A comparative study of human glomerular basement membrane thickness using direct measurement and orthogonal intercept methods

Introduction Here we report estimates of glomerular basement membrane (GBM) thickness in the Brazilian population performed using direct (DM) and orthogonal interception methods (OIM), and comment on potential sources of variation among estimates made by different laboratories. Methodology A total of 38 patients, ranging from 3 to 78 years of age, 26 (68%) males and 12 (32%) females, were submitted to kidney biopsy procedures for renal disease diagnosis. Glomeruli were diagnosed with minor histological changes by conventional, immunofluorescence and electron microscopy. GBM thickness was estimated using both DM and OIM methods. Results Estimates of GBM thickness obtained using DM were higher than those obtained by OIM. However, the application of a correction for non-perpendicular membrane sectioning to DM estimates yielded similar results to those obtained under OIM. The estimated GMB thickness using DM after correction was 289 + 44 nm, versus 287 + 48 nm by OIM. No statistically significant differences were detected in GMB thickness, nor with respect to patient age or sex. Conclusions GBM thickness in the studied Brazilian population measured approximately 290 nm. The application of criteria for estimating the shortest distance between the endothelial and podocyte cell membranes with correction for non-perpendicular membrane sectioning can increase the accuracy of GBM thickness estimates using DM and OIM.

Renoprotective Effects of Metformin and Its Relationship with Oxidative Stress in Type 2 Diabetic Mice

Background: Oxidative stress has previously been shown to play critical roles in the development of diabetes and its complications. The purpose of this research was to observe the reno-protective effect of metformin and its effect on oxidative stress in type 2 diabetic mice renal tissue.Methods: Type 2 diabetes mellitus mice model was established by High-fat feed combined with small-dose STZ and randomly divided into diabetes model group, Metformin [MET, 250mg/(kg.d)] group, Glibenclamide (GLIB) [GLIB, 2.5mg/(kg.d)] group, and normal control group (NC). After 8 weeks of intervention, blood and urine samples were collected for detection of FBG, HbA1c, urine albumin (Alb), retinol-binding protein (RBP), podocalyxin (PCX), 8-OHdG, 8-iso-PG, and creatinine (Cr). Renal tissue specimens were preserved for observing renal glomerular basement membrane thickness (GBMT) and foot process fusion rate (FPFR) under electron microscopy.Results: Compared with the NC group, FBG, HbA1c, urinary Alb/Cr (UACR), RBP/Cr (URCR), PCX/Cr (UPCR), 8-OHdG /Cr (UOHCR), and 8-iso-PG /Cr (UISOCR) significantly increased in the T2DM group (P <0.05). Compared with the T2DM group, FBG, HbA1c, UACR, URCR, UPCR, UOHCR, and UISOCR were significantly reduced in the GLIB group and MET group (P <0.05). Compared to the GLIB group, UACR, URCR, UPCR, UOHCR, and UISOCR decreased in the MET group (P <0.05), but FBG and HbA1c were not differenced statistically between the two groups. GBMT and FPFR increased in the T2DM group (P <0.05), which were reduced in the MET group and lighter than those in the GLIB group (P <0.05).Conclusion: Metformin intervention can play a reno-protective effect in type 2 diabetic mice, which may be related to its effect in inhibiting enhanced oxidative stress in vivo.

MO052COL4A3/COL4A4 AS A CAUSE OF MULTICYSTIC KIDNEY DISEASE

Background and Aims Thin basement membrane nephropathy (TBMN), the most common cause of persistent microhaematuria (mH), is due to mutations in genes codifying alfa-3 and alfa-4 collagen IV chains (COL4A4/COL4A3). Initially considered as a benign condition, subsequent studies have shown that an important number of patients develop proteinuria and CKD. We reported in a previous small study the presence of multicystic kidney disease (MCD) in some TBMD patients. In this study we aimed to evaluate the presence of MCD in a larger cohort of TBMD patients and analyze its association with renal outcomes. Method We collected 50 patients with a diagnosis of TBMD based on the presence of persistent mH (&gt;5 erythocytes per high power field in more than 90% of urinary sediments and radiological examinations to exclude other causes of mH) and at least one first-degree relative with persistent mH. TBMD diagnosis was confirmed by renal biopsy (glomerular basement membrane thickness less than 150nm) in 18 patients and by genetic test (pathogenic mutations in COL4A3/COL4A4) in 6 patients. MCD was diagnosed by the presence of uncountable cysts on renal ultrasonography. Results Mean age at diagnosis was 43.7 years, 34% were males and 18% had hypertension. At baseline, serum creatinine (SCr) was 0.9 mg/dL, proteinuria 0.48 gr/24h and 9 patients (18%) had CKD (estimated glomerular filtration rate -eGFR- lower than &lt;60 mL/min/1.73m2). 7 patients (14%) had CKD G3 and 2 (4%) CKD G4. Kidney cysts were found in 34 patients (68%) and 19 (38%) met MCD criteria. After a mean follow-up of 14.7±11.5 years, 23 patients (46%) had CKD. Among them, 17 patients (34%) had CKD G3, 2 (4%) CKD G4, and 4 (8%) CKD G5. Hypertension was more frequent among CKD patients as compared with no-CKD patients (39 vs 0%, p 0.00), proteinuria was higher (0.58±0.68 vs 0.39±0.58 g/24h, p 0.05) and MCD more frequent (65.2% vs 14.8%, p 0.00). Patients with MCD had higher SCr (2.1 vs 1.1 mg/dL, p 0.004) and lower eGFR (41.7 vs 77.2 mL/min/1.73m2, p 0.00) at the end of follow-up, and MCD was the only risk factor for the occurrence of CKD (OR 6.49, 95% CI 1.3-31.6) by multivariable analysis that included age, hypertension and proteinuria. Conclusion MCD is frequently observed in TBMD patients and is a risk factor for the progression of CKD.

Reticular Basement Membrane Thickness Is Associated with Growth- and Fibrosis-Promoting Airway Transcriptome Profile-Study in Asthma Patients

Airway remodeling in asthma is characterized by reticular basement membrane (RBM) thickening, likely related to epithelial structural and functional changes. Gene expression profiling of the airway epithelium might identify genes involved in bronchial structural alterations. We analyzed bronchial wall geometry (computed tomography (CT)), RBM thickness (histology), and the bronchial epithelium transcriptome profile (gene expression array) in moderate to severe persistent (n = 21) vs. no persistent (n = 19) airflow limitation asthmatics. RBM thickness was similar in the two studied subgroups. Among the genes associated with increased RBM thickness, the most essential were those engaged in cell activation, proliferation, and growth (e.g., CDK20, TACC2, ORC5, and NEK5) and inhibiting apoptosis (e.g., higher mRNA expression of RFN34, BIRC3, NAA16, and lower of RNF13, MRPL37, CACNA1G). Additionally, RBM thickness correlated with the expression of genes encoding extracellular matrix (ECM) components (LAMA3, USH2A), involved in ECM remodeling (LTBP1), neovascularization (FGD5, HPRT1), nerve functioning (TPH1, PCDHGC4), oxidative stress adaptation (RIT1, HSP90AB1), epigenetic modifications (OLMALINC, DNMT3A), and the innate immune response (STAP1, OAS2). Cluster analysis revealed that genes linked with RBM thickness were also related to thicker bronchial walls in CT. Our study suggests that the pro-fibrotic profile in the airway epithelial cell transcriptome is associated with a thicker RBM, and thus, may contribute to asthma airway remodeling.

CORRELATION BETWEEN OXIDATIVE STRESS AND ANTIOXIDANT IN DIABETIC NEPHROPATHY

Background: Diabetic Nephropathy is consider as one of the major micro-vascular problems of diabetes mellitus and has become the most general single factor of end stage of kidney disease. It is defined traditionally by kidney morphological and modification like: glomerular hyper filtration, glomerular and kidney hypertrophy, increased urinary albumin excretion (> 300 mg/24 hours), increased GBM (Glomerular Basement Membrane) thickness and mesangial expansion and also accumulation of extracellular proteins comprising laminin, collagens and fibronectin worldwide. Oxidative stress (OS) has been characterized as the imbalance between reactive oxygen species (ROS) yielding and the possessive antioxidant defense system. Objective of the study: Correlation between oxidative stress and antioxidant in diabetic nephropathy. Materials and methods: The investigation was conducted on 100 DN subjects of both sex and aged 20 or more and 100 age and sex matched healthy control subjects. MDA, SOD and Catalase of each subject was measured. Results: the present investigation shows that the MDA was elevated significantly and SOD and Catalase level was found to be significantly low in DN individuals as compared to controls. Conclusion: This study concluded that the MDA could be better marker for early recognition of DN. Keywords: Diabetic nephropathy (DN), MDA, SOD, Catalase, Kidney disease

Active integrins regulate white adipose tissue insulin sensitivity and brown fat thermogenesis

Reorganization of the extracellular matrix is a prerequisite for healthy adipose tissue expansion, whereas fibrosis is a key feature of adipose dysfunction and inflammation. However, very little is known about the direct effects of impaired cell-matrix interaction in adipocyte function and insulin sensitivity. Using adipose selective deletion of β1 integrin (Itgb1adipo-cre) and Kindlin-2 (Kind2adipo-cre), we demonstrate here that active β1 and β3 integrins directly interact with the insulin receptor to regulate white adipocyte insulin action and systemic metabolism. Consequently, loss of adipose integrin activity, similar to loss of adipose insulin receptors, results in lipodystrophy and systemic insulin resistance. Conversely, we find that brown adipose tissue of Kind2adipo-cre and Itgb1adipo-cre mice is chronically hyperactivated, and has increased substrate delivery, reduced endothelial basement membrane thickness, and increased endothelial vesicular transport. Thus, we establish integrin-extracellular matrix interactions as key regulators of white and brown adipose tissue function and whole body metabolism.

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.

Targeting NF-κB by the Cell-Permeable NEMO-Binding Domain Peptide Improves Albuminuria and Renal Lesions in an Experimental Model of Type 2 Diabetic Nephropathy

Diabetic nephropathy (DN) is a multifactorial disease characterized by hyperglycemia and close interaction of hemodynamic, metabolic and inflammatory factors. Nuclear factor-κB (NF-κB) is a principal matchmaker linking hyperglycemia and inflammation. The present work investigates the cell-permeable peptide containing the inhibitor of kappa B kinase γ (IKKγ)/NF-κB essential modulator (NEMO)-binding domain (NBD) as therapeutic option to modulate inflammation in a preclinical model of type 2 diabetes (T2D) with DN. Black and tan, brachyuric obese/obese mice were randomized into 4 interventions groups: Active NBD peptide (10 and 6 µg/g body weight); Inactive mutant peptide (10 µg/g); and vehicle control. In vivo/ex vivo fluorescence imaging revealed efficient delivery of NBD peptide, systemic biodistribution and selective renal metabolization. In vivo administration of active NBD peptide improved albuminuria (>40% reduction on average) and kidney damage, decreased podocyte loss and basement membrane thickness, and modulated the expression of proinflammatory and oxidative stress markers. In vitro, NBD blocked IKK-mediated NF-κB induction and target gene expression in mesangial cells exposed to diabetic-like milieu. These results constitute the first nephroprotective effect of NBD peptide in a T2D mouse model that recapitulates the kidney lesions observed in DN patients. Targeting IKK-dependent NF-κB activation could be a therapeutic strategy to combat kidney inflammation in DN.

Independent apical and basal mechanical systems determine cell and tissue shape in the Drosophila wing disc

How cell shape and mechanics are organized in three dimensions during tissue morphogenesis is poorly understood. In the Drosophila wing imaginal disc, we examined the mechanical processes that determine the shape of epithelial cells. Since it has been known that basement membrane influences the mechanics intracellularly, we reexamined the material properties of the basement membrane with fluorescence and transmission electron microscopy in its native environment. Further, we investigated the effect on cell shape and tissue mechanics when disruptions were instigated at three different time scales: (1) short (seconds with laser cutting), (2) medium (minutes with drug treatments), and (3) long (days with RNAi interference). We found regions in which the basement membrane is much thicker and heterogeneous than previously reported. Disrupting the actin cytoskeleton through drug treatment affects cell shape only at the apical surface, while the shapes in the medial and basal surfaces were not altered. In contrast, when integrin function was inhibited via RNAi or basement membrane integrity was disrupted by drug treatment, the medial and basal cell shapes were affected. We propose that basement membrane thickness patterns determine the height and basal surface area of cells and the curvature of folds in the wing disc. Based on these findings and previous studies, we propose a model of how cell shapes and tissue properties were determined by highly local, modular apical and basal mechanics.Graphical abstract