Areas Of Interstitial Fibrosis Research Articles

Effect of growth hormone on endometrium growth of intrauterine adhesion and the underlying mechanism.

The main treatment for intrauterine adhesion (IUA) is hysteroscopic adhesiolysis (HA), which most of treatment frequently employs estrogen and progesterone cycle therapy. The growth and coverage of endometrium after operation is a difficult problem, and several hospitals in China have performed growth hormone (GH) in empirically treating IUA, which has achieved excellent curative effects. Unfortunately, the mechanism of action has not yet been clearly elucidated. In previous study, an IUA animal model after surgical abortion and curettage in pregnant rats has been successfully established. In this experiment, the IUA animal model after surgical abortion and curettage in pregnant rats, which is more in line with the mechanism of human intrauterine adhesion, was used for the first time to investigate the therapeutic effect of GH on IUA in the pregnant rat curettage model. The expression of signal transducers and activators of transcription 3(STAT3), phosphorylated STAT3 (p-STAT3), STAT5 and p-STAT5 content were detected by immunohistochemistry to preliminarily explore the possible mechanism of GH involving in promoting endometrial growth of IUA, and to provide a theoretical basis for clinical medication and treatment. Pregnant rats were anesthetized, and the bilateral embryos were removed completely. Then the rat endometrium was scraped with a curette in 4 different directions (front, back, left, and right). After the IUA animal model was established, the rats were randomly divided into 3 groups (n=5): a control group, a GH group, and a GH + AG490 group. Normal saline (0.4 mL/100 g) was injected subcutaneously at the 7th day after curettage in the control group；0.15 U/100 g of GH was injected subcutaneously at the 7th day after curettage in the GH group; 0.15 U/100 g of GH was injected subcutaneously and 1 mg/100 g AG490 was injected intraperitoneally at the 7th day after curettage in the GH+ AG490 group. All the rats were injected continuously for 5 days. The rats in each group were sacrificed at the 14th day. The uterus of rats in each group was stained with HE staining to explore the endometrial morphology and the number of endometrial glands in each group, and Masson staining was utilized to observe the degree of endometrial fibrosis. The levels of STAT3, p-STAT3, STAT5 and p-STAT5 were detected by immunohistochemistry. 1) The number of glands in the GH group was more than that in the control group on the 14th day, with statistical difference (P<0.05). However, the number of endometrial glands in the AG490+GH group was decreased compared with the GH group on the 14th day (P<0.05). 2) The fibrosis ratio in the GH group was less than that in the control group at the 14th day after operation (P<0.05). However, the area of endometrial interstitial fibrosis in the AG490+GH group was much higher than that in the GH group 14 days after operation (P<0.05). 3) Compared with the control group, there was not significant difference in the levels of STAT3 and STAT5 in GH group (both P>0.05), while the levels of protein p-STAT3 and p-STAT5 were increased in the GH group (both P<0.05). Compared with the GH group, there was not significant difference in the levels of STAT3 and STAT5 in the AG490+GH group (both P>0.05), while the levels of p-STAT3 and p-STAT5 were decreased in the AG490+GH group (both P<0.05). GH can not only promote the growth of endometrial glands in the IUA model, but also reduce the degree of fibrosis and play a role in the treatment of IUA, which may be related to the activation of the Janus kinase (JAK), JAK/STAT3 and STAT5 signaling pathways.

Abstract 10279: Three-Dimensional Deep-Tissue Imaging of the Right Ventricle Reveals the Complex Remodeling of the Microvascular Network in Right Heart Failure

Introduction: Right ventricular (RV) failure is the leading cause of death in patients with pulmonary arterial hypertension (PAH). Capillary rarefaction has been proposed as the hallmark of RV failure, yet our understanding of the capillary architecture is limited by 2D image analysis. Three-dimensional (3D) confocal deep tissue imaging offers the unparalleled opportunity to characterize the architectural microvascular changes in relationship to cardiomyocytes in pressure-overloaded RV failure. Methods and Results: 250 μm heart tissue slices from mice with RV failure, 7-weeks after Pulmonary Artery Banding (PAB) were harvested and the microvascular network was assessed for density, segment length, diameter, orientation, as well as microvasculature-cardiomyocyte contact area in regions with and without interstitial fibrosis using confocal 3D imaging. Human heart tissue from end-stage PAH patients was compared to controls with normal RV function. In PAB mice, 3D imaging of the RV free wall revealed tortuous, shorter, thicker and higher-branched microvascular segments as well as development of fibrosis compared to Sham animals (Fig A). When corrected for fibrosis, the microvascular density was preserved even in advanced stages of RV failure. Physical contact between the microvasculature and cardiomyocytes was preserved in areas without fibrosis, yet significantly impaired in areas with interstitial fibrosis (Fig B), accompanied by an overexpression of β-myosin heavy chain suggesting local ischemia (Fig C). 3D imaging of human RV tissue of end-stage PAH patients revealed architectural microvascular remodeling that varied depending on etiologies and length of disease yet showed preserved microvascular density. (Fig. D). Conclusions: 3D deep tissue imaging revealed compensatory changes of the microvascular network to maintain a stable cardiomyocyte coverage in pressure-overloaded RV failure, which was impaired in areas of interstitial fibrosis.

315.8: The Molecular Nature of the Banff iIFTA Lesion

Introduction: Inflammation within areas of interstitial fibrosis and tubular atrophy (iIFTA) have been linked to adverse outcomes in kidney transplantation through an association with T-cell immunity leading to altered renal parenchymal structures. To date there is no literature that describes changes in the transcriptome in transplant recipients with iIFTA lesions. Further, molecular evidence for the progression of the banff iIFTA lesion, within the first year of transplantation, remains absent from the literature. Herein we define molecular changes within kidney allografts with an iIFTA lesion. We further characterise molecular signatures that accurately describe the progression of iIFTA at 12-months on a 3-month protocol biopsy. Methods: We performed RNA sequencing of 113 protocol biopsies with all samples scored by the same histopathologist. Differentially expressed genes were deemed significant if they had a Benjamini-Hochberg adjusted P < 0.05. We then performed a gene set enrichment analysis using the gene ontology database to identify biological pathways that were enriched in iIFTA lesions. We also compared the transcriptomic profiles of patients with antibody- and T-cell mediated rejection with the iIFTA lesion. We further interrogated the same cohort for the progression of their iIFTA score at 12-months. Differential gene expression, and pathway analysis were employed to identify risk genes in patients that developed high iIFTA at 12-months post-transplant. Results: Of the 113 biopsies with RNA sequencing, 37 demonstrated iIFTA by histopathology. Following transcriptomic analysis, 336 genes were differentially expressed in biopsies with an iIFTA diagnosis. The top tanking genes and pathways were all involved in the formation of immunoglobin and B-cell activation. Further, transcriptomic changes in the iIFTA lesion closely resembled those found in antibody-mediated rejection. Finally, we demonstrate that genomic information, from both peripheral blood and kidney biopsy 3-months post-transplant, provides better prediction power than clinical data in identifying patients with iIFTA lesions.Conclusion: RNA sequencing enabled us to identify novel transcriptomic changes in patients with iIFTA. This pathology is primarily driven by immune-based changes, closely resembling antibody-mediated rejection, which is at odds with the current concept of iIFTA mediated by T-cell activation. Further, this study aims to build a foundation for using molecular technology in identifying the progression of the Banff iIFTA lesion.

Automated Prediction of Kidney Failure in IgA Nephropathy with Deep Learning from Biopsy Images.

Digital pathology and artificial intelligence offer new opportunities for automatic histologic scoring. We applied a deep learning approach to IgA nephropathy biopsy images to develop an automatic histologic prognostic score, assessed against ground truth (kidney failure) among patients with IgA nephropathy who were treated over 39 years. We assessed noninferiority in comparison with the histologic component of currently validated predictive tools. We correlated additional histologic features with our deep learning predictive score to identify potential additional predictive features. Training for deep learning was performed with randomly selected, digitalized, cortical Periodic acid-Schiff-stained sections images (363 kidney biopsy specimens) to develop our deep learning predictive score. We estimated noninferiority using the area under the receiver operating characteristic curve (AUC) in a randomly selected group (95 biopsy specimens) against the gold standard Oxford classification (MEST-C) scores used by the International IgA Nephropathy Prediction Tool and the clinical decision supporting system for estimating the risk of kidney failure in IgA nephropathy. We assessed additional potential predictive histologic features against a subset (20 kidney biopsy specimens) with the strongest and weakest deep learning predictive scores. We enrolled 442 patients; the 10-year kidney survival was 78%, and the study median follow-up was 6.7 years. Manual MEST-C showed no prognostic relationship for the endocapillary parameter only. The deep learning predictive score was not inferior to MEST-C applied using the International IgA Nephropathy Prediction Tool and the clinical decision supporting system (AUC of 0.84 versus 0.77 and 0.74, respectively) and confirmed a good correlation with the tubolointerstitial score (r=0.41, P<0.01). We observed no correlations between the deep learning prognostic score and the mesangial, endocapillary, segmental sclerosis, and crescent parameters. Additional potential predictive histopathologic features incorporated by the deep learning predictive score included (1) inflammation within areas of interstitial fibrosis and tubular atrophy and (2) hyaline casts. The deep learning approach was noninferior to manual histopathologic reporting and considered prognostic features not currently included in MEST-C assessment. This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2022_07_26_CJN01760222.mp3.

Convolutional Neural Networks for the Evaluation of Chronic and Inflammatory Lesions in Kidney Transplant Biopsies

In kidney transplant biopsies, both inflammation and chronic changes are important features that predict long-term graft survival. Quantitative scoring of these features is important for transplant diagnostics and kidney research. However, visual scoring is poorly reproducible and labor intensive. The goal of this study was to investigate the potential of convolutional neural networks (CNNs) to quantify inflammation and chronic features in kidney transplant biopsies. A structure segmentation CNN and a lymphocyte detection CNN were applied on 125 whole-slide image pairs of periodic acid-Schiff- and CD3-stained slides. The CNN results were used to quantify healthy and sclerotic glomeruli, interstitial fibrosis, tubular atrophy, and inflammation within both nonatrophic and atrophic tubuli, and in areas of interstitial fibrosis. The computed tissue features showed high correlation with Banff lesion scores of five pathologists (A.A., A.Dend., J.H.B., J.K., and T.N.). Analyses on a small subset showed a moderate correlation toward higher CD3+ cell density within scarred regions and higher CD3+ cell count inside atrophic tubuli correlated with long-term change of estimated glomerular filtration rate. The presented CNNs are valid tools to yield objective quantitative information on glomeruli number, fibrotic tissue, and inflammation within scarred and non-scarred kidney parenchyma in a reproducible manner. CNNs have the potential to improve kidney transplant diagnostics and will benefit the community as a novel method to generate surrogate end points for large-scale clinical studies.

The Potential Diagnostic Value of Immune-Related Genes in Interstitial Fibrosis and Tubular Atrophy after Kidney Transplantation.

Background Inflammation within areas of interstitial fibrosis and tubular atrophy (IF/TA) is associated with kidney allograft failure. The aim of this study was to reveal new diagnostic markers of IF/TA based on bioinformatics analysis. Methods Raw data of IF/TA samples after kidney transplantation and control samples after kidney transplantation were extracted from the Gene Expression Omnibus (GEO) database (GSE76882 and GSE120495 datasets), and genes that were differentially expressed between the two groups (DEGs) were screened. Gene Set Enrichment Analysis (GSEA), ESTIMATE and single sample GSEA (ssGSEA), least absolute shrinkage and selection operator (LASSO) regression analysis, and competing endogenous RNA (ceRNA) network were used to analyze the data. Results The results of GSEA revealed that multiple immune-related pathways were enriched in the IF/TA group, and subsequent immune landscape analysis also showed that the IF/TA group had higher immune and stromal scores and up to 15 types of immune cells occupied them, such as B cells, cytotoxic cells, and T cells. LASSO regression analysis selected 6 (including ANGPTL3, APOH, LTF, FCGR2B, HLA-DQA2, and EGF) out of 14 DE-IRGs as diagnostic genes to construct a diagnostic model. Then, receiver operating characteristic (ROC) curve analysis showed the powerful diagnostic value of the model, and the area under the curve (AUC) of a single diagnostic gene was greater than 0.75. The results of ingenuity pathway analysis (IPA) also indicated that DEGs were involved in the immune system and kidney disease-related pathways. Finally, we found multiple miRNAs that could regulate diagnostic genes from the ceRNA network. Conclusion This study identified 6 IF/TA-related genes, which might be used as a new diagnosis model in the clinical practice.

MO174: Proliferative Glomerulonephritis with Monoclonal Immunoglobulin Deposits: Rare Disease with Poor Prognosis?

Abstract BACKGROUND AND AIMS Proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMIDs) is a recently described disease among the spectrum of monoclonal gammopathy of renal significance (MGRS). The disease is renal limited and manifests with chronic glomerular disease, altered renal function and albuminuria, sometimes in the nephrotic range. Kidney injury results from direct glomerular deposition of the monoclonal immunoglobulin. Of note, in approximately 70% of the cases of PGNMID, a clone cannot be detected. METHOD Clinical case of PGNMID. RESULTS A 36-year-old woman, with a past history of acute glomerulonephritis, was treated with mycophenolate mofetil 2 g and concomitant methylprednisolone 2 mg from January 2020 to September 2021 (kidney biopsy was not performed). After 3 months, she showed recurrence of nephrotic syndrome: peripheral edema, severe weakness, shortness of breath, decreased diuresis up to oliguria and increased blood pressure. Blood analysis has shown: GFR 127 mL/min/1.73 m2, total protein 44.4 g/L, albumin 16.2 g/L; autoimmune markers—negative; kappa free light chains 182 mg/L (3.3–19.4 mg/L); lambda free light chains 112 mg/L (5.71–26.3 mg/L), ratio of kappa/lambda 1.63 (0.26 to 1.65). Urine analysis has shown: total protein excretion rate 3.5 g/24 h; albuminuria 1000 mg/g; urine microscopy—normal; kappa free light chains ++++; lambda free light chains +++. A kidney biopsy has shown up to 12 glomerulies, which have different sizes, some are enlarged, as due to diffuse proliferation of mesangial cells, oedema and segmental proliferation of endothelial cells, as fibrinoid changes; some of the capillary loops are infiltrated with monocytes, an uneven increase in the mesangial matrix, there are small areas of segmental sclerosis that tend to be nodular in some glomerulies. Degenerative changes (granular, vacuolar, in some areas hyaline-droplet dystrophy), necrosis of epithelial cell groups, areas of epithelial atrophy with thickening of the tubular basement membrane (up to 10% of the biopsy volume) are sharply expressed in the tubules. In the tubules—protein masses, exfoliated epithelium, the lumen of the tubules is sharply reduced and/or absent due to pronounced degenerative changes in the epithelium. In the stroma—edema, areas of interstitial fibrosis, including perivascular (up to 10% of the biopsy volume), focal lymphohistiocytic cell infiltrates. By definition, there is evidence of monoclonal deposits by immunofluorescence: IgA (predominantly), IgG, IgM, С3 deposits; С1q, CD20 (В- lymphocytes), AA amyloidosis -negative expression; kappa free light chains (predominantly) +++; lambda free light chains ++ (Figure 1). Finally, we reached a clinical diagnosis of CKD G1, A3: PGNMIDs (IgM, IgA, IgG; LC Ķ˃λ). Nephrotic syndrome. Light Chain Deposition Disease. At that point (January 2021), we have started clone-targeted chemotherapy following the data sheet, with urinary and haematological response after 2 weeks, and normal kidney function with complete remission of nephrotic syndrome after 4 weeks, as well as a negativization of kappa and lambda free light chains deposits (Table 1). Three months after admission, the patient maintained haematological and kidney remission up to now. CONCLUSION MGRS is not an independent kidney disease, not ‘chronic glomerulonephritis’, but a condition in which kidney damage is secondary to clonal B-cell proliferation. In other words, MGRS is a precancerous disease combined with chronic kidney disease that requires immediate treatment. Renal prognosis is poor, with progression to ESKD in 25% of patients within 30 months and frequent early recurrence on the renal allograft. However, recent studies indicate that clone-targeted chemotherapy may significantly improve renal outcomes, opening future perspectives for the management of this rare disease.

Cyclin-dependent kinase 4-related tubular epithelial cell proliferation is regulated by Paired box gene 2 in kidney ischemia-reperfusion injury

Paired box 2 (Pax2) is a transcription factor essential for kidney development and is reactivated in proximal tubular epithelial cells (PTECs) during recovery from kidney injury. However, the role of Pax2 in this process is still unknown. Here the role of Pax2 reactivation during injury was examined in the proliferation of PTECs using an ischemia-reperfusion injury (IRI) mouse model. Kidney proximal tubule-specific Pax2 conditional knockout mice were generated by mating kidney androgen-regulated protein-Cre and Pax2 flox mice. The degree of cell proliferation and fibrosis was assessed and a Pax2 inhibitor (EG1) was used to evaluate the role of Pax2 in the hypoxic condition of cultured PTECs (O2 5%, 24 hours). The number of Pax2-positive cells and Pax2 mRNA increased after IRI. Sirius red staining indicated that the area of interstitial fibrosis was significantly larger in knockout mice 14 days after IRI. The number of Ki-67-positive cells (an index of proliferation) was significantly lower in knockout than in wild-type mice after IRI, whereas the number of TUNEL-positive cells (an index of apoptotic cells) was significantly higher in knockout mice four days after IRI. Expression analyses of cell cycle-related genes showed that cyclin-dependent kinase 4 (CDK4) was significantly less expressed in the Pax2 knockout mice. Invitro data showed that the increase in CDK4 mRNA and protein expression induced by hypoxia was attenuated by EG1. Thus, Pax2 reactivation may be involved in PTEC proliferation by activating CDK4, thereby limiting kidney fibrosis.

Persistence and shedding of senecavirus A in naturally infected boars.

Senecavirus A (SVA) infection in pigs causes vesicular disease and results in a short viremia and transient shedding of the virus, mainly in oral fluids and feces. Here we describe the consistent prolonged shedding of SVA in the semen of 2 boars, and persistence of SVA within the tonsils and testes of 3 adult boars. Two SVA-infected boars that were identified on a Minnesota sow farm in 2017 shed SVA RNA in semen for >3 mo after an outbreak of vesicular disease had occurred on the farm. SVA was isolated from 1 semen sample collected 9 d after clinical disease began on the farm. The third SVA-infected boar was identified on an Indiana sow farm in 2020. All boars had SVA RNA detected in the testes and tonsils by RT-rtPCR, with lower Ct values obtained for the testes than from the tonsils. All boars had multifocal lymphocytic orchitis with segmental degeneration and atrophy of the germinal epithelium within the seminiferous tubules. One boar also had areas of seminiferous tubule collapse and interstitial fibrosis within the testes. In all boars, in situ hybridization demonstrated the presence of SVA mRNA within cells located basally in the seminiferous tubules of the testes, and within the basal surface epithelial cells, crypt epithelial cells, and subepithelial and parafollicular lymphocytes and histiocytes of the tonsil.

Ginkgobiloba leaf extract mitigates cisplatin-induced chronic renal interstitial fibrosis by inhibiting the epithelial-mesenchymal transition of renal tubular epithelial cells mediated by the Smad3/TGF-\u03b21 and Smad3/p38 MAPK pathways

BackgroundOur previous study indicated that Ginkgo biloba leaf extract (EGb) could protect against cisplatin-induced acute kidney injury in rabbits. The present study aimed to determine the effects and potential molecular mechanisms of EGb on chronic renal interstitial fibrosis induced by cisplatin using in vivo and in vitro models.MethodsRats received a single dose of cisplatin on Day 1, and a subset of rats was intraperitoneally injected with EGb daily between Days 22–40. In vitro, HK-2 cells were treated with cisplatin, and a subset of cells was cultivated with EGb or SIS3 (Smad3 inhibitor) for 48 h. Renal function of rats was assessed by detecting the levels of serum creatinine (Scr), blood urea nitrogen (BUN) and urinary N-acetyl-β-D-glucosaminidase (NAG). Hematoxylin and eosin staining and Masson’s trichrome staining were used to evaluate the damage and fibrosis of renal tissue. Western blotting, immunohistochemistry and immunofluorescence were used to detect the protein levels of fibrosis-associated proteins and signaling pathway-related proteins. RT–qPCR analysis was used to examine the mRNA levels of related indicators.ResultsEGb significantly decreased the increased levels of Scr, BUN and urinary NAG and attenuated renal damage and the relative area of renal interstitial fibrosis induced by cisplatin. Additionally, EGb decreased the protein levels of α-SMA, Col I, TGF-β1, smad2/3, phosphorylated (p)-smad2/3, p38 MAPK, and p-p38 MAPK; the ratio of p-p38 MAPK/p38 MAPK; and the mRNA level of p38 MAPK in renal tissues induced by cisplatin. In agreement with in vivo studies, EGb significantly reduced the increased protein levels of these indicators. Additionally, EGb significantly reduced the increased protein levels of vimentin, TIMP-1, and CTGF, as well as the mRNA levels of α-SMA, vimentin, and TGF-β1, while it significantly increased the reduced E-cadherin protein level and the MMP-1/TIMP-1 ratio in HK-2 cells induced by cisplatin. It’s worth noting that the effects of SIS3 in changing the above indicators were similar to those of EGb.ConclusionOur study demonstrated that EGb improved cisplatin-induced chronic renal interstitial fibrosis, and its mechanisms were associated with inhibiting the epithelial-mesenchymal transition of renal tubular epithelial cells via the Smad3/TGF-β1 and Smad3/p38 MAPK pathways.

Abstract 11271: Three-Dimensional Deep-Tissue Imaging of the Right Ventricle Reveals Decreased Capillary-Cardiomyocyte Contact Surface in Decompensated Right Heart Failure

Introduction: Right ventricular (RV) function determines the prognosis of pulmonary hypertension. Capillary rarefaction using 2D imaging has been proposed as the hallmark of RV failure, although this paradigm is controversially discussed. By applying 3D deep-tissue imaging, we characterized the adaptive and maladaptive response of the capillaries in the pressure-overloaded RV in hypertrophy and failure. Methods and Results: Mice subjected to pulmonary artery banding (PAB) were harvested after 3 weeks (w) (compensated RV hypertrophy) and 7w post-surgery (decompensated RV failure). Hearts were sectioned into 250 μm and capillaries were stained with isolectin B4. Samples were imaged with confocal microscopy and reconstructed into 3D using Imaris imaging software (Fig. A, n=4). In the RV free wall, mean capillary length decreased, and branching, tortuosity, mean and total capillary diameter increased 3w post-PAB. Mean and total diameter, and total length decreased at 7w resulting in a reduced capillary volume normalized to heart volume at 7w vs 3w, but not significantly reduced vs baseline suggesting that this is not the mechanism of RV failure (Fig. B). However, most strikingly we uncovered by sparsely labeling cardiomyocytes that the capillary-cardiomyocyte contact surface was maintained at w3, yet significantly decreased in areas of interstitial fibrosis in the decompensated RV at 7w compared to baseline and 3w (Fig. C, D). Conclusions: 3D deep-tissue imaging uncovered the remarkable adaptive response of the RV microvasculature in the pressure-overloaded RV to maintain an adequate capillary-cardiomyocyte contact by increasing capillary branching, length and diameter. These mechanisms failed in the decompensating RV when interstitial fibrosis impaired the capillary-cardiomyocyte interaction. Our method offers the unprecedented opportunity to study microvascular adaption in the pressure overloaded RV and is readily transferable to human tissue.

Abstract 14247: Myocardial Histopathology In Atrial Cardiomyopathy

Background: Atrial cardiomyopathy (ACM) is characterized by multiple histopathological features but the entity lacks systematic validation. Objectives: We sought to correlate atrial histopathology with clinical characteristics of patients with ACM and AF and outcomes following cardiac surgery. Methods: The pathological findings of the atrial myocardial biopsies from 178 patients who underwent any cardiac surgery between 2016-2018. Atrial cell nuclear structure and morphology, myocyte hypertrophy and interstitial fibrosis were graded and analyzed quantitatively. Study population was divided in three groups based on preoperative ECG: 1) patients in sinus rhythm with no signs of ACM (P wave duration &lt;120ms)(SRn); 2) patients in sinus rhythm and ACM (P&gt;=120 or biphasic waves in inferior leads)(SRa); and 3) atrial fibrillation (AF). Results: SRa patients had decreased number of nuclei/area and smaller total cardiomyocyte area compared to SRn patients (p=0.04 forboth). Patients with AF had bigger, less solid and circular nuclei compared to SRn patients (p&lt;0.01 for all). AF patients had increased area of interstitial fibrosis (p&lt;0.01) compared to SRn patients. Conclusions: Histopathological characteristics of ACM include changes in nuclear chromatin structure.

Assessment of the Banff Working Group classification of definitive BK polyomavirus nephropathy.

Polyomavirus associated nephropathy (PyVAN) continues to be a burden in renal transplantation leading to allograft insufficiency or graft failure. A presumptive diagnosis of PyVAN is made based on the presence of BK polyomavirus in patients' plasma; however, kidney biopsy remains the gold standard to establish a definitive diagnosis. The Banff Working Group on PyVAN proposed a novel classification of definitive PyVAN based on polyomavirus replication/load level and the extent of interstitial fibrosis. The aim of our study was to test the newly defined classes of PyVAN using independent cohorts of 124 kidney transplant patients with PyVAN with respect to the initial presentation and outcome, and to compare our analysis to that previously reported. Detailed analysis of our cohort revealed that the proposed classification of PyVAN did not stratify or identify patients at increased risk of allograft failure. Specifically, while class 3 was associated with the worst prognosis, there was no significant difference between the outcomes in classes 1 and 2. We also found that the timing post-transplantation and inflammation in areas of interstitial fibrosis and tubular atrophy might be additional factors contributing to an unfavorable allograft outcome in patients with PyVAN.

Sclerosing Sialadenitis Is Associated With Salivary Gland Hypofunction and a Unique Gene Expression Profile in Sjögren's Syndrome.

PurposeTo develop a novel method to quantify the amount of fibrosis in the salivary gland and to investigate the relationship between fibrosis and specific symptoms associated with Sjögren’s syndrome (SS) using this method.Materials and MethodsParaffin-embedded labial salivary gland (LSG) slides from 20 female SS patients and their clinical and LSG pathology data were obtained from the Sjögren’s International Collaborative Clinical Alliance. Relative interstitial fibrosis area (RIFA) in Masson’s trichrome-stained LSG sections was quantified from digitally scanned slides and used for correlation analysis. Gene expression levels were assessed by microarray analysis. Core promoter accessibility for RIFA-correlated genes was determined using DNase I hypersensitive sites sequencing analysis.ResultsRIFA was significantly correlated with unstimulated whole saliva flow rate in SS patients. Sixteen genes were significantly and positively correlated with RIFA. In a separate analysis, a group of differentially expressed genes was identified by comparing severe and moderate fibrosis groups. This combined set of genes was distinct from differentially expressed genes identified in lung epithelium from idiopathic pulmonary fibrosis patients compared with controls. Single-cell RNA sequencing analysis of salivary glands suggested most of the RIFA-correlated genes are expressed by fibroblasts in the gland and are in a permissive chromatin state.ConclusionRIFA quantification is a novel method for assessing interstitial fibrosis and the impact of fibrosis on SS symptoms. Loss of gland function may be associated with salivary gland fibrosis, which is likely to be driven by a unique set of genes that are mainly expressed by fibroblasts.

Renal allograft DARCness in subclinical acute and chronic active ABMR.

SummaryGene expression profiling of renal allograft biopsies revealed the Duffy antigen receptor for chemokines (DARC) as being strikingly upregulated in antibody‐mediated rejection (ABMR). DARC has previously been shown to be associated with endothelial injury. This study aimed at assessing the value of DARC immunohistochemistry as diagnostic marker in ABMR. The study was performed on 82 prospectively collected biopsies of a clinically well‐defined population (BORTEJECT trial, NCT01873157) of DSA‐positive patients with gene expression data available for all biopsies. Diagnostic histologic assessment of biopsies was performed according to the Banff diagnostic scheme. DARC expression was focally accentuated, on peritubular capillaries (PTC) mostly in areas of interstitial fibrosis and/or inflammation. DARC positivity was associated with diagnosis of ABMR and correlated with DARC gene expression levels detected by microarray analysis. Still, as previously described, a substantial number of biopsies without signs of rejection showed DARC‐positive PTC. We did not observe significantly reduced graft survival in cases showing histologic signs of ABMR and being DARC‐positive, as compared to DARC‐negative ABMR. In summary, the upregulation of DARC, detected by immunohistochemistry, is associated with but not specific for ABMR. We did not observe reduced graft survival in DARC‐positive patients.

MO566THE BONE EXPRESSION OF WNT INHIBITORS IN THE EXPERIMENTAL MODEL OF EARLY CHRONIC KIDNEY DISEASE

Abstract Background and Aims Molecular mechanisms implicated in the initial stages of inorganic phosphate (Pi) imbalance in chronic kidney disease (CKD) remain poorly understood.The aim of the study was to evaluate whether canonical Wnt pathway inhibitors (iWnt) involved in early response to Pi retention in CKD. Methods Mild CKD was induced by 3/4 nephrectomy (NE) in spontaneously hypertensive rats (SHR) fed rat chow diet containing 0.6 % phosphate. Controls were sham operated SHR (SO). Duration of experimental exposure (NE or SO) was 2 and 6 months. Serum levels of creatinine (Cr), inorganic phosphate (Pi), fractional Pi excretion (FEPi), intact parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), alfa-Klotho (KL), sclerostin (SOST) and Dickkopf-1 (DKK1) were measured. The following morphological characteristics by light microscopy of bone metaphysis and kidney tissues: the area of renal interstitial fibrosis (RF) (Masson's trichrome), bone matrix volume (MV), the active osteoblasts to trabecular cells number ratio (aOB/cells), eroded surface to bone surface ratio (ES/BS) (hematoxylin &amp; eosin), and bone SOST and DKK1 proteins expression (by IHC) were analyzed and calculated quantitatively. Statistical comparisons among groups were performed using Mann–Whitney U-test and Kruskal-Wallis H-test. Results Serum Cr, RF and indices of Pi exchange in the experimental model corresponded to early CKD (Table). Pi elevated in NE6 suggestive for its renal retention. KL level decreased (Table) in all experimental groups vs control. No differences were observed in serum levels FGF23 (p=0.62) and PTH (p=0.63). Serum SOST and DKK1 levels were significantly higher in NE6 group compared to SO6 (Table). The bone SOST and DKK1 expression increased in NE6 compared to SO6 (Figure). aOB/cells were lower in NE2, SO6 and NE6 vs SO2 (all p-values&amp;lt;=0.041). ES/BS increased in NE2 (vs SO2) while being lowest in NE6 and SO6 animals (Table). SOST and DKK1 metaphyseal expression increased in NE6 compared to SO2, SO6, NE2 (Figure). Osteocyte SOST expression increased in SO6 compared to SO2 and NE2 without differences in later groups. Osteoblast SOST expression was also higher in SO6 vs SO2 (Figure). Conclusion Increased serum levels of sclerostin and Dickkopf-1 and their bone expression are apparent in early stages of experimental CKD associating with hyperphosphatemia. Alterations of bone resorption and osteoblast depopulation occurred before the increase of serum Pi likely reflecting incipient stages of renal Pi retention. These molecular and cellular events seem to be independent of systemic FGF23 and PTH response.

Involvement of chronic inflammation via monocyte chemoattractant protein-1 in uraemic cardiomyopathy: a human biopsy study.

AimsPatients undergoing dialysis, even those without coronary artery disease or valvular abnormalities, sometimes present with reduced heart function, which resembles dilated cardiomyopathy (DCM). This condition is known as uraemic cardiomyopathy (UCM). The mechanisms of UCM development are not fully understood. Previous studies demonstrated that the balance between placental growth factor (PlGF) and fms‐like tyrosine kinase‐1 (Flt‐1) is correlated with renal function, and PlGF/Flt‐1 signalling is involved in the development of cardiovascular diseases in patients with chronic kidney disease. This study was conducted to evaluate the pathogenesis of UCM and clarify the differences in the mechanisms of UCM and DCM by using human endomyocardial biopsy and blood samples.Methods and resultsThe clinical and pathological features of 30 patients on dialysis with reduced cardiac function [left ventricular ejection fraction (LVEF) ≤50%] (UCM group; mean age: 58.5 ± 9.4 years and LVEF: 39.1 ± 7.2%), 196 DCM patients (DCM group; mean age: 62.7 ± 14.0 years and LVEF: 33.5 ± 8.8%) as controls with reduced cardiac function (LVEF ≤ 45%), and 21 patients as controls with normal cardiac function (control group; mean age: 56.2 ± 19.3 years and LVEF: 67.5 ± 6.7%) were analysed. The percentage of the interstitial fibrosis area in the UCM group was greater than that in the DCM group (P = 0.045). In UCM patients, the percentage of the interstitial fibrosis area was positively correlated with the duration of renal replacement therapy (P < 0.001). The number of infiltrated CD68‐positive macrophages in the myocardium and expression of monocyte chemoattractant protein‐1 (MCP‐1) in cardiomyocytes were significantly greater in the UCM group than in the other groups (P < 0.001, respectively). Furthermore, while the serum level of soluble form of Flt‐1, an endogenous inhibitor of PlGF, in the UCM group was lower compared with that in the DCM group (P < 0.001), the serum levels of PlGF and PlGF/soluble form of Flt‐1 ratio and plasma level of MCP‐1 in the UCM group were higher than those in the DCM group (P < 0.001, respectively).ConclusionsThese results suggest that activated PlGF/Flt‐1 signalling and subsequent macrophage‐mediated chronic non‐infectious inflammation via MCP‐1 in the myocardium are involved in the pathogenesis of UCM.

Myocardial work index: a marker of left ventricular contractility in pressure‐ or volume overload‐induced heart failure

AimsWhile global longitudinal strain (GLS) is considered to be a sensitive marker of left ventricular (LV) function, it is significantly influenced by loading conditions. We hypothesized that global myocardial work index (GMWI), a novel marker of LV function, may show better correlation with load‐independent markers of LV contractility in rat models of pressure‐induced or volume overload‐induced heart failure.Methods and resultsMale Wistar rats underwent either transverse aortic constriction (TAC; n = 12) or aortocaval fistula creation (ACF; n = 12), inducing LV pressure or volume overload, respectively. Sham procedures were performed to establish control groups (n = 12/12). Echocardiographic loops were obtained to determine GLS and GMWI. Pressure‐volume analysis with transient occlusion of the inferior caval vein was carried out to calculate preload recruitable stroke work (PRSW), a load‐independent ‘gold‐standard’ parameter of LV contractility. Myocardial samples were collected to assess interstitial and perivascular fibrosis area and also myocardial atrial‐type natriuretic peptide (ANP) and brain‐type natriuretic peptide (BNP) relative mRNA expression. Compared with controls, GLS was substantially lower in the TAC group (−7.0 ± 2.8 vs. −14.5 ± 2.5%; P < 0.001) and was only mildly reduced in the ACF group (−13.2 ± 2.4 vs. −15.4 ± 2.0%, P < 0.05). In contrast with these findings, PRSW and GMWI were comparable with sham in TAC (110 ± 26 vs. 116 ± 68 mmHg; 1687 ± 275 mmHg% vs. 1537 ± 662 mmHg%; both P = NS), while it was found to be significantly reduced in ACF (58 ± 14 vs. 111 ± 40 mmHg; 1328 ± 411 vs. 1934 ± 308 mmHg%, both P < 0.01). In the pooled population, GMWI (r = 0.70; P < 0.001) but not GLS (r = −0.23; P = 0.12) showed a strong correlation with PRSW. GLS correlated with interstitial (r = 0.61; P < 0.001) and perivascular fibrosis area (r = 0.54; P < 0.001), and also with myocardial ANP (r = 0.85; P < 0.001) and BNP relative mRNA expression (r = 0.75; P < 0.001), while GMWI demonstrated no or only marginal correlation with these parameters.ConclusionsBeing significantly influenced by loading conditions, GLS may not be a reliable marker of LV contractility in heart failure induced by pressure or volume overload. GMWI better reflects contractility in haemodynamic overload states, making it a more robust marker of systolic function, while GLS should be considered as an integrative marker, incorporating systolic function, haemodynamic loading state, and adverse tissue remodelling of the LV.

Centella asiatica Extract Attenuates Kidney Fibrosis Through Reducing Mesenchymal Transition and Inflammation in Ureteral Ligation Model in Mice

Background: Kidney fibrosis is the common final pathway of chronic kidney disease (CKD), and is characterized by inflammation, mesenchymal transition with myofibroblast formation and epithelial to mesenchymal transition (EMT). Centella asiatia (CeA) is an herb that has a reno-protective effect. However, its mechanism of action in kidney fibrosis has not been elucidated. Aim: To elucidate the effect of CeA in amelioration of kidney fibrosis in a unilateral ureteral obstruction (UUO) model and focus on mesenchymal transition and inflammation. Methods: Unilateral ureteral obstruction was performed in male Swiss-background mice (age: 2–3 months, weight: 30–40 g, UUO group n = 6) to induce kidney fibrosis. Two doses of CeA extract with oral administration, 210 and 840 mg/kg body weight were added in UUO (U+C210 and U+C840 groups, each n = 6). The sham operation procedure was performed for the control group (SO, n = 6). The mice were euthanized at day-14 after operation. Tubular injury and interstitial fibrosis area fractions in kidney tissues of the mice were quantified based on periodic acid-Schiff (PAS) and Sirius Red (SR) staining. Immunostaining was performed for examination of fibroblast (PDGFR-β), myofibroblast (α-SMA), Monocyte Chemoattractant Protein-1 (MCP-1) and macrophage (CD68), meanwhile double immunofluorescence was performed with PDGFR-β and α-SMA. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to examine mRNA expression of TGF-β, Collagen-1, Snail, E-cadherin, vimentin, fibroblast-specific protein 1 (FSP-1), CD68, toll-like receptor 4 (TLR4), and MCP-1. Results: We observed a significantly higher interstitial fibrosis area fraction and tubular injury (p < 0.001) with fibroblast expansion and myofibroblast formation in the UUO group than in the SO group. These findings were associated with higher mRNA expression of TGF-β, Collagen-1, Snail, vimentin, FSP-1, CD68, TLR4, and MCP-1 and lower mRNA expression of E-cadherin. The U+C840 group had a significantly lower tubular injury score and interstitial fibrosis area fraction, which associated with downregulation of mRNA expression of TGF-β, Collagen-1, Snail, vimentin, FSP-1, CD68, TLR4, and MCP-1, with upregulation of mRNA expression of E-cadherin. Immunostaining observation revealed the U+C840 group demonstrated reduction of macrophage infiltration and myofibroblast expansion. Conclusion: CeA treatment with dose-dependently ameliorates mesenchymal transition and inflammation in kidney fibrosis in mice.

Ginaton injection alleviates cisplatin-induced renal interstitial fibrosis in rats via inhibition of apoptosis through regulation of the p38MAPK/TGF-β1 and p38MAPK/HIF-1α pathways.

Ginaton injection (Ginkgo biloba extract; GBE) has been reported to protect against cisplatin-induced acute renal failure in rats. In the present study, the effects and molecular mechanisms of GBE on cisplatin-induced renal interstitial fibrosis were evaluated using a rat model. The rats were intraperitoneally injected with cisplatin once on the first day and a subset of rats were treated with GBE or SB203580 (SB; a specific p38 MAPK inhibitor) daily from days 22 to 40. The levels of N-acetyl-β-D-Glucosaminidase (NAG) in the urine, and of urea nitrogen (BUN) and creatinine (Scr) in the blood were assessed. The damage and fibrosis of renal tissues were evaluated using hematoxylin and eosin staining, as well as Masson's trichrome staining, respectively. Apoptosis in renal tissues was detected using a TUNEL assay. The protein expression levels of α-smooth muscle actin (SMA), collagen 1 (Col I), Bax, Bcl-2, caspase-3/cleaved caspase-3, hypoxia-inducible factor-1α (HIF-1α), TGF-β1 and p38MAPK, as well as the mRNA levels of p38MAPK in renal tissues were investigated. The results showed that GBE markedly reduced the levels of urinary NAG, Scr and BUN, and renal expression of α-SMA and Col I levels were also reduced. Furthermore, GBE significantly reduced renal tissue injury and the relative area of renal interstitial fibrosis induced by cisplatin. GBE effectively reduced the apoptotic rate of renal tissues, the protein expression levels of Bax, cleaved caspase-3, phospho-p38MAPK, TGF-β1 and HIF-1α, as well as the mRNA expression levels of p38MAPK in renal tissues induced by cisplatin, whereas GBE significantly increased Bcl-2 protein expression. SB exhibited similar effects to GBE, although it was not as effective. In summary, the present study is the first to show that GBE significantly alleviated renal interstitial fibrosis following cisplatin-induced acute renal injury. The mechanisms by which GBE exhibited its effects were associated with the inhibition of apoptosis via downregulation of the p38MAPK/TGF-β1 and p38MAPK/HIF-1α signaling pathways.