated that attenuation of CCN2 expression sufficed to alleviate early kidney damage. However, little is known about the role of CCN2 in fibrosis of severely damaged and more chronically injured kidneys. Therefore, we examined the effects of CCN2 haploinsufficiency on the progression of renal scarring in long-term STZ-induced diabetic nephropathy, in a more advanced stage of obstructive nephropathy following unilateral ureteric obstruction (UUO), and in severe aristolochic acid (AA)-induced tubulotoxic nephritis. Methods: Wild-type (WT, CCN2(+/+)) and hemizygous CCN2(+/-) C57BI/6 mice were studied. In the diabetes experiment, streptozotocin-injected and control mice were followed for 6 months, with regular blood pressure, glycaemia and albuminuria recordings. In the UUO experiment, the left ureter was obstructed for 14 days with the contralateral kidney serving as control. For the AA experiment, mice were followed for 25 days after 5 intraperitoneal injections with AA and compared to control mice injected with buffer alone. Organs were harvested for histology, mRNA and protein measurements. Collagen content was determined by HPLC and expressed as hydroxyproline/proline ratio. Results: CCN2 expression was significantly increased in the damaged as compared to control kidneys. In all three models, CCN2 levels in the damaged kidneys of CCN2(+/-) mice averaged about 50% of those in damaged WT kidneys. After 6 months of diabetes, albuminuria was increased 2.5-fold in WT mice, compared to 1.5-fold in CCN2(+/-) mice, mesangial matrix was expanded 5-fold in WT and 4.4-fold in CCN2(+/-) mice and the glomerular basement membrane was thickened 1.3-fold in WT and 1.5-fold in CCN2(+/-) mice (all differences between WT and CCN2(+/-) mice are NS). Tubular damage and interstitial fibrosis scores were also not different between Wt and CCN2(+/-) mice in the diabetes (1.8 vs. 1.7), UUO (2.8 vs. 2.6). and AA (1.4 vs. 1.2) models, as was the case for macrophage influx and collagen content in these three models. Conclusion: Unlike in mild and relatively early STZ-induced diabetic nephropathy, scarring of severely and chronically damaged kidneys is not attenuated by a 50% reduction of CCN2 to (near) normal levels. This suggests that CCN2 is either redundant in severe and chronic kidney disease, or that it is a limiting factor only at subnormal concentrations requiring further reduction by available or emerging therapies to prevent fibrosis of the severely injured kidney. (c) 2012 Elsevier B.V. All rights reserve

Mutational analysis using Sanger and next generation sequencing in sporadic spindle cell hemangiomas : a study of 19 cases

Spindle cell hemangioma (SCH) is a distinct vascular soft-tissue lesion characterized by cavernous blood vessels and a spindle cell component mainly occurring in the distal extremities of young adults. The majority of cases harbor heterozygous mutations in IDH1/2 sporadically or rarely in association with Maffucci syndrome. However, based on mosaicism and accordingly a low percentage of lesional cells harboring a mutant allele, detection can be challenging. We tested 19 sporadic SCHs by Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), conventional next generation sequencing (NGS), and NGS using a single molecule molecular inversion probes (smMIP)-based library preparation to compare their diagnostic value. Out of 10 cases tested by Sanger sequencing and 2 analyzed using MLPA, 4 and 1, respectively, revealed a mutation in IDH1 (p.R132C). The 7 remaining negative cases and additional 6 cases were investigated using smMIP/NGS, showing hot spot mutations in IDH1 (p.R132C) (8 cases) and IDH2 (3 cases; twice p.R172S and once p.R172G, respectively). One case was negative. Owing to insufficient DNA quality and insufficient coverage, 2 cases were excluded. In total, in 16 out of 17 cases successfully tested, an IDH1/2 mutation was found. Given that IDH1/2 mutations were absent in 161 other vascular lesions tested by smMIP/NGS, the mutation can be considered as highly specific for SCH

Connective tissue growth factor is involved in structural retinal vascular changes in long-term experimental diabetes

Early retinal vascular changes in the development of diabetic retinopathy (DR) include capillary basal lamina (BL) thickening, pericyte loss and the development of acellular capillaries. Expression of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family member CCN2 or connective tissue growth factor (CTGF), a potent inducer of the expression of BL components, is upregulated early in diabetes. Diabetic mice lacking one functional CTGF allele (CTGF⁺/⁻) do not show this BL thickening. As early events in DR may be interrelated, we hypothesized that CTGF plays a role in the pathological changes of retinal capillaries other than BL thickening. We studied the effects of long-term (6-8 months) streptozotocin-induced diabetes on retinal capillary BL thickness, numbers of pericytes and the development of acellular capillaries in wild type and CTGF⁺/⁻ mice. Our results show that an absence of BL thickening of retinal capillaries in long-term diabetic CTGF⁺/⁻ mice is associated with reduced pericyte dropout and reduced formation of acellular capillaries. We conclude that CTGF is involved in structural retinal vascular changes in diabetic rodents. Inhibition of CTGF in the eye may therefore be protective against the development of D

Prognostic value of histopathological DCIS features in a large-scale international interrater reliability study

PURPOSE: For optimal management of ductal carcinoma in situ (DCIS), reproducible histopathological assessment is essential to distinguish low-risk from high-risk DCIS. Therefore, we analyzed interrater reliability of histopathological DCIS features and assessed their associations with subsequent ipsilateral invasive breast cancer (iIBC) risk. METHODS: Using a case-cohort design, reliability was assessed in a population-based, nationwide cohort of 2767 women with screen-detected DCIS diagnosed between 1993 and 2004, treated by breast-conserving surgery with/without radiotherapy (BCS ± RT) using Krippendorff's alpha (KA) and Gwet's AC2 (GAC2). Thirty-eight raters scored histopathological DCIS features including grade (2-tiered and 3-tiered), growth pattern, mitotic activity, periductal fibrosis, and lymphocytic infiltrate in 342 women. Using majority opinion-based scores for each feature, their association with subsequent iIBC risk was assessed using Cox regression. RESULTS: Interrater reliability of grade using various classifications was fair to moderate, and only substantial for grade 1 versus 2 + 3 when using GAC2 (0.78). Reliability for growth pattern (KA 0.44, GAC2 0.78), calcifications (KA 0.49, GAC2 0.70) and necrosis (KA 0.47, GAC2 0.70) was moderate using KA and substantial using GAC2; for (type of) periductal fibrosis and lymphocytic infiltrate fair to moderate estimates were found and for mitotic activity reliability was substantial using GAC2 (0.70). Only in patients treated with BCS-RT, high mitotic activity was associated with a higher iIBC risk in univariable analysis (Hazard Ratio (HR) 2.53, 95% Confidence Interval (95% CI) 1.05-6.11); grade 3 versus 1 + 2 (HR 2.64, 95% CI 1.35-5.14) and a cribriform/solid versus flat epithelial atypia/clinging/(micro)papillary growth pattern (HR 3.70, 95% CI 1.34-10.23) were independently associated with a higher iIBC risk. CONCLUSIONS: Using majority opinion-based scores, DCIS grade, growth pattern, and mitotic activity are associated with iIBC risk in patients treated with BCS-RT, but interrater variability is substantial. Semi-quantitative grading, incorporating and separately evaluating nuclear pleomorphism, growth pattern, and mitotic activity, may improve the reliability and prognostic value of these features.status: publishe

Prognostic value of histopathological DCIS features in a large-scale international interrater reliability study

Purpose: For optimal management of ductal carcinoma in situ (DCIS), reproducible histopathological assessment is essential to distinguish low-risk from high-risk DCIS. Therefore, we analyzed interrater reliability of histopathological DCIS features and assessed their associations with subsequent ipsilateral invasive breast cancer (iIBC) risk. Methods: Using a case-cohort design, reliability was assessed in a population-based, nationwide cohort of 2767 women with screen-detected DCIS diagnosed between 1993 and 2004, treated by breast-conserving surgery with/without radiotherapy (BCS ± RT) using Krippendorff’s alpha (KA) and Gwet’s AC2 (GAC2). Thirty-eight raters scored histopathological DCIS features including grade (2-tiered and 3-tiered), growth pattern, mitotic activity, periductal fibrosis, and lymphocytic infiltrate in 342 women. Using majority opinion-based scores for each feature, their association with subsequent iIBC risk was assessed using Cox regression. Results: Interrater reliability of grade using various classifications was fair to moderate, and only substantial for grade 1 versus 2 + 3 when using GAC2 (0.78). Reliability for growth pattern (KA 0.44, GAC2 0.78), calcifications (KA 0.49, GAC2 0.70) and necrosis (KA 0.47, GAC2 0.70) was moderate using KA and substantial using GAC2; for (type of) periductal fibrosis and lymphocytic infiltrate fair to moderate estimates were found and for mitotic activity reliability was substantial using GAC2 (0.70). Only in patients treated with BCS-RT, high mitotic activity was associated with a higher iIBC risk in univariable analysis (Hazard Ratio (HR) 2.53, 95% Confidence Interval (95% CI) 1.05–6.11); grade 3 versus 1 + 2 (HR 2.64, 95% CI 1.35–5.14) and a cribriform/solid versus flat epithelial atypia/clinging/(micro)papillary growth pattern (HR 3.70, 95% CI 1.34–10.23) were independently associated with a higher iIBC risk. Conclusions: Using majority opinion-based scores, DCIS grade, growth pattern, and mitotic activity are associated with iIBC risk in patients treated with BCS-RT, but interrater variability is substantial. Semi-quantitative grading, incorporating and separately evaluating nuclear pleomorphism, growth pattern, and mitotic activity, may improve the reliability and prognostic value of these features

Loss of Endogenous Bone Morphogenetic Protein-6 Aggravates Renal Fibrosis

Bone morphogenetic protein-6 (BMP-6) suppresses inflammatory genes in renal proximal tubular cells and regulates iron metabolism by inducing hepcidin. In diabetic patients, an increase of myofibroblast progenitor cells (MFPCs), also known as fibrocytes, was found to be associated with decreased BMP-6 expression. We hypothesized that loss of endogenous BMP-6 would aggravate renal injury and fibrosis. Wild type (WT) and BMP-6 null mice underwent unilateral ureteral obstruction. In WT mice, ureteral obstruction down-regulated BMP-6. Obstructed kidneys of BMP-6 null mice showed more casts (1.5-fold), epithelial necrosis (1.4-fold), and brush border loss (1.3-fold). This was associated with more inflammation (1.8-fold more CD45+ cells) and more pronounced overexpression of profibrotic genes for αSMA (2.0-fold), collagen I (6.8-fold), fibronectin (4.3-fold), CTGF (1.8-fold), and PAI-1 (3.8-fold), despite similar BMP-7 expression. Also, 1.3-fold more MFPCs were obtained from BMP-6 null than from WT mononuclear cell cultures, but in vivo only very few MFPCs were observed in obstructed kidneys, irrespective of BMP-6 genotype. The obstructed kidneys of BMP-6 null mice showed 2.2-fold more iron deposition, in association with 3.3-fold higher expression of the oxidative stress marker HO-1. Thus, ureteral obstruction leads to down-regulation of BMP-6 expression, and BMP-6 deficiency aggravates tubulointerstitial damage and fibrosis independent of BMP-7. This process appears to involve loss of both direct anti-inflammatory and antifibrotic action and indirect suppressive effects on renal iron deposition, oxidative stress, and MFPCs