Glomerular Tuft Research Articles

The Protective Effect of Annona Extracts on Renal Histopathology Induced by Ethylene Glycol in Male Rabbits

Annona is one of the many plant extracts that have been explored owing to theiranti-inflammatory and anticancer effects. Male locale rabbit were separated into five groups, Group Iconsider as control, The 4th other groups were treated orally with 0.75% ethylene glycol (EG) to inducerenal damage this treatment done till 30th days. Group III to V animals were served as curative regimenand received flavonoids, glycosides and alkaloids extract of the plant of Annona at a dose of 100 mg/kgbody weight from 15th day to 30th day. The extracts was administered twice daily by oral route. significantkidney injury in EG animals as demonstrated by a significant elevation (P= 0.05) in serum activities urea,creatinine, total protein and calculated globulin levels. Meanwhile there was significant drop (P=0.05) inalbumin, and A/G ratio compared to the control and treated group , The levels of lipid peroxidation weresignificantly higher in the Serum of the groups of animals treated with EG , while the levels of SOD,CAT and GPx were significantly lower than animals were treatment of EG group with Annona significantlyelevated the antioxidant mechanisms differ with that before treatment. Renal Histopathological changesincludes deposition of oxalates in the renal tubules lumen, degeneration and necrosis of epithelium of renaltubules , congestion of blood cells , infiltration of inflammatory cells and atrophy in glomerular tuft leadingto expansion of bowmans spaces. While Histopathological changes in animals group treated with Annonaextract and ethylene glycol were limited dilatation of renal tubules with vascular changes with blood vesselscongestion and interstitial hemorrhage , reduced renal damage revealed by less degenerative and necroticlesion in tubular epithelium and no inflammatory reaction and the glomeruli appeared normal compared withethylene glycol sections.

Protective Effect of Earthworms (Pheretima hawayana Rosa and Allolobophora caliginosa Savigny) Methanolic Extract on Acrylamide Induced Hepatorenal Toxicity

The present study was carried out to evaluate the protective potentials of earthworm extract (EE) against acrylamide-induced hepatorenal toxicity. Forty rats were randomly divided into 4 groups (n=10). G І (control): were received distilled water (D.W); G ІІ: rats were administered acrylamide (ACR; 5 mg/kg B.W in D.W) orally for 3 weeks; G ІІІ: rats were given EE (300 mg/kg B.W in D.W) orally for 3 weeks; G ІV: rats were pretreated with EE for 3 weeks, then co-treated by EE and ACR for an additional 3 weeks. The results showed that acrylamide increased serum ALT, AST, total bilirubin, urea, and creatinine, while decreased albumin and total proteins. Acrylamide resulted in congestion of central veins and hepatic sinusoids, and hydropic degeneration of most hepatocytes. Also, ACR caused inflammatory cells infiltration in hepatic sinusoids and areas of coagulative necrosis infiltrated by inflammatory cells. Furthermore, ACR lead to alteration in kidney tissue via shrinkage of glomerular tuft with increased Bowman's space. Also, ACR lead to congestion and inflammatory cells infiltration of interstitial tissue, renal tubules with desquamated renal epithelium in their lumen forming epithelial casts. Acrylamide caused degenerative changes of some epithelial cells lining the renal tubules, other cells showed necrosis. EE recovered hepatic and renal histopathological and biochemical alterations induced by ACR. In Conclusion: Earthworm extract ameliorated acrylamide-induced hepatorenal toxicity.

Abstract P212: Kidney Injury Caused By Preeclamptic Pregnancy Improves Postpartum In A Transgenic Rat Model

Preeclamptic pregnancies involve mild renal injuries. However, there has been evidence that women with a history of preeclampsia (PE) have an increased risk to develop kidney disease in association to high blood pressure later in life. This study aims to characterize renal injury during pregnancy and postpartum in an established transgenic rat model for PE. Female Sprague-Dawley rats transgenic for the human angiotensinogen gene develop a PE phenotype in pregnancy, including cardiac remodeling, when mated with male rats harboring the human renin gene. Postpartum, blood pressure restores but cardiac remodeling persists. We hypothesize that PE during pregnancy mediates kidney injuries but does not fully restore after ending of the high blood pressure (postpartum). The renal alterations were analyzed by histological staining, gene expression and urine analysis. PE rats have elevated mean arterial blood pressure during pregnancy (PE d19 148.4 ± 20.7 mmHg) compared to normotensive values in control animals (WT d19 105.3 ± 4.6 mmHg). During PE increased expression of kidney injury marker 1 ( Kim-1/18S : PE d21 4.14 ± 3.26; WT d21 0.08 ± 0.02), neutrophil gelatinase associated lipocalin 2 ( Ngal/18S : PE d21 1.64 ± 0.83; WT d21 0.59 ± 0.28) and connective-tissue growth factor ( Ctgf/18S: PE d21 1.51 ± 0.46; WT d21 0.92 ± 0.32) were detected. Kidneys of PE rats showed mild glomerular (PAS-positive glomerular area PE d21 86.2 ± 4.4%; WT d21 79.1 ± 3.2%) and tubular changes during PE pregnancy resulting in albuminuria (albumin/creatinine ratio PE d19 2193.8 ± 1878.2; WT d19 290.4 ± 252.0). However, 4 weeks after pregnancy (approx. 2 years in humans) most of the PE related renal damages were absent including albuminuria and elevated expression of biomarkers ( Kim-1/18S : PE d50 0.09 ± 0.05; WT d50 0.23 ± 0.13; Ctgf/18S : PE d50 0.78 ± 0.25; WT d50 0.8 ± 0.25; Ngal/18S : PE d50 0.37 ± 0.17; WT 50 0.47 ± 0.11). Only mild enlargement of glomerular tuft area (PE d50 7523.8 ± 418.7 μm 2 ; WT d50 7058.4 ± 198.8 μm 2 ) was detected. Overall, the glomerular and tubular injuries are present during pregnancy in this transgenic PE rat. Most restore postpartum, speculating long-term kidney failure observed in humans is associated to hypertension and additional cardiovascular events.

Utility of glomerular morphometry in diagnosing pediatric renal disease

BackgroundMorphometry has now become a useful adjunct to the diagnostic armamentarium of light, immunofluorescence, and electron microscopy, as it provides a deep insight into quantitative parameters of nephropathies. There has been a limited study on its utility especially in diagnosing pediatric renal diseases. This study is probably the first in India to assess the contribution of this diagnostic modality in pediatric renal disease to the best of authors’ knowledge. MethodsIt's a retrospective cross-sectional study covering a period of 05 years at a tertiary care hospital. The study includes 28 cases of pediatric (age till 14 years) nephropathies. The diseases were divided into two groups—nephrotic presentation and nephritic presentation. Glomerular morphometry was performed and mean was calculated for Bowman's capsule area, glomerular capillary tuft area, and Bowman's space area; for the three groups, respectively. Renal parameters serum creatinine, blood urea, 24 h urine protein were studied along with hemoglobin and serum cholesterol for the cases. Data were analyzed using SPSS software, version 25, for one-way ANOVA comparing mean in the three groups. ResultsWe found a positive and significant correlation between Bowman's capsule area with proteinuria, blood urea, and serum creatinine. There was positive and significant correlation between glomerular capillary tuft area and serum creatinine and Bowman's space area and proteinuria in both the groups. ConclusionGlomerular morphometry may contribute to the diagnosis of some glomerulopathies and the association between glomerular morphometric parameters and laboratory data may promote better understanding of the prognosis of these patients.

Pyrethroid-Induced Organ Toxicity and Anti-Oxidant-Supplemented Amelioration of Toxicity and Organ Damage: The Protective Roles of Ascorbic Acid and α-Tocopherol.

The pyrethroid toxicants, fatal at high doses, are found as remnants of crop pesticides and ingredients of commercially available insecticides. The toxic effects of high-content insecticidal pyrethroid formulations are available in 0.05 g, 1.17 g, and 0.04 g pyrethroid-instilled products, namely burning coils, pyrethroid-soaked mats, and liquid formulations of pyrethroids that release pyrethroid vapor/smoke upon heating. They provided 5.46 g/kg, 21.15 g/kg, and 4.24 g/kg of toxicants to the experimental animals over a total of 3 weeks/5 h per os (p.o.) administration, producing necrosis, hyperemia, and fatty changes in the liver; fiber separation in cardiac muscles; atrophy, lymphatic infiltration, blood vessel congestion, and hyperemia in the heart tissues of the experimental animals. The glomerular tuft necrosis, cytoplasmic degeneration of renal tubular cells, necrotic tubules, congestion, and dilatation of blood vessels were observed in the kidney tissue of intoxicated animals. Air-space enlargement, interstitial inflammation, lymphocyte infiltration aggregates, connective tissue infiltration by inflammatory cells, and hyperemia were found in the lung tissues. The pyrethroid toxicants also produced nervous tissue degeneration and decreased neurons in the brain, which were observed through histopathological examinations of the brain, lungs, heart, kidneys, and liver. The protective effects of ascorbic acid (AA/vitamin C) and α-tocopherol (E307/vitamin E) at 100 mg/kg oral doses administered daily for the entire period of the toxicant exposure of three weeks to the experimental mice, aged between 3–4 months and weighing ≈30 g, ameliorated the tissue damage, as observed through the histopathological examinations. The ascorbic acid caused recovery of the liver, kidney, brain, and heart tissue damage, while α-tocopherol was effective at ameliorating the damage in the kidneys and lung tissue compared with the control groups. The high levels of tissue damage recovery suggested a prophylactic effect of the concurrent use of ascorbic acid and α-tocopherol for the subjects under the exposure of pyrethroids.

Development and evaluation of deep learning–based segmentation of histologic structures in the kidney cortex with multiple histologic stains

The application of deep learning for automated segmentation (delineation of boundaries) of histologic primitives (structures) from whole slide images can facilitate the establishment of novel protocols for kidney biopsy assessment. Here, we developed and validated deep learning networks for the segmentation of histologic structures on kidney biopsies and nephrectomies. For development, we examined 125 biopsies for Minimal Change Disease collected across 29 NEPTUNE enrolling centers along with 459 whole slide images stained with Hematoxylin & Eosin (125), Periodic Acid Schiff (125), Silver (102), and Trichrome (107) divided into training, validation and testing sets (ratio 6:1:3). Histologic structures were manually segmented (30048 total annotations) by five nephropathologists. Twenty deep learning models were trained with optimal digital magnification across the structures and stains. Periodic Acid Schiff-stained whole slide images yielded the best concordance between pathologists and deep learning segmentation across all structures (F-scores: 0.93 for glomerular tufts, 0.94 for glomerular tuft plus Bowman’s capsule, 0.91 for proximal tubules, 0.93 for distal tubular segments, 0.81 for peritubular capillaries, and 0.85 for arteries and afferent arterioles). Optimal digital magnifications were 5X for glomerular tuft/tuft plus Bowman’s capsule, 10X for proximal/distal tubule, arteries and afferent arterioles, and 40X for peritubular capillaries. Silver stained whole slide images yielded the worst deep learning performance. Thus, this largest study to date adapted deep learning for the segmentation of kidney histologic structures across multiple stains and pathology laboratories. All data used for training and testing and a detailed online tutorial will be publicly available.

Nicorandil prevents the nephrotoxic effect of cyclosporine-A in albino rats through modulation of HIF-1α/VEGF/eNOS signaling.

Despite that cyclosporine-A (CsA) is a widely used immunosuppressive drug, its nephrotoxic effect limits its long-term administration. Herein we tried to investigate its renal effect on endothelial dysfunction targeting the hypoxia-inducible factor (HIF-1α)/ vascular endothelial growth factor (VEGF)/ endothelial nitric oxide synthase (eNOS) pathway and the possible modulation by nicorandil. Eight groups of adult male Wistar rats were included: (1) control; (2) vehicle group (received oil); (3) glibenclamide 5mg·kg-1·day-1 administered orally; (4) nicorandil 10mg·kg-1·day-1 administered orally; (5) CsA 25mg·kg-1·day-1 administered orally; (6) combined administration of CsA and nicorandil; (7) glibenclamide was added to CsA; and (8) both CsA and nicorandil were combined with glibenclamide. The treatment continued for sixweeks. Combined nicorandil with CsA improved renal function deterioration initiated by CsA. CsA decreased the renal expression levels (P< 0.001) of HIF-1α, eNOS, and VEGF, inducing endothelial dysfunction and triggering inflammation, and upregulated the profibrotic marker transforming growth factor (TGF-β). Nicorandil fixed the disturbed HIF-1α/VEGF/eNOS signaling. Nicorandil corrected the renal functions, confirmed by the improved histological glomerular tuft retraction that was obvious in the CsA group, without significant influence by glibenclamide. Proper protection from CsA-induced nephrotoxicity was achieved by nicorandil. Nicorandil reversed the disturbed HIF-1α/VEGF/eNOS pathway created by CsA.

Human carnosinase 1 overexpression aggravates diabetes and renal impairment in BTBROb/Ob mice

ObjectiveTo assess the influence of serum carnosinase (CN1) on the course of diabetic kidney disease (DKD).MethodshCN1 transgenic (TG) mice were generated in a BTBROb/Ob genetic background to allow the spontaneous development of DKD in the presence of serum carnosinase. The influence of serum CN1 expression on obesity, hyperglycemia, and renal impairment was assessed. We also studied if aggravation of renal impairment in hCN1 TG BTBROb/Ob mice leads to changes in the renal transcriptome as compared with wild-type BTBROb/Ob mice.ResultshCN1 was detected in the serum and urine of mice from two different hCN1 TG lines. The transgene was expressed in the liver but not in the kidney. High CN1 expression was associated with low plasma and renal carnosine concentrations, even after oral carnosine supplementation. Obese hCN1 transgenic BTBROb/Ob mice displayed significantly higher levels of glycated hemoglobin, glycosuria, proteinuria, and increased albumin-creatinine ratios (1104 ± 696 vs 492.1 ± 282.2 μg/mg) accompanied by an increased glomerular tuft area and renal corpuscle size. Gene-expression profiling of renal tissue disclosed hierarchical clustering between BTBROb/Wt, BTBROb/Ob, and hCN1 BTBROb/Ob mice. Along with aggravation of the DKD phenotype, 26 altered genes have been found in obese hCN1 transgenic mice; among them claudin-1, thrombospondin-1, nephronectin, and peroxisome proliferator–activated receptor-alpha have been reported to play essential roles in DKD.ConclusionsOur data support a role for serum carnosinase 1 in the progression of DKD. Whether this is mainly attributed to the changes in renal carnosine concentrations warrants further studies.Key messagesIncreased carnosinase 1 (CN1) is associated with diabetic kidney disease (DKD).BTBROb/Ob mice with human CN1 develop a more aggravated DKD phenotype.Microarray revealed alterations by CN1 which are not altered by hyperglycemia.These genes have been described to play essential roles in DKD.Inhibiting CN1 could be beneficial in DKD.

Nephrotic syndrome due to minimal-change disease superimposed on anti-glomerular basement membrane antibody positive glomerulonephritis; a case report

BackgroundThe prognosis for renal function in anti-GBM glomerulonephritis (anti-GBM GN) is extremely poor, and when renal impairment progresses severely, it is difficult to expect improvement. In addition, it is also known that once the disease activity can be controlled by aggressive treatment, its recurrence is rare. We experienced an anti-GBM GN that improved from severe renal dysfunction and relapsed. A possible cause was the superimpose of nephrotic syndrome due to minimal change disease (MCD).Case presentationA 30-year-old man was admitted to our hospital because of general malaise, fever, oliguria and renal dysfunction. The patient’s laboratory data showed serum creatinine as high as 6.6 mg/dl, and severe inflammation (C-reactive protein 20.6 mg/dl). Anti-glomerular basement membrane antibody (anti-GBM Ab) was detected in his serum, which led to the diagnosis of anti-GBM GN. Treatment was initiated with high-dose glucocorticoid (GC) and plasma exchange therapy (PE), and the patient’s renal function and oliguria improved rapidly and he was discharged 40 days after admission. Renal biopsy findings showed cellular crescents associated with linear IgG depositions along the glomerular tufts compatible with anti-GBM GN, but only about one-third of the glomeruli was involved, suggesting that it still remains an early stage of the disease. However, 2 months after discharge, he had a relapse and was readmitted due to severe proteinuria with positive anti-GBM Ab. On the second admission, after high-dose GC and PE combined with intravenous cyclophosphamide, and remission was achieved. Despite the relatively minor renal biopsy findings, the patient showed rapid renal dysfunction and relatively rapid improvement with our treatment. Electron microscopy of the renal biopsy tissue showed significant foot process effacement on podocytes in the apparently normal glomeruli, without electron dense deposits.ConclusionOn the basis of clinical course and renal pathology, it is suggested that the present case was a rare complication of an early stage of anti-GBM GN and minimal change nephrotic syndrome. Although the simultaneous development of anti-GBM GN and MCD with anti-GBM antibody is unclear, it might have been precipitated by influenza infection or some unknown factor.

Ivabradine Ameliorates Kidney Fibrosis in L-NAME-Induced Hypertension.

Hypertension-induced renal injury is characterized by structural kidney alterations and function deterioration. Therapeutics for kidney protection are limited, thus novel renoprotectives in hypertension are being continuously sought out. Ivabradine, an inhibitor of the If current in the sinoatrial node reducing heart rate (HR), was shown to be of benefit in various cardiovascular pathologies. Yet, data regarding potential renoprotection by ivabradine in hypertension are sparse. Thirty-six adult male Wistar rats were divided into non-diseased controls and rats with NG-nitro-L-arginine methyl ester (L-NAME)-induced hypertension to assess ivabradine's site-specific effect on kidney fibrosis. After 4 weeks of treatment, L-NAME increased the average systolic blood pressure (SBP) (by 27%), decreased glomerular density (by 28%) and increased glomerular tuft area (by 44%). Moreover, L-NAME induced glomerular, tubulointerstitial, and vascular/perivascular fibrosis by enhancing type I collagen volume (16-, 19- and 25-fold, respectively). L-NAME also increased the glomerular type IV collagen volume and the tubular injury score (3- and 8-fold, respectively). Ivabradine decreased average SBP and HR (by 8 and 12%, respectively), increased glomerular density (by 57%) and reduced glomerular tuft area (by 30%). Importantly, ivabradine decreased type I collagen volume at all three of the investigated sites (by 33, 38, and 72%, respectively) and enhanced vascular/perivascular type III collagen volume (by 67%). Furthermore, ivabradine decreased the glomerular type IV collagen volume and the tubular injury score (by 63 and 34%, respectively). We conclude that ivabradine attenuated the alterations of glomerular density and tuft area and modified renal fibrosis in a site-specific manner in L-NAME-hypertension. It is suggested that ivabradine may be renoprotective in hypertensive kidney disease.

Correlation-Guided Network for Fine-Grained Classification of Glomerular lesions in Kidney Histopathology Images.

Chronic Kidney Disease has become a worldwide public health problem which demands careful assessments by pathologists. In this paper, we propose a novel architecture for fine-grained classification of glomerular lesions in renal pathology images sampling from patients with IgA nephropathy. The adversarial correlation loss is innovatively presented to guide a parallel convolutional neural network. In this well- designed loss function, bias between the prediction and the label was take into account while the relationship among different categories is well-aligned. Glomerular lesions in this study are divided into five subcategories, Neg (Negative samples such as tubule and artery), SS (sclerosis involving a portion of the glomerular tuft), GS (sclerosis involving 100% of the tuft), C (build-up of more than two layers of cells within Bowman's space, often with fibrin and collagen deposition) and NOA (none of above). Our model with 93.0% accuracy and 92.9% Fl-score for these five categories has proved superior to other models through experimental results.

Effect of Age on Histological Changes of Urogenital Tract in Albino Rats

This study was conducted to investigate the histologic changes of kidney, urinary bladder, testis, seminal vesicle and prostate gland of rats of various ages (6,12 and 18 months old). Results illustrated variable changes of kidney represented by congestion of renal capillaries, acute tubular necrosis, shrinkage of glomerular tuft, degeneration in tubular epithelium of the 6,12 and 18 months old rats, the morphometric changes of the glomerular diameter decreases with advanced age. The lesions of urinary bladder characterized by hyperplasia of the epithelial lining, papillae into the lumen, hypertrophy of muscle fibers, desquamation of epithelium, cystitis in addition to thickening of the mucosa and hyalinization of muscle fibers of the 6,12 and 18 months old rats. The thickness of the bladder wall showed a significant changes increase at (P≤0.05) with age, while morphometric analysis did not show any age related changes in the bladder muscle thickness. In testis there were congestion of blood vessels, a spermia, degeneration of the spermatocytes and spermatozoa, thickening of interstitial and hyperplasia of leydig cells of the 6,12 and 18 months old rats. The wall thickness of Seminiferous tubule increases with age and basement membrane thickness and tubular diameter decreased with age. The seminal vesicle of the 6,12 and 18 months old rats revealed hyperplasia of glandular epithelium. Lesions of the prostate gland of 6,12 and 18 months old rats showed epithelial hyperplasia which extended as finger-like projections and presence of variable amounts of colloid substance.

DIAGNOSIS OF MYXOBOLUS BRAMAE (MYXOSPOREA: MYXOBOLIDAE) IN THE KIDNEYS TISSUE OF CARASOBARBUS LUTEUS AND HISTOPATHOLOGICAL CHANGES ASSOCIATED WITH INFECTION

This study was conducted during the period from March till the end of October 2018, to study the histopathological changes of Myxobolus bramae in kidney tissue of Carasobarbus luteus caught from Tigris River passing through Baghdad city. During the period of this study, a total of 60 fishes belonging to Carasobarbus luteus species from the family Cyprinidae were collected. The prevalence of infection with these protozoa was determinate (5.00%). Histopathological study due to M. bramae in the kidney tissue of C. luteus was done by using three types of stain: Hemotoxylin and eosin, giemsa and acid fast stain to observe plasmodia cyst and structures of spores. These changes characterized by tubular degeneration, necrosis, hyalinization of glomerular tuft, mild distension of Bowman᾿s space with a reduction in haemopoitic tissue together with inflammatory response, and accumulation of melanomacrophages at the site of infection. The results of this study revealed that Carasobarbus luteus from Tigris River at Baghdad city, it infected with Myxobolus bramae and this parasite cause severe histopathological changes in the infected kidneys tissue.

Proteomic Analysis Identifies Distinct Glomerular Extracellular Matrix in Collapsing Focal Segmental Glomerulosclerosis.

The mechanisms leading to extracellular matrix (ECM) replacement of areas of glomerular capillaries in histologic variants of FSGS are unknown. This study used proteomics to test the hypothesis that glomerular ECM composition in collapsing FSGS (cFSGS) differs from that of other variants. ECM proteins in glomeruli from biopsy specimens of patients with FSGS not otherwise specified (FSGS-NOS) or cFSGS and from normal controls were distinguished and quantified using mass spectrometry, verified and localized using immunohistochemistry (IHC) and confocal microscopy, and assessed for gene expression. The analysis also quantified urinary excretion of ECM proteins and peptides. Of 58 ECM proteins that differed in abundance between cFSGS and FSGS-NOS, 41 were more abundant in cFSGS and 17 in FSGS-NOS. IHC showed that glomerular tuft staining for cathepsin B, cathepsin C, and annexin A3 in cFSGS was significantly greater than in other FSGS variants, in minimal change disease, or in membranous nephropathy. Annexin A3 colocalized with cathepsin B and C, claudin-1, phosphorylated ERK1/2, and CD44, but not with synaptopodin, in parietal epithelial cells (PECs) infiltrating cFSGS glomeruli. Transcripts for cathepsins B and C were increased in FSGS glomeruli compared with normal controls, and urinary excretion of both cathepsins was significantly greater in cFSGS compared with FSGS-NOS. Urinary excretion of ECM-derived peptides was enhanced in cFSGS, although in silico analysis did not identify enhanced excretion of peptides derived from cathepsin B or C. ECM differences suggest that glomerular sclerosis in cFSGS differs from that in other FSGS variants. Infiltration of activated PECs may disrupt ECM remodeling in cFSGS. These cells and their cathepsins may be therapeutic targets.

Toll-like receptor-2 mediates systemic inflammation in gentamicin-induced rat nephrotoxicity.

Gentamicin is an aminoglycoside antibiotic commonly administrated to patients with Gram-negative infections. Gentamicin induced nephrotoxicity by functional and structural impairment. Toll-like receptors (TLRs) as key mediators in the innate and adaptive immune system response involved in gentamicin-induced nephrotoxicity. The present study aimed to investigate the gene expression of TLR2 and pro-inflammatory cytokines in the renal tissues and buffy coat of the whole blood in gentamicin-treated rats. Twenty adult male Sprague Dawley rats weighing 180-200 were randomly divided into gentamicin (100mg/kg, i.p) and control groups (n=10). After 10days, the serum creatinine (Cr) levels and blood urea nitrogen (BUN) were measured. The mRNA levels of TLR2, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and monocyte chemoattractant peptide (MCP)-1 were investigated in the renal tissue and buffy coat by qRT-PCR. Kidney histological analysis performed by hematoxylin-eosin (H&E) staining. Functional disturbance is characterized by a significant increase in the serum levels of Cr and BUN in the gentamicin group. Renal tissue slides of the gentamicin group indicated severe glomerular and tubular damage including lobulation of the glomerular tuft, Bowman's space enlargement, acute tubular necrosis, and proximal tubular destruction. The mRNA levels of IL-1β, TNF-α, MCP-1, and TLR2 increased in the buffy coat, but all of them except TLR2 decreased in the renal tissues in the gentamicin group compared with controls. Gentamicin administration induced relative systemic inflammation, which may be related to an increase in the mRNA levels of TLR2 results in gene expression of pro-inflammatory chemokines and cytokines including IL-1β, TNF-α, and MCP-1 in immune cells.

P0420PARTIAL PHARMACOGENETIC PODOCYTE DEPLETION IN A HIGH-THROUGHPUT ZEBRAFISH MODEL RESEMBLES HUMAN FOCAL AND SEGMENTAL GLOMERULOSCLEROSIS

Abstract Background and Aims Although focal and segmental glomerulosclerosis (FSGS) has been in the scientific focus for many years, it is still a massive burden for patients with no causal therapeutic option. In FSGS, glomerular podocytes are injured, parietal epithelial cells (PECs) are activated and engage in the formation of cellular lesions leading to progressive glomerular scarring. Therefore, novel drug-screening assays are needed. Unfortunately, simple cellular in vitro-based screening assays are not ideal as glomerular architecture and crosstalk between glomerular cells is insufficiently modelled. Therefore, reliable animal models are still required for drug development, which unfortunately are not ideal for high-throughput applications. To date, due to its size, easy maintenance and breeding, zebrafish larvae are the simplest vertebrate model that are used in high-content screenings. Until today, it was unclear whether zebrafish can be used as a model for human FSGS. We therefore aimed to investigate whether partial podocyte-depletion in larval zebrafish leads to formation of FSGS-like disease and if the model can be used for screening purposes. Method We used a transgenic zebrafish model of pharmacogenetic podocyte depletion: In the Tg(nphs2:GAL4), Tg(UAS:Eco.nfsb-mCherry) strain, podocytes express the bacterial nitroreductase under control of the podocin promotor and can be dose-dependently ablated upon administration of metronidazole. Proteinuria was quantified using in vivo confocal laser scanning microscopy of intravenously administered high-molecular-weight fluorescent dextran. Plastic-embedded larvae where histologically and morphometrically assessed using HE, PAS and Jone’s silver staining after metronidazole washout. Glomerular ultrastructure was assessed using transmission electron microscopy of ultrathin sections. Immunofluorescence staining was carried out on kryosections to investigate extracellular matrix deposition (collagen-1, laminin), cellular proliferation (pcna) as well as parietal cell origin and activation (pax2a). Results To partially deplete podocytes, larvae where treated with 80 µM metronidazole from 4-6 days post fertilization, so that a subset of podocytes was depleted. In contrast to controls, podocyte-depleted larvae developed severe whole-body edema (Fig. A). Dynamic in vivo imaging of intravascular 500 kDa fluorescent dextran revealed massive leakage of the glomerular filtration barrier. Ultrastructural and immunofluorescent evaluation showed broad foot process effacement of remaining podocytes (Fig. D) and massive decrease of the slit diaphragm component podocin. Moreover, we found numerous sub-podocyte space pseudocysts (asterisk in Fig. D), microvillous transformation and formation of podocytic tight junctions as well as parietovisceral adhesions of the two layers of Bowman’s capsule. Parietal epithelial cells where activated, changed their phenotype towards a cuboidal shape, began to proliferate as demonstrated by pcna immunofluorescence and where recruited to cellular lesions on the glomerular tuft as demonstrated by the presence of cuboidal pax2a+ cells on the glomerular tuft (arrowheads Fig. B). Moreover, we found significant extracellular matrix deposition by the pax2a+ cells as demonstrated by Jone’s silver staining and laminin immunofluorescence (Fig. C). Conclusion Herein we show that upon podocyte-depletion, zebrafish larvae develop important functional and morphological features of human FSGS such as severe proteinuria and edema, podocyte foot process effacement, activation of parietal epithelial cells which contribute to cellular lesions and deposit extracellular matrix on the glomerular tuft. We conclude that this model resembles the human disease in important features and therefore propose its applicability for a high-throughput drug screening assay for FSGS.

P0110DPP4- INHIBITOR LINAGLIPTIN ACCELERATES RESOLUTION OF CRESCENT FORMATION IN A RAT MODEL OF ANTI-GBM GLOMERULONEPHRITIS

Abstract Background and Aims Crescent formation is a severe glomerular damage in the pathogenesis of different kidney diseases. However the exact pathomechanism of crescent formation is unclear up to now. In human kidney biopsies we observed high dipeptidyl peptidase 4 (DPP4) expression in early crescent formation. Therefore, we investigated the potential involvement of DPP4 in the pathogenesis of crescentic anti-GBM nephritis in a rat model using a DPP4- inhibitor independent of his present use in the treatment of type 2 diabetes. Method Anti-GBM nephritis was induced in Wistar Kyoto rats for investigation of short and long term effects of the DPP4- inhibitor linagliptin treatment (3 mg/kg bw). In the short term experiment (endpoint 2 weeks) linagliptin treatment was started either with model induction or one week later. In the long term experiment (endpoint 8 weeks) linagliptin treatment was started either on day 0 or on week 4. In both experiments we included an untreated healthy and anti-GBM nephritic group. All groups consisted of 11 animals. Kidney function was monitored on weeks 1, 2, 3, 5 and 8. Kidney tissue was used to determine DPP4 expression and activity, morphologic changes and mRNA expression. Results Similarly to our observation in human kidney biopsies the in situ DPP4 activity and DPP4 expression were both increased in nephritic glomeruli especially within the crescents. Linagliptin could almost completely inhibit all renal DPP4 activity without any effect on DPP4 expression. Short-term treatment had no significant effect on crescent formation. However, long-term preventive treatment resulted in reduced number of crescents (51±3% vs 65±3%), glomerulosclerosis (score 1.2±0.07 vs 1.6±0.1), tubulointerstitial injury (score 1.2±0.1 vs 1.8±0.2), renal fibrosis (score 1.3±0.13 vs 1.9±0.14) and proteinuria (265±29 vs 363±22 mg/24h) compared to untreated nephritic rats. Therapeutic intervention with linagliptin resulted in weaker amelioration of renal disease at week 8, but also significantly reduced renal fibrosis (score 1.4±0.13 vs. 1.9±0.14) and crescent formation (52±4% vs. 65±3%) compared with vehicle. About 20% of all glomeruli showed Pax8+ parietal epithelial cells (PECs) on the tuft in short term treatment, being significantly reduced by therapeutic linagliptin treatment on day 14. In the long term experiment the number of Pax8+ PECs on glomerular tuft was reduced by more than 50% in both treatment groups and confirmed by quantitative mRNA analysis using isolated glomeruli. Despite the number of Pax8-positive cells on glomerular tuft was reduced by linagliptin therapy, a subpopulation of PECs positive for the transcription factor SOX9 was significantly increased on glomerular tuft compared to healthy and non-treated rats on day 14. Conclusion DPP4- inhibition with linagliptin reduced Pax8+ cells on glomerular tuft, indicating accelerated resolution of formed crescents and improvement in renal injury. This DDP4 effect might be mediated by differential regulation of a subpopulation of PECs expressing the transcription factor SOX9.

P0719SRC KINASES AGGRAVATE DIABETIC KIDNEY INJURY THROUGH ACTIVATION OF ENDOPLASMIC RETICULUM STRESS

Abstract Background and Aims Diabetic kidney disease (DKD) is the major cause of end-stage kidney disease which is characterized by prominent kidney fibrosis. Src family kinases (SFKs), a family of proto-oncogenes, has been acknowledged to mediate the development of kidney fibrosis. While, several studies in liver and skeletal muscle suggested the role of Src kinases in activating endoplasmic reticulum (ER) stress. The present study aimed to investigate the mechanism of Src kinases-ER stress in mediating the progression of DKD. Method Type 1 diabetes was induced by a single 60 mg/kg i.p injection of streptozotocin (STZ) in 7-week-old male, Sprague-Dawley rats. Diabetic rats received 8-week-treatment of either KF-1607 (30 mg/kg/day), a pharmacological inhibitor of SFKs, or losartan (1 mg/kg/day), a standard treatment for patients with DKD. Results Among SFKs, Fyn and Lyn kinases were particularly increased in the diabetic kidney. Inhibition of Src kinases by KF-1607 improved kidney function and inhibited tubular injury, presented by decreased serum creatinine, albuminuria, and urinary KIM-1 excretion. Pathological changes in the kidney, such as enhanced glomerular volume, tuft area, and fractional mesangial area, were ameliorated in KF-treated rats. Highly-accumulated collagen network as well as increased TGF-β and α-SMA mRNA levels in the diabetic kidney were also significantly reduced in response to KF treatment. Furthermore, it consistently attenuated kidney inflammation and oxidative stress. The renoprotective effects of KF were interestingly similar to those of losartan. We showed increases in protein levels of phosphorylated IRE-1α, ATF6, GRP78 as well as CHOP indicating an exacerbated ER stress in the diabetic kidney. These ER stress markers were significantly decreased in KF treated mice. Conclusion Altogether, Src kinases through activation of ER stress aggravates kidney injury in STZ-induced diabetic rats.

CD44 impacts glomerular parietal epithelial cell changes in the aged mouse kidney.

CD44 contributes to the activation of glomerular parietal epithelial cells (PECs). Although CD44 expression is higher in PECs of healthy aged mice, the biological role of CD44 in PECs in this context remains unclear. Accordingly, young (4 months) and aged (24 months) CD44−/− mice were compared to age‐matched CD44+/+ mice, both aged in a nonstressed environment. Parietal epithelial cell densities were similar in both young and aged CD44+/+ and CD44−/− mice. Phosphorylated ERK 1/2 (pERK) was higher in aged CD44+/+ mice. Vimentin and α‐SMA, markers of changes to the epithelial cell phenotype, were present in PECs in aged CD44+/+ mice, but absent in aged CD44−/− mice in both outer cortical (OC) and juxtamedullary (JM) glomeruli. Because age‐related glomerular hypertrophy was lower in CD44−/− mice, mTOR activation was assessed by phospho‐S6 ribosomal protein (pS6RP) staining. Parietal epithelial cells and glomerular tuft staining for pS6RP was lower in aged CD44−/− mice compared to aged CD44+/+ mice. Podocyte density was higher in aged CD44−/− mice in both OC and JM glomeruli. These changes were accompanied by segmental and global glomerulosclerosis in aged CD44+/+ mice, but absent in aged CD44−/− mice. These results show that the increase in CD44 in PECs in aged kidneys contributes to several changes to the glomerulus during healthy aging in mice, and may involve ERK and mTOR activation.

The Diagnostic Value of the Changes in the Renal Glomerulus in the Postmortem Diagnosis of Freshwater Drownings.

Drowning is one of the major causes of unintentional injury death worldwide. As there are no pathomorphological findings specific to the diagnosis of drowning, definitive postmortem diagnosis of drowning continues to be a significant problem in forensic medicine. This study aims to present an additional diagnostic sign in cases of freshwater drowning by investigating the histopathological changes of renal tissue in the postmortem diagnosis of freshwater drowning. For this purpose, 103 cases were investigated in which the causes of death were freshwater drowning (n = 45), traffic accidents (n = 33) and acute myocardial infarction (n = 25). Renal corpuscular structures of selected cases were examined stereologically and histopathologically. Renal corpuscle diameter, renal corpuscle surface area, glomerular tuft surface area, and Bowman space were calculated by stereological method. When compared with the glomeruli of the control group, renal corpuscle diameter, renal corpuscle surface area, glomerular tuft surface area, and Bowman space values of kidney tissues of all freshwater drowning cases were found to be decreased (P < 0.001). These changes may be considered as an essential histopathological finding in postmortem diagnosis of freshwater drowning cases.