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1.  Whole slide images for primary diagnostics of urinary system pathology: a feasibility study 
Introduction: During the last decade, whole slide images (WSI) have been used in many areas of pathology such as teaching, research, digital archiving, teleconsultation and quality assurance testing. However, WSI have as yet not much been used for upfront diagnostics because of the lack of validation studies.
Objectives: The aim of this study was to test the feasibility of WSI for primary diagnosis of urinary tract pathology.
Materials and Methods: 100 consecutive urinary tract biopsies and resections which had been diagnosed conventionally between the years 2008-2009 were scanned at 20× magnification, and rediagnosed by two pathologists on WSI, having the original clinical information available, but blinded to the original diagnoses. Original and WSI diagnoses were compared and classified as concordant, slightly discordant (without clinical consequences) and discordant.
Results: Original and WSI based rediagnosis were concordant in 87% of the cases. Original and WSI diagnosis were slightly discordant in 8% of cases. Major discrepancies with clinical or prognostic implications were founded in only 5 cases. However, for 6 out of the 13 discrepancies, WSI based diagnoses were considered to be better than the original diagnoses.
Conclusion: Primary diagnostics of urinary tract specimens can be reliably done on WSI. Further improvements of image resolution may help to increase diagnostic accuracy and WSI acceptance in routine pathology.
doi:10.12861/jrip.2014.26
PMCID: PMC4301392  PMID: 25610886
Diagnostics; Digital pathology; Urinary pathology; Validation; Whole slide images
2.  Nephronophthisis-Associated CEP164 Regulates Cell Cycle Progression, Apoptosis and Epithelial-to-Mesenchymal Transition 
PLoS Genetics  2014;10(10):e1004594.
We recently reported that centrosomal protein 164 (CEP164) regulates both cilia and the DNA damage response in the autosomal recessive polycystic kidney disease nephronophthisis. Here we examine the functional role of CEP164 in nephronophthisis-related ciliopathies and concomitant fibrosis. Live cell imaging of RPE-FUCCI (fluorescent, ubiquitination-based cell cycle indicator) cells after siRNA knockdown of CEP164 revealed an overall quicker cell cycle than control cells, although early S-phase was significantly longer. Follow-up FACS experiments with renal IMCD3 cells confirm that Cep164 siRNA knockdown promotes cells to accumulate in S-phase. We demonstrate that this effect can be rescued by human wild-type CEP164, but not disease-associated mutants. siRNA of CEP164 revealed a proliferation defect over time, as measured by CyQuant assays. The discrepancy between accelerated cell cycle and inhibited overall proliferation could be explained by induction of apoptosis and epithelial-to-mesenchymal transition. Reduction of CEP164 levels induces apoptosis in immunofluorescence, FACS and RT-QPCR experiments. Furthermore, knockdown of Cep164 or overexpression of dominant negative mutant allele CEP164 Q525X induces epithelial-to-mesenchymal transition, and concomitant upregulation of genes associated with fibrosis. Zebrafish injected with cep164 morpholinos likewise manifest developmental abnormalities, impaired DNA damage signaling, apoptosis and a pro-fibrotic response in vivo. This study reveals a novel role for CEP164 in the pathogenesis of nephronophthisis, in which mutations cause ciliary defects coupled with DNA damage induced replicative stress, cell death, and epithelial-to-mesenchymal transition, and suggests that these events drive the characteristic fibrosis observed in nephronophthisis kidneys.
Author Summary
Nephronophthisis is a leading inherited cause of renal failure in children and young adults. This work contributes to understanding of the disease mechanism of nephronophthisis, which is characterized by multi-cystic and fibrotic kidneys. The genes mutated in patients with nephronophthisis all seem to encode proteins involved in cilia function, and some of them are recently reported to also function in DNA damage signaling. We investigated how loss of cilia and impaired DNA damage signaling could cause the excessive fibrosis seen in nephronophthisis. Studies during the past decade have focused on treating the cysts of this early-onset renal disease. However, we think that understanding and curing the fibrosis seen in these patients will provide new treatment opportunities. Our work gives insight into the orchestration of downstream effects on the cellular level after loss of nephronophthisis gene CEP164 as a result of loss of cilia and accumulating DNA damage signaling.
doi:10.1371/journal.pgen.1004594
PMCID: PMC4207587  PMID: 25340510
3.  Nephrectomy in Autosomal Dominant Polycystic Kidney Disease: A Patient with Exceptionally Large, Still Functioning Kidneys 
Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease. It is characterized by progressive cyst formation in both kidneys, often leading to end-stage kidney disease. Indications for surgical removal of an ADPKD kidney include intractable pain, hematuria, infection, or exceptional enlargement and small abdominal cavity hampering implantation of a donor kidney. We report the case of an extraordinarily large ADPKD kidney weighing 8.7 kg (19.3 lb) with a maximal length of 48 cm (19 inch), and with cysts filled with both clear and bloody fluid.
doi:10.1159/000363378
PMCID: PMC4086038  PMID: 25028584
Autosomal dominant polycystic kidney disease; Nephrectomy; Transplantation
4.  Left Ventricular Mass in Dialysis Patients, Determinants and Relation with Outcome. Results from the COnvective TRansport STudy (CONTRAST) 
PLoS ONE  2014;9(2):e84587.
Background and Objectives
Left ventricular mass (LVM) is known to be related to overall and cardiovascular mortality in end stage kidney disease (ESKD) patients. The aims of the present study are 1) to determine whether LVM is associated with mortality and various cardiovascular events and 2) to identify determinants of LVM including biomarkers of inflammation and fibrosis.
Design, Setting, Participants, & Measurements
Analysis was performed with data of 327 ESKD patients, a subset from the CONvective TRAnsport STudy (CONTRAST). Echocardiography was performed at baseline. Cox regression analysis was used to assess the relation of LVM tertiles with clinical events. Multivariable linear regression models were used to identify factors associated with LVM.
Results
Median age was 65 (IQR: 54–73) years, 203 (61%) were male and median LVM was 227 (IQR: 183–279) grams. The risk of all-cause mortality (hazard ratio (HR) = 1.73, 95% CI: 1.11–2.99), cardiovascular death (HR = 3.66, 95% CI: 1.35–10.05) and sudden death (HR = 13.06; 95% CI: 6.60–107) was increased in the highest tertile (>260grams) of LVM. In the multivariable analysis positive relations with LVM were found for male gender (B = 38.8±10.3), residual renal function (B = 17.9±8.0), phosphate binder therapy (B = 16.9±8.5), and an inverse relation for a previous kidney transplantation (B = −41.1±7.6) and albumin (B = −2.9±1.1). Interleukin-6 (Il-6), high-sensitivity C-reactive protein (hsCRP), hepcidin-25 and connective tissue growth factor (CTGF) were not related to LVM.
Conclusion
We confirm the relation between a high LVM and outcome and expand the evidence for increased risk of sudden death. No relationship was found between LVM and markers of inflammation and fibrosis.
Trial Registration
Controlled-Trials.com ISRCTN38365125
doi:10.1371/journal.pone.0084587
PMCID: PMC3914777  PMID: 24505249
5.  CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress 
CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2−/− chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.
Electronic supplementary material
The online version of this article (doi:10.1007/s12079-013-0201-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s12079-013-0201-y
PMCID: PMC3709047  PMID: 23666466
Cartilage; CCN2/CTGF; Chondrocytes; Cellular stress; Nuclear factor κB; Autophagy
6.  Vitreous TIMP-1 levels associate with neovascularization and TGF-β2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy 
In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor; CTGF) cause blindness by neovascularization and subsequent fibrosis. This angio-fibrotic switch is associated with a shift in the balance between vitreous levels of CCN2 and VEGF in the eye. Here, we investigated the possible involvement of other important mediators of fibrosis, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor (TGF)-β2, and of the matrix metalloproteinases (MMP)-2 and MMP-9, in the natural course of PDR. TIMP-1, activated TGF-β2, CCN2 and VEGF levels were measured by ELISA in 78 vitreous samples of patients with PDR (n = 28), diabetic patients without PDR (n = 24), and patients with the diabetes-unrelated retinal conditions macular hole (n = 10) or macular pucker (n = 16), and were related to MMP-2 and MMP-9 activity on zymograms and to clinical data, including degree of intra-ocular neovascularization and fibrosis. TIMP-1, CCN2 and VEGF levels, but not activated TGF-β2 levels, were significantly increased in the vitreous of diabetic patients, with the highest levels in PDR patients. CCN2 and the CCN2/VEGF ratio were the strongest predictors of degree of fibrosis. In diabetic patients with or without PDR, activated TGF-β2 levels correlated with TIMP-1 levels, whereas in PDR patients, TIMP-1 levels, MMP-2 and proMMP-9 were associated with degree of neovascularization, like VEGF levels, but not with fibrosis. We confirm here our previous findings that retinal fibrosis in PDR patients is significantly correlated with vitreous CCN2 levels and the CCN2/VEGF ratio. In contrast, TIMP-1, MMP-2 and MMP-9 appear to have a role in the angiogenic phase rather than in the fibrotic phase of PDR.
doi:10.1007/s12079-012-0178-y
PMCID: PMC3590360  PMID: 23054594
Diabetic retinopathy; CCN2; VEGF; TGF-β; TIMP-1; MMP-2; MMP-9; Neovascularization; Fibrosis
7.  FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair 
Nature genetics  2012;44(8):910-915.
SUMMARY
Chronic kidney disease (CKD) represents a major health burden1. Its central feature of renal fibrosis is not well understood. By whole exome resequencing in a model disorder for renal fibrosis, nephronophthisis (NPHP), we identified mutations of Fanconi anemia-associated nuclease 1 (FAN1) as causing karyomegalic interstitial nephritis (KIN). Renal histology of KIN is indistinguishable from NPHP except for the presence of karyomegaly2. FAN1 has nuclease activity, acting in DNA interstrand crosslinking (ICL) repair within the Fanconi anemia pathway of DNA damage response (DDR)3–6. We demonstrate that cells from individuals with FAN1 mutations exhibit sensitivity to the ICL agent mitomycin C. However, they do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from patients with Fanconi anemia. We complement ICL sensitivity with wild type FAN1 but not mutant cDNA from individuals with KIN. Depletion of fan1 in zebrafish revealed increased DDR, apoptosis, and kidney cysts akin to NPHP. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms of renal fibrosis and CKD.
doi:10.1038/ng.2347
PMCID: PMC3412140  PMID: 22772369
8.  Compensatory Growth of Congenital Solitary Kidneys in Pigs Reflects Increased Nephron Numbers Rather Than Hypertrophy 
PLoS ONE  2012;7(11):e49735.
Background
Patients with unilateral MultiCystic Kidney Dysplasia (MCKD) or unilateral renal agenesis (URA) have a congenital solitary functioning kidney (CSFK) that is compensatory enlarged. The question whether this enlargement is due to increased nephron numbers and/or to nephron hypertrophy is unresolved. This question is of utmost clinical importance, since hypertrophy is associated with a risk of developing hypertension and proteinuria later in life with consequent development of CKD and cardiovascular disease.
Methodology/Principal Findings
In a cohort of 32,000 slaughter pigs, 7 congenital solitary functioning kidneys and 7 control kidneys were identified and harvested. Cortex volume was measured and with a 3-dimensional stereologic technique the number and volume of glomeruli was determined and compared. The mean total cortex volume was increased by more than 80% and the mean number of glomeruli per kidney was 50% higher in CSFKs than in a single control kidney, equaling 75% of the total nephron number in both kidneys of control subjects. The mean total glomerular volume in the CSFKs was not increased relative to the controls.
Conclusions/Significance
Thus, in pigs, compensatory enlargement of a CSFK is based on increased nephron numbers. Extrapolation of these findings to the human situation suggests that patients with a CSFK might not be at increased risk for developing hyperfiltration-associated renal and cardiovascular disease in later life due to a lower nephron number.
doi:10.1371/journal.pone.0049735
PMCID: PMC3504144  PMID: 23185419
9.  Gain of glycosylation in integrin α3 causes lung disease and nephrotic syndrome 
The Journal of Clinical Investigation  2012;122(12):4375-4387.
Integrins are transmembrane αβ glycoproteins that connect the extracellular matrix to the cytoskeleton. The laminin-binding integrin α3β1 is expressed at high levels in lung epithelium and in kidney podocytes. In podocytes, α3β1 associates with the tetraspanin CD151 to maintain a functional filtration barrier. Here, we report on a patient homozygous for a novel missense mutation in the human ITGA3 gene, causing fatal interstitial lung disease and congenital nephrotic syndrome. The mutation caused an alanine-to-serine substitution in the integrin α3 subunit, thereby introducing an N-glycosylation motif at amino acid position 349. We expressed this mutant form of ITGA3 in murine podocytes and found that hyperglycosylation of the α3 precursor prevented its heterodimerization with β1, whereas CD151 association with the α3 subunit occurred normally. Consequently, the β1 precursor accumulated in the ER, and the mutant α3 precursor was degraded by the ubiquitin-proteasome system. Thus, these findings uncover a gain-of-glycosylation mutation in ITGA3 that prevents the biosynthesis of functional α3β1, causing a fatal multiorgan disorder.
doi:10.1172/JCI64100
PMCID: PMC3533548  PMID: 23114595
10.  A shift in the balance of vascular endothelial growth factor and connective tissue growth factor by bevacizumab causes the angiofibrotic switch in proliferative diabetic retinopathy 
Introduction
In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) may cause blindness by neovascularisation followed by fibrosis of the retina. It has previously been shown that a shift in the balance between levels of CTGF and VEGF in the eye is associated with this angiofibrotic switch. This study investigated whether anti-VEGF agents induce accelerated fibrosis in patients with PDR, as predicted by this model.
Methods
CTGF and VEGF levels were measured by ELISA in 52 vitreous samples of PDR patients, of which 24 patients had received intravitreal bevacizumab 1 week to 3 months before vitrectomy, and were correlated with the degree of vitreoretinal fibrosis as determined clinically and intra-operatively.
Results
CTGF correlated positively, and VEGF correlated negatively with the degree of fibrosis. The CTGF/VEGF ratio was the strongest predictor of fibrosis. Clinically, increased fibrosis was observed after intravitreal bevacizumab.
Conclusions
These results confirm that the CTGF/VEGF ratio is a strong predictor of vitreoretinal fibrosis in PDR, and show that intravitreal anti-VEGF treatment causes increased fibrosis in PDR patients. These findings provide strong support for the model that the balance of CTGF and VEGF determines the angiofibrotic switch, and identify CTGF as a possible therapeutic target in the clinical management of PDR.
doi:10.1136/bjophthalmol-2011-301005
PMCID: PMC3308470  PMID: 22289291
Angiogenesis; choroid; CTGF; diabetic retinopathy; drugs; fibrosis; imaging; macula; retina; VEGF; vitreous
11.  ACE Inhibition in Anti-Thy1 Glomerulonephritis Limits Proteinuria but Does Not Improve Renal Function and Structural Remodeling 
Nephron Extra  2012;2(1):9-16.
Background/Aims
ACE inhibitor (ACE-I) treatment effectively inhibits proteinuria and ameliorates the course of various renal diseases. In experimental glomerulonephritis, however, angiotensin II (AngII) infusion has also been shown to be renoprotective. We evaluated the long-term (28 days) course of anti-Thy1 glomerulonephritis in animals with suppressed AngII formation by ACE-I treatment.
Methods
Brown Norway rats received perindopril (2.8 mg/kg/day, n = 12), dihydropyridine calcium-antagonist amlodipine (Ca-A; 13 mg/kg/day, n = 6) or were left untreated (n = 14). All animals were monitored for blood pressure, proteinuria, and creatinine clearance after anti-Thy1 injection. Renal histology was assessed at day 7 and 28.
Results
Systolic blood pressure was equally reduced by ACE-I and Ca-A treatment. AngII suppression prevented development of proteinuria, but did not protect against glomerular microaneurysm formation or reduction in creatinine clearance. After resolution of the microaneurysms, animals with suppressed AngII production showed a modest increase in glomerulosclerosis and vasculopathic thickening of intrarenal vessels.
Conclusions
In anti-Thy1 glomerulonephritis, suppression of AngII formation does not protect against the induction of glomerular damage and is associated with mild aggravation of adverse renal fibrotic remodeling. Proteinuria, however, is effectively prevented by ACE-I treatment. Ca-A treatment did not affect the course of glomerulonephritis, indicating that ACE-I effects are blood pressure independent.
doi:10.1159/000335750
PMCID: PMC3318936  PMID: 22479264
Anti-Thy1 glomerulonephritis; Angiotensin II; ACE inhibitor; Glomerulosclerosis
12.  The CTGF -945GC polymorphism is not associated with plasma CTGF and does not predict nephropathy or outcome in type 1 diabetes 
The -945GC polymorphism (rs6918698) in the connective tissue growth factor gene promoter (CTGF/CCN-2) has been associated with end organ damage in systemic sclerosis. Because CTGF is important in progression of diabetic kidney disease, we investigated whether the -945GC polymorphism is associated with plasma CTGF level and outcome in type 1 diabetes.
The study cohort consisted of 448 diabetic nephropathy patients and 419 normoalbuminuric diabetic patients with complete data concerning renal function and cardiovascular characteristics. Genomic DNA was genotyped by a QPCR-based SNP assay. We observed no relation between the -945GC polymorphism and plasma CTGF level, and the genotype frequencies were not different in nephropathy patients vs. normoalbuminuric controls. General and cardiovascular mortality, and renal function decline was similar in patients with CC, CG or GG genotypes.
In conclusion, the -945GC SNP does not affect plasma CTGF levels, incidence and prognosis of diabetic nephropathy, and cardiovascular outcome.
doi:10.1186/1477-5751-10-4
PMCID: PMC3112427  PMID: 21548990
13.  Modulation of TGF-β/BMP-6 expression and increased levels of circulating smooth muscle progenitor cells in a type I diabetes mouse model 
Background
Diabetic patients experience exaggerated intimal hyperplasia after endovascular procedures. Recently it has been shown that circulating smooth muscle progenitor cells (SPC) contribute to intimal hyperplasia. We hypothesized that SPC differentiation would be increased in diabetes and focused on modulation of TGF-β/BMP-6 signaling as potential underlying mechanism.
Methods
We isolated SPC from C57Bl/6 mice with streptozotocin-induced diabetes and controls. SPC differentiation was evaluated by immunofluorescent staining for αSMA and collagen Type I. SPC mRNA expression of TGF-β and BMP-6 was quantified using real-time PCR. Intima formation was assessed in cuffed femoral arteries. Homing of bone marrow derived cells to cuffed arterial segments was evaluated in animals transplanted with bone marrow from GFP-transgenic mice.
Results
We observed that SPC differentiation was accelerated and numeric outgrowth increased in diabetic animals (24.6 ± 8.8 vs 8.3 ± 1.9 per HPF after 10 days, p < 0.05). Quantitative real-time PCR showed increased expression of TGF-β and decreased expression of the BMP-6 in diabetic SPC. SPC were MAC-3 positive, indicative of monocytic lineage. Intima formation in cuffed arterial segments was increased in diabetic mice (intima/media ratio 0.68 ± 0.15 vs 0.29 ± 0.06, p < 0.05). In GFP-chimeric mice, bone marrow derived cells were observed in the neointima (4.4 ± 3.3 cells per section) and particularly in the adventitia (43.6 ± 9.3 cells per section). GFP-positive cells were in part MAC-3 positive, but rarely expressed α-SMA.
Conclusions
In conclusion, in a diabetic mouse model, SPC levels are increased and SPC TGF-β/BMP-6 expression is modulated. Altered TGF-β/BMP-6 expression is known to regulate smooth muscle cell differentiation and may facilitate SPC differentiation. This may contribute to exaggerated intimal hyperplasia in diabetes as bone marrow derived cells home to sites of neointima formation.
doi:10.1186/1475-2840-9-55
PMCID: PMC2954908  PMID: 20858224
14.  Deletion of Irs2 causes reduced kidney size in mice: role for inhibition of GSK3β? 
Background
Male Irs2-/- mice develop fatal type 2 diabetes at 13-14 weeks. Defects in neuronal proliferation, pituitary development and photoreceptor cell survival manifest in Irs2-/- mice. We identify retarded renal growth in male and female Irs2-/- mice, independent of diabetes.
Results
Kidney size and kidney:body weight ratio were reduced by approximately 20% in Irs2-/- mice at postnatal day 5 and was maintained in maturity. Reduced glomerular number but similar glomerular density was detected in Irs2-/- kidney compared to wild-type, suggesting intact global kidney structure. Analysis of insulin signalling revealed renal-specific upregulation of PKBβ/Akt2, hyperphosphorylation of GSK3β and concomitant accumulation of β-catenin in Irs2-/- kidney. Despite this, no significant upregulation of β-catenin targets was detected. Kidney-specific increases in Yes-associated protein (YAP), a key driver of organ size were also detected in the absence of Irs2. YAP phosphorylation on its inhibitory site Ser127 was also increased, with no change in the levels of YAP-regulated genes, suggesting that overall YAP activity was not increased in Irs2-/- kidney.
Conclusions
In summary, deletion of Irs2 causes reduced kidney size early in mouse development. Compensatory mechanisms such as increased β-catenin and YAP levels failed to overcome this developmental defect. These data point to Irs2 as an important novel mediator of kidney size.
doi:10.1186/1471-213X-10-73
PMCID: PMC2910663  PMID: 20604929
15.  Allelic Depletion of grem1 Attenuates Diabetic Kidney Disease 
Diabetes  2009;58(7):1641-1650.
OBJECTIVE
Gremlin (grem1) is an antagonist of the bone morphogenetic protein family that plays a key role in limb bud development and kidney formation. There is a growing appreciation that altered grem1 expression may regulate the homeostatic constraints on damage responses in diseases such as diabetic nephropathy.
RESEARCH DESIGN AND METHODS
Here we explored whether knockout mice heterozygous for grem1 gene deletion (grem1+/−) exhibit protection from the progression of diabetic kidney disease in a streptozotocin-induced model of type 1 diabetes.
RESULTS
A marked elevation in grem1 expression was detected in the kidneys and particularly in kidney tubules of diabetic wild-type mice compared with those of littermate controls. In contrast, diabetic grem1+/− mice displayed a significant attenuation in grem1 expression at 6 months of diabetes compared with that in age- and sex-matched wild-type controls. Whereas the onset and induction of diabetes were similar between grem1+/− and wild-type mice, several indicators of diabetes-associated kidney damage such as increased glomerular basement membrane thickening and microalbuminuria were attenuated in grem1+/− mice compared with those in wild-type controls. Markers of renal damage such as fibronectin and connective tissue growth factor were elevated in diabetic wild-type but not in grem1+/− kidneys. Levels of pSmad1/5/8 decreased in wild-type but not in grem1+/− diabetic kidneys, suggesting that bone morphogenetic protein signaling may be maintained in the absence of grem1.
CONCLUSIONS
These data identify grem1 as a potential modifier of renal injury in the context of diabetic kidney disease.
doi:10.2337/db08-1365
PMCID: PMC2699858  PMID: 19401426
16.  Loss of the BMP antagonist USAG-1 ameliorates disease in a mouse model of the progressive hereditary kidney disease Alport syndrome 
The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney. Mutations involving any of the collagen IV genes (COL4A3, COL4A4, and COL4A5) affect GBM assembly and cause Alport syndrome, a progressive hereditary kidney disease with no definitive therapy. Previously, we have demonstrated that the bone morphogenetic protein (BMP) antagonist uterine sensitization–associated gene-1 (USAG-1) negatively regulates the renoprotective action of BMP-7 in a mouse model of tubular injury during acute renal failure. Here, we investigated the role of USAG-1 in renal function in Col4a3–/– mice, which model Alport syndrome. Ablation of Usag1 in Col4a3–/– mice led to substantial attenuation of disease progression, normalization of GBM ultrastructure, preservation of renal function, and extension of life span. Immunohistochemical analysis revealed that USAG-1 and BMP-7 colocalized in the macula densa in the distal tubules, lying in direct contact with glomerular mesangial cells. Furthermore, in cultured mesangial cells, BMP-7 attenuated and USAG-1 enhanced the expression of MMP-12, a protease that may contribute to GBM degradation. These data suggest that the pathogenetic role of USAG-1 in Col4a3–/– mice might involve crosstalk between kidney tubules and the glomerulus and that inhibition of USAG-1 may be a promising therapeutic approach for the treatment of Alport syndrome.
doi:10.1172/JCI39569
PMCID: PMC2827946  PMID: 20197625
17.  The Angio-Fibrotic Switch of VEGF and CTGF in Proliferative Diabetic Retinopathy 
PLoS ONE  2008;3(7):e2675.
Background
In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) cause blindness by neovascularization and subsequent fibrosis, but their relative contribution to both processes is unknown. We hypothesize that the balance between levels of pro-angiogenic VEGF and pro-fibrotic CTGF regulates angiogenesis, the angio-fibrotic switch, and the resulting fibrosis and scarring.
Methods/Principal Findings
VEGF and CTGF were measured by ELISA in 68 vitreous samples of patients with proliferative DR (PDR, N = 32), macular hole (N = 13) or macular pucker (N = 23) and were related to clinical data, including degree of intra-ocular neovascularization and fibrosis. In addition, clinical cases of PDR (n = 4) were studied before and after pan-retinal photocoagulation and intra-vitreal injections with bevacizumab, an antibody against VEGF. Neovascularization and fibrosis in various degrees occurred almost exclusively in PDR patients. In PDR patients, vitreous CTGF levels were significantly associated with degree of fibrosis and with VEGF levels, but not with neovascularization, whereas VEGF levels were associated only with neovascularization. The ratio of CTGF and VEGF was the strongest predictor of degree of fibrosis. As predicted by these findings, patients with PDR demonstrated a temporary increase in intra-ocular fibrosis after anti-VEGF treatment or laser treatment.
Conclusions/Significance
CTGF is primarily a pro-fibrotic factor in the eye, and a shift in the balance between CTGF and VEGF is associated with the switch from angiogenesis to fibrosis in proliferative retinopathy.
doi:10.1371/journal.pone.0002675
PMCID: PMC2443281  PMID: 18628999
18.  Antimyeloperoxidase antibodies in the Churg-Strauss syndrome 
Thorax  1991;46(1):70-71.
Antibodies to myeloperoxidase were detected in the serum of three patients with the Churg-Strauss syndrome.
PMCID: PMC1020922  PMID: 1651566

Results 1-18 (18)