Glomerular Basement Membrane Research Articles

Exploration on the protective effect of liraglutide on renal injury in diabetic nephropathy rats based on ROS-NLRP3 inflammasomes

Objective: To investigate the effect of liraglutide on diabetic nephropathy in rats and its regulatory mechanism. Methods: The diabetic nephropathy rat model was constructed with high-glucose-high-fat diet in combination with STZ, and was randomly divided into normal saline group and liraglutide group. The rats in liraglutide group were given sc 200 μg/kg of liraglutide, and the rats in normal saline group were given sc 20 mg/kg of normal saline twice a day for 4 weeks. The normal control group was not treated with any treatment. The biochemical indexes such as rat body weight, 24-hour urine total protein quantification (UTP), fasting blood glucose (FPG), triglyceride (TG), cholesterol (TC), blood urea nitrogen (BUN) and serum creatinine (Scr) were measured. HE staining, Masson staining and PAS staining were used to observe the pathologically morphological changes in renal tissues. Western-blot was used to detect the expression of NLRP3 inflammasome-related protein in renal tissues. Elisa was used to measure the serum levels of interleukin-18 (IL-18) and IL-1β. SPSS 26.0 statistical software and Graph Prism 9.0 software were used for analysis and mapping. The t-test was used for the comparison of measurement data between two groups, one-way ANOVA was used for the comparison among multiple groups, and Tukey’s test was used for the comparison in the same group. Results: Compared with the normal saline group, FBG, UTP, BUN, Scr, TC and TG in the liraglutide group were significantly decreased (p < .01), the glomerular basement membrane was slightly thickened, with the tubular lumen slightly dilated and the lesion damage alleviated; the expression levels of NLRP3, ASC and caspase-1 inflammasome-related proteins were decreased (p < .01), and the levels of IL-18 and IL-1β were decreased (p < .01).Conclusion: Liraglutide can inhibit the activation of NLRP3 inflammasome mediated by oxidative stress in renal tissues through ROS-NLRP3 inflammasome pathway, thereby inhibiting the inflammation, and finally playing an anti-diabetic nephropathy renal injury role.

Digital pathology assessment of kidney glomerular filtration barrier ultrastructure in an animal model of podocytopathy.

Transmission electron microscopy (TEM) images can visualize kidney glomerular filtration barrier ultrastructure, including the glomerular basement membrane (GBM) and podocyte foot processes (PFP). Podocytopathy is associated with glomerular filtration barrier morphological changes observed experimentally and clinically by measuring GBM or PFP width. However, these measurements are currently performed manually. This limits research on podocytopathy disease mechanisms and therapeutics due to labor intensiveness and inter-operator variability. We developed a deep learning-based digital pathology computational method to measure GBM and PFP width in TEM images from the kidneys of Integrin-Linked Kinase (ILK) podocyte-specific conditional knockout (cKO) mouse, an animal model of podocytopathy, compared to wild-type (WT) control mouse. We obtained TEM images from WT and ILK cKO littermate mice at 4 weeks old. Our automated method was composed of two stages: a U-Net model for GBM segmentation, followed by an image processing algorithm for GBM and PFP width measurement. We evaluated its performance with a 4-fold cross-validation study on WT and ILK cKO mouse kidney pairs. Mean (95% confidence interval) GBM segmentation accuracy, calculated as Jaccard index, was 0.73 (0.70-0.76) for WT and 0.85 (0.83-0.87) for ILK cKO TEM images. Automated and manual GBM width measurements were similar for both WT (p=0.49) and ILK cKO (p=0.06) specimens. While automated and manual PFP width measurements were similar for WT (p=0.89), they differed for ILK cKO (p<0.05) specimens. WT and ILK cKO specimens were morphologically distinguishable by manual GBM (p<0.05) and PFP (p<0.05) width measurements. This phenotypic difference was reflected in the automated GBM (p<0.05) more than PFP (p=0.06) widths. These results suggest that certain automated measurements enabled via deep learning-based digital pathology tools could distinguish healthy kidneys from those with podocytopathy. Our proposed method provides high-throughput, objective morphological analysis and could facilitate podocytopathy research and translate into clinical diagnosis.

Fabry nephropathy: a treatable cause of chronic kidney disease

Fabry disease is a rare X-linked inborn error of metabolism that has a high prevalence of chronic kidney disease (CKD) and renal failure. It is due to the deficiency of the α-galactosidase A (α-Gal) lysosomal enzyme with subsequent accumulation of globotriaosylceramide (Gb3) in lysosomes. In the kidney, the podocyte is the main target of this disease, although all cell types are involved. The podocyte, being terminally differentiated, does not replicate and thus accumulates Gb3 throughout life. Podocytes are injured by Gb3, leading to their detachment from the glomerular basement membrane and subsequent loss in the urine. Albuminuria starts in childhood and progresses to overt proteinuria in the teens and 20 s. CKD ensues with adults starting dialysis at an average age of 42 years. Patients have a high prevalence of stroke and cardiomyopathy with hypertrophic change, heart failure, and dysrhythmias. Patient survival is limited in both genders. Diagnosis is based on the demonstration of a low α-Gal activity and a pathogenic GLA mutation. Clinical features are highly variable, which makes recognition of this condition difficult. Treatment with intravenous recombinant human enzyme replacement therapy (ERT) and oral pharmacologic chaperone are available. Control of proteinuria to 0.5 g/day or less is of critical importance to limit progression to end-stage renal disease. Early initiation of treatment gives the best results, but the optimal age to start is uncertain. Fabry nephropathy remains a challenge due to its multisystem nature, difficult diagnosis, and complicated management. It is important as a treatable cause of CKD.

Quantifiable and reproducible phenotypic assessment of a constitutive knockout mouse model for congenital nephrotic syndrome of the Finnish type

Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of childhood chronic kidney disease. Congenital nephrotic syndrome of the Finnish type (CNF) (MIM# 256300) is caused by biallelic variants in the gene NPHS1, encoding nephrin, an integral component of the kidney filtration barrier. No causal treatments exist, and children inevitably require kidney replacement therapy. In preparation for gene replacement therapy (GRT) in CNF, we established a quantifiable and reproducible phenotypic assessment of the nephrin-deficient CNF mouse model: 129/Sv-Nphs1tm1Rkl/J. We assessed the phenotypic spectrum of homozygous mice (Nphs1tm1Rkl/Nphs1tm1Rkl) compared to heterozygous controls (Nphs1tm1Rkl/Nphs1WT) by the following parameters: 1. cohort survival, 2. podocyte foot process (FP) density per glomerular basement membrane (GBM) using transmission electron microscopy, 3. tubular microcysts in brightfield microscopy, and 4. urinary albumin/creatinine ratios. Nphs1tm1Rkl/Nphs1tm1Rkl mice exhibited: 1. perinatal lethality with median survival of 1 day, 2. FP effacement with median FP density of 1.00 FP/µm GBM (2.12 FP/µm in controls), 3. tubular dilation with 65 microcysts per section (6.5 in controls), and 4. increased albumin/creatinine ratio of 238 g/g (4.1 g/g in controls). We here established four quantifiable phenotyping features of a CNF mouse model to facilitate future GRT studies by enabling sensitive detection of phenotypic improvements.

Four novel mutations identified in the COL4A3, COL4A4 and COL4A5 genes in 10 families with Alport syndrome

BackgroundAlport syndrome (AS) is an inherited nephropathy caused by mutations in the type IV collagen genes. It is clinically characterized by damage to the eyes, ears and kidneys. Diagnosis of AS is hampered by its atypical clinical picture, particularly when the typical features, include persistent hematuria and microscopic changes in the glomerular basement membrane (GBM), are the only clinical manifestations in the patient.MethodsWe screened 10 families with suspected AS using whole exome sequencing (WES) and analyzed the harmfulness, conservation, and protein structure changes of mutated genes. In further, we performed in vitro functional analysis of two missense mutations in the COL4A5 gene (c.2359G > C, p.G787R and c.2605G > A, p.G869R).ResultsWe identified 11 pathogenic variants in the type IV collagen genes (COL4A3, COL4A4 and COL4A5). These pathogenic variants include eight missense mutations, two nonsense mutations and one frameshift mutation. Notably, Family 2 had digenic mutations in the COL4A3 (p.G1170A) and UMOD genes (p.M229K). Family 3 had a digenic missense mutation (p.G997E) in COL4A3 and a frameshift mutation (p.P502L fs*151) in COL4A4. To our knowledge, four of the 11 mutations are novel mutations. In addition, we found that COL4A5 mutation relation mRNA levels were significantly decreased in HEK 293 T cell compared to control, while the cellular localization remained the same.ConclusionsOur research expands the spectrum of COL4A3-5 pathogenic variants, which is helpful for clinical and scientific research.

A Comparative Histological Study Between the Protective and the Therapeutic Effect of Probiotic on Cisplatin Induced Nephrotoxicity in Adult Male Albino Rat

Abstract Introduction Although cisplatin (CP) is one of the most effective chemotherapies, it is associated with significant nephrotoxicity (Acute kidney injury). Lactobacillus plantarum is often used as a probiotic on its own for its beneficial properties and anti-inflammatory activity. Aim to compare the protective and therapeutic role of probiotics on the microscopic structure of the kidney in a model of cisplatin-induced nephrotoxicity. Material and Methods thirty-five adult male albino rats weighing (200-220gm) were divided into 4 groups. Group I (control group), Group II (Cisplatin group): rats received intraperitoneal injection of CP (7.5 mg/kg) for 3 days. Group III (protective group): Rats received 1 mL of probiotic orally for 7 days. From the fifth day, the rats received intraperitoneal injections of CP (7.5mg/kg) for 3 days. Group IV (Therapeutic group): Rats received intraperitoneal injection of CP for 3 days. From the fourth day, the rats received probiotic L. plantarum orally at a dose of (1 mL/day) for 14 days. The concentration of probiotics in group III and group IV was 109 Colony forming units (CFU)/mL/100 g of body weight. Kidney specimens were proceeded for light microscopic examination, as well as Morphometrical and statistical analyses. Results H&amp;E-stained sections of Group II showed marked renal affection in which most glomeruli showed atrophy. Renal tubules were mostly distorted, necrotic, and degenerative with interstitial hemorrhage, congestion, and cellular infiltration. PAS-stained sections showed marked increased reaction in glomerular and tubular Basement membranes with an apparent loss of Brush borders of most of the PCTs. Masson`s trichrome-stained sections showed a marked increase in collagen fibers in renal interstitia mostly around blood vessels. Immunohistochemically stained sections showed a marked increase in positive cytoplasmic reaction to caspase -3 antibodies mostly in renal tubules. On the other hand, the renal structure of The protective group showed marked improvement which appeared nearly comparable to that of the control group while the therapeutic group showed minimal improvement. Conclusion The protective effect of probiotics on cisplatin-induced Acute Kidney Injury (Nephrotoxicity) was markedly better than the therapeutic effect as detected by microscopic, morphometric, and statistical studies.

Human umbilical cord mesenchymal stem cells attenuate diabetic nephropathy through the IGF1R-CHK2-p53 signalling axis in male rats with type 2 diabetes mellitus.

Diabetes mellitus (DM) is a disease syndrome characterized by chronic hyperglycaemia. A long-term high-glucose environment leads to reactive oxygen species (ROS) production and nuclear DNA damage. Human umbilical cord mesenchymal stem cell (HUcMSC) infusion induces significant antidiabetic effects in type 2 diabetes mellitus (T2DM) rats. Insulin-like growth factor 1 (IGF1) receptor (IGF1R) is important in promoting glucose metabolism in diabetes; however, the mechanism by which HUcMSC can treat diabetes through IGF1R and DNA damage repair remains unclear. In this study, a DM rat model was induced with high-fat diet feeding and streptozotocin (STZ) administration and rats were infused four times with HUcMSC. Blood glucose, interleukin-6 (IL-6), IL-10, glomerular basement membrane, and renal function were examined. Proteins that interacted with IGF1R were determined through coimmunoprecipitation assays. The expression of IGF1R, phosphorylated checkpoint kinase 2 (p-CHK2), and phosphorylated protein 53 (p-p53) was examined using immunohistochemistry (IHC) and western blot analysis. Enzyme-linked immunosorbent assay (ELISA) was used to determine the serum levels of 8-hydroxydeoxyguanosine (8-OHdG). Flow cytometry experiments were used to detect the surface markers of HUcMSC. The identification of the morphology and phenotype of HUcMSC was performed by way of oil red "O" staining and Alizarin red staining. DM rats exhibited abnormal blood glucose and IL-6/10 levels and renal function changes in the glomerular basement membrane, increased the expression of IGF1 and IGF1R. IGF1R interacted with CHK2, and the expression of p-CHK2 was significantly decreased in IGF1R-knockdown cells. When cisplatin was used to induce DNA damage, the expression of p-CHK2 was higher than that in the IGF1R-knockdown group without cisplatin treatment. HUcMSC infusion ameliorated abnormalities and preserved kidney structure and function in DM rats. The expression of IGF1, IGF1R, p-CHK2, and p-p53, and the level of 8-OHdG in the DM group increased significantly compared with those in the control group, and decreased after HUcMSC treatment. Our results suggested that IGF1R could interact with CHK2 and mediate DNA damage. HUcMSC infusion protected against kidney injury in DM rats. The underlying mechanisms may include HUcMSC-mediated enhancement of diabetes treatment via the IGF1R-CHK2-p53 signalling pathway.

The Functional Role of microRNAs and mRNAs in Diabetic Kidney Disease: A Review.

Diabetes is a group of diseases marked by poor control of blood glucose levels. Diabetes mellitus (DM) occurs when pancreatic cells fail to make insulin, which is required to keep blood glucose levels stable, disorders, and so on. High glucose levels in the blood induce diabetic effects, which can cause catastrophic damage to bodily organs such as the eyes and lower extremities. Diabetes is classified into many forms, one of which is controlled by hyperglycemia or Diabetic Kidney Disease (DKD), and another that is not controlled by hyperglycemia (nondiabetic kidney disease or NDKD) and is caused by other factors such as hypertension, hereditary. DKD is associated with diabetic nephropathy (DN), a leading cause of chronic kidney disease (CKD) and end-stage renal failure. The disease is characterized by glomerular basement membrane thickening, glomerular sclerosis, and mesangial expansion, resulting in a progressive decrease in glomerular filtration rate, glomerular hypertension, and renal failure or nephrotic syndrome. It is also represented by some microvascular complications such as nerve ischemia produced by intracellular metabolic changes, microvascular illness, and the direct impact of excessive blood glucose on neuronal activity. Therefore, DKD-induced nephrotic failure is worse than NDKD. MicroRNAs (miRNAs) are important in the development and progression of several diseases, including diabetic kidney disease (DKD). These dysregulated miRNAs can impact various cellular processes, including inflammation, fibrosis, oxidative stress, and apoptosis, all of which are implicated during DKD. MiRNAs can alter the course of DKD by targeting several essential mechanisms. Understanding the miRNAs implicated in DKD and their involvement in disease development might lead to identifying possible therapeutic targets for DKD prevention and therapy. Therefore, this review focuses specifically on DKD-associated DN, as well as how in-silico approaches may aid in improving the management of the disease.

Emerging Therapeutics Targeting Cellular Stress Pathways to Mitigate End-Organ Damage in Type 1 Diabetes

Type 1 diabetes (T1D) is an autoimmune disease characterized by insulin deficiency and impaired glucose regulation. While daily insulin therapy is life-saving, many patients struggle to achieve optimal glycemic control, leading to microvascular complications affecting various organs, including the kidneys and the liver. This review aims to summarize the current state of knowledge regarding the pathogenesis of hepatic and renal complications in T1D, highlight recent advances in potential therapeutic targets, and provide evidence-based recommendations for mitigating end-organ damage. Chronic hyperglycemia drives diabetic complications through several interrelated mechanisms, including increased polyol pathway flux, advanced glycation end-product (AGE) formation, protein kinase C activation, and mitochondrial reactive oxygen species overproduction. In the liver, these processes contribute to non-alcoholic fatty liver disease, with up to 50% of T1D patients developing hepatic steatosis. Diabetic nephropathy, affecting 25%–40% of long-term T1D patients, is characterized by glomerular basement membrane thickening, mesangial expansion, and tubulointerstitial fibrosis. Recent innovations in T1D management include genomics and precision medicine approaches, gut microbiome modulation, nanomedicine, and artificial intelligence-driven glucose monitoring systems. Emerging immunotherapies aim to fundamentally modify the autoimmune response in T1D. Mitigating T1D complications requires intensive glycemic control, targeted pharmacotherapy, and lifestyle modifications. Emerging therapies and precision medicine approaches offer promising avenues. Ongoing research into molecular mechanisms remains crucial for developing novel interventions and improving long-term outcomes in T1D patients.

Glomerulus-Targeted ROS-Responsive Polymeric Nanoparticles for Effective Membranous Nephropathy Therapy.

Membranous nephropathy (MN) is a common immune-mediated glomerular disease that requires the development of safe and highly effective therapies. Celastrol (CLT) has shown promise as a therapeutic molecule candidate, but its clinical use is currently limited due to off-target toxicity. Given that excess levels of reactive oxygen species (ROS) contributing to podocyte damage is a key driver of MN progression to end-stage renal disease, we rationally designed ROS-responsive cationic polymeric nanoparticles (PPS-CPNs) with a well-defined particle size and surface charge by employing poly(propylene sulfide)-polyethylene glycol (PPS-PEG) and poly(propylene sulfide)-polyethylenimine (PPS-PEI) to selectively deliver CLT to the damaged glomerulus for MN therapy. Experimental results show that PPS-CPNs successfully crossed the fenestrated endothelium, accumulated in the glomerular basement membrane (GBM), and were internalized by podocytes where rapid drug release was triggered by the overproduction of ROS, thereby outperforming nonresponsive CLT nanotherapy to alleviate subepithelial immune deposits, podocyte foot process effacement, and GBM expansion in a rat MN model. Moreover, the ROS-responsive CLT nanotherapy was associated with significantly lower toxicity to major organs than free CLT. These results suggest that encapsulating CLT into PPS-CPNs can improve efficacy and reduce toxicity as a promising treatment option for MN.

Loss of 3-O-sulfotransferase enzymes, Hs3st3a1 and Hs3st3b1, reduces kidney and glomerular size and disrupts glomerular architecture

Heparan sulfate (HS) is an important component of the kidney anionic filtration barrier, the glomerular basement membrane (GBM). HS chains attached to proteoglycan protein cores are modified by sulfotransferases in a highly ordered series of biosynthetic steps resulting in immense structural diversity due to negatively charged sulfate modifications. 3-O-sulfation is the least abundant modification generated by a family of seven isoforms but creates the most highly sulfated HS domains. We analyzed the kidney phenotypes in the Hs3st3a1, Hs3st3b1 and Hs3st6 -knockout (KO) mice, the isoforms enriched in kidney podocytes. Individual KO mice show no overt kidney phenotype, although Hs3st3b1 kidneys were smaller than wildtype (WT). Furthermore, Hs3st3a1-/-; Hs3st3b1-/- double knockout (DKO) kidneys were smaller but also had a reduction in glomerular size relative to wildtype (WT). Mass spectrometry analysis of kidney HS showed reduced 3-O-sulfation in Hs3st3a1-/- and Hs3st3b1-/-, but not in Hs3st6-/- kidneys. Glomerular HS showed reduced HS staining and reduced ligand-and-carbohydrate engagement (LACE) assay, a tool that detects changes in binding of growth factor receptor-ligand complexes to HS. Interestingly, DKO mice have increased levels of blood urea nitrogen, although no differences were detected in urinary levels of albumin, creatinine and nephrin. Finally, transmission electron microscopy showed irregular and thickened GBM and podocyte foot process effacement in the DKO compared to WT. Together, our data suggest that loss of 3-O-HS domains disrupts the kidney glomerular architecture without affecting the glomerular filtration barrier and overall kidney function.

Heavy-chain deposition disease associated with multiple myeloma: a case report

Heavy-chain deposition disease (HCDD) is a rare disorder characterized by the deposition of monoclonal immunoglobulin. Due to the disease's rarity and diagnostic challenges, its prognosis is generally poor. Herein, we report a case of successful treatment of HCDD coexisting with multiple myeloma. A 56-year-old man presented at an outpatient clinic with complaints of whole-body pain, edema, and dyspnea that had persisted for 2 weeks. Diagnostic tests confirmed nephrotic syndrome, hematuria, and progressive renal failure. Serum immunofixation electrophoresis identified the presence of IgG kappa paraprotein. A diagnosis of multiple myeloma was established following a bone marrow biopsy. A renal biopsy revealed antibodies specific to the heavy chains of IgG on immunofluorescence, and electron microscopy showed diffuse electron-dense "powdery" densities in the glomerular basement membrane. Based on these findings, the patient was diagnosed with both HCDD and multiple myeloma. Following the diagnosis, the patient immediately began anti-plasma cell therapy using bortezomib, lenalidomide, and dexamethasone. Intermittent hemodialysis was initiated due to persistent azotemia during the diagnostic process; however, renal function improved significantly after only 1 month of therapy, allowing the discontinuation of dialysis. Early intervention with anti-plasma cell therapy, such as bortezomib, is known to improve prognosis in the early stages of the disease. This case report is presented to enhance understanding of HCDD and underscore the importance of prompt diagnosis and treatment in managing this rare condition.

Rosehip tea as a possible cause of acute interstitial nephritis

BackgroundAcute interstitial nephritis (AIN) is characterized by an inflammatory infiltrate in the renal interstitium. The etiology of AIN is broad with the most common cause being medications. Treatment includes removal of the offending agent, glucocorticoid therapy, and immunosuppression. Rosehips are a fruit produced from wild roses that are often used in teas and herbal supplements. They are reported to have anti-inflammatory effects and sought after for this purported benefit.Case presentationWe present a case of a 67-year-old woman who presented with acute kidney injury after two months of excessive rosehip tea consumption. She was dialyzed for 1 month and treated with oral steroids. Her initial renal biopsy revealed AIN, a thin glomerular basement membrane, and five nodular sclerosed glomeruli. Her work-up was negative for known causes of AIN and the etiology was determined to be rosehip tea. Two months after initial presentation she continued to have clinical and pathologic signs of AIN. She was started on mycophenolate mofetil (MMF) with clinical improvement.ConclusionRosehip tea is known to have diuretic properties and, when consumed, may lead to a self-perpetuating cycle of intake. Excessive consumption may lead to increased concentrations of arachidonic acid and its metabolites. These metabolites have been shown to perpetuate an inflammatory response capable of causing AIN. Providers should be aware when counseling patients that excessive intake of rosehip tea may lead to AIN.

Element Concentrations and Histopathology of Liver and Kidney in West Greenland Ringed Seals (Pusa hispida).

Ringed seals are consumed in Greenland and are therefore included as a key biomonitoring species with the focus on pollution exposure and health effects. Ringed seals in Central West Greenland (Qeqertarsuaq) and in North West Greenland (Qaanaaq) were analyzed for metal concentrations in the liver and histological changes in the liver and kidney. The mean liver concentration of mercury in Qaanaaq was 3.73 ± 5.01 µg/g ww (range: 0.28-23.29 µg/g ww), and the mean cadmium concentration was 7.80 ± 8.95 µg/g ww (range: 0.013-38.79 µg/g ww). For Qeqertarsuaq, the liver concentration of mercury was 1.78 ± 1.70 µg/g ww (range: 0.45-8.00 µg/g ww) and the mean cadmium concentration was 11.58 ± 6.32 µg/g ww (range: 0.11-25.45 µg/g ww). Age had a positive effect on the liver concentrations of metals, while no effect was found for sex or histological changes. The prevalence of histological changes in liver tissue decreased in the following order: random pattern mononuclear cell infiltration (92.1%), portal cell infiltration (68.4%), hepatic intracellular fat (18.4%), portal fibrosis (7.9%), focal hepatic fibrosis (7.9%), bile duct hyperplasia/fibrosis (7.9%) and lipid granuloma (2.6%). For kidney tissue, the prevalence of histological changes decreased in the following order: glomerular mesangial deposits (54.1%) > glomerular basement membrane thickening (45.9%) > THD (40%) > tubular hyaline casts (14.0%) > glomerular atrophy (13.5%) > dilated tubules (13.5%) > glomerular hyper-cellularity (10.8%) > mononuclear cell infiltrations (8.1%).

Glomerular Endothelial Cell Receptor Adhesion G-Protein-Coupled Receptor F5 (ADGRF5) and the Integrity of the Glomerular Filtration Barrier.

Background: Glomerular endothelial cells are recognized to be important for maintaining the glomerular filtration barrier. ADGRF5, an adhesion G protein-coupled receptor, has been suggested to be involved in endothelial cell function. However, the role of ADGRF5 in the glomerular filtration barrier integrity remains elusive. Methods: Cellular expression of ADGRF5 in mouse glomerulus was determined by histological analyses. The impact of ADGRF5 deletion on the glomerular morphology, kidney function, and glomerular endothelial gene/protein expression was then analyzed using ADGRF5 knockout (Adgrf5 −/−) mice and human primary glomerular endothelial cells. Results: ADGRF5 was specifically expressed in the capillary endothelial cells within the glomerulus. Adgrf5 −/− mice developed albuminuria and impaired kidney function with morphological defects in the glomeruli, namely glomerular hypertrophy, glomerular basement membrane splitting and thickening, diaphragmed fenestration and detachment of the glomerular endothelial cells, and mesangial interposition. These defects were accompanied by the altered expression of genes responsible for glomerular basement membrane organization (type IV collagens and laminins) and Krüppel-like factor 2 (Klf2) in glomerular endothelial cells. Moreover, ADGRF5 knockdown decreased COL4A3 and COL4A4 expression and increased KLF2 expression in human primary glomerular endothelial cells. Conclusions: The loss of ADGRF5 resulted in altered gene expression in glomerular endothelial cells, and perturbed the structure and permselectivity of the glomerular filtration barrier.

Monoclonal immunoglobulin deposition disease a spectrum? The treatment of an unusual biopsy presentation

Introduction: Monoclonal gammopathy of renal significance (MGRS) is characterized by clonal cell deposition of monoclonal immunoglobulin causing renal manifestations without overt malignancy. MIDD is a subtype of MGRS characterized by deposition of monoclonal light (LCDD), heavy chains (HCDD), or both (LHCDD) along the renal glomerular and tubular basement membranes. Virtually all organs can be involved, and if left untreated, MIDD can progress to MM and end stage renal disease. Although these patients classically did not receive treatment, current MIDD management is primarily focused on controlling the underlying clonal plasma cell disorder. Case description: We present a 56-year-old male with atypical MIDD treated successfully with plasma cell directed therapy consisting of daratumumab, cyclophosphamide, bortezomib, and dexamethasone (DARA-CYBOR-D). Despite a primary diagnosis of diabetic kidney disease, renal biopsy suggested myeloma-related MIDD based on linear immunofluorescent staining for IgG and kappa light chains along the tubular and glomerular basement membranes. Notably there were no electron dense deposits in the mesangium or tubular basement membranes typical to MIDD electron microscopy. Despite the presentation of MIDD on IF only, DARA-CYBOR-D treatment led to hematologic and renal improvement and remission from MIDD, with a GFR increase from 22 to 59 mL/min/BSA and a drop in urine protein from up to 9 to 0.6 g/dL. Conclusions: In conclusion, our case illustrates the efficacy of the DARA-CYBOR-D regimen in a patient with IF-only MIDD. We highlight the importance of obtaining a kidney biopsy when there are other potential causes for real decline and enhance our understanding of pathological and clinical spectrum of MIDD.

Role of connexin 43 in a rat model of periodontitis-induced renal injury.

This study aims to investigate the role of gap junction mediated by connexin 43 (Cx43) in renal injury induced by periodontitis in rats. Twelve SPF-grade Wistar male rats were divided into a control group and a periodontitis group by using a completely random number table method, with six rats in each group. The control group rats were not treated, while the periodontitis group rats were subjected to wire ligation of the neck of their bilateral maxillary first molars to construct a periodontitis model. After 8 weeks of modeling, the rats were examined for clinical indicators of the periodontium. micro-CT scanning of the maxilla reconstructed its 3D structure and analyzed the absorption of alveolar bone. Histopathological changes in periodontal and renal tissues were detected. MitoSOX red reagent was used to determine reactive oxygen species (ROS) content in renal tissues. A biochemical reagent kit was used to detect serum oxidative stress biomarkers. Real-time fluorescent quantitative-polymerase chain reaction (qRT-PCR) was employed to determine Cx43, nuclear factor kappa-B (NF-κB) , interleukin (IL)-1β, IL-6, BCL2-Associated X (Bax), B-lymphomatoma-2 gene (Bcl-2), and Caspase-3 mRNA were determined. Western blot analysis was used to detect Cx43, NF-κB, IL-1β, Bax, Bcl-2 and Caspase-3 protein. micro-CT 3D reconstruction showed significant bone resorption of the first molar alveolar bone in the periodontitis group rats and decreased height of the alveolar ridge. The distance from the enamel cementum boundary to the top of the alveolar ridge in the periodontitis group was significantly higher than that inthe control group. The histopathological results showed a large number of inflammatory cells that infiltrated the periodontal tissue of the periodontitis group, and the alveolar bone was significantly absorbed. Rats in the periodontitis group also exhibited mild thickening of the glomerular basement membrane, dilation of the Bowman's capsule, and destruction of the brush-like edge of the renal tubules in the renal tissue. The MitoSOX red staining results showed a significant increase in ROS content in the renal tissue of the periodontitis group. The biochemical test results showed that the levels of superoxide dismutase and glutathione in the serum of rats with periodontitis decreased, while that of malondialdehyde increased. The results of qRT-PCR and Western blot showed that the expression levels of Cx43, IL-1β, IL-6, Bax, Caspase-3 mRNA and Cx43, IL-1β, NF-κB, Bax, Caspase-3 proteins in the periodontitis group significantly increased compared with those in the control group, while the expression levels of Bcl-2 mRNA and protein decreased. Periodontitis may activate NF-κB signaling molecules by upregulating the expression of Cx43 in rat kidney tissues, leading to increased levels of inflammation and apoptosis and ultimately inducing kidney injury.

Podocyte Pathogenic Bone Morphogenetic Protein-2 Pathway and Immune Cell Behaviors in Primary Membranous Nephropathy.

Primary membranous nephropathy (PMN) is one of the leading causes of end-stage renal disease, and the most frequent cause of massive proteinuria in nondiabetic adults, resulting in fatal complications. However, the underlying pathomechanisms of PMN remain largely unclear. Here, single-cell RNA sequencing is employed to analyze kidney biopsies from eleven PMN patients and seven healthy subjects. Profiling 44060 cells from patients allowed us to characterize the cellular composition and cell-type-specific gene expression in the PMN kidney. The complement-induced BMP2/pSMAD1/COL4 pathway is identified as the pathogenic pathway in podocytes, bridging two key events, i.e., complement system activation and glomerular basement membrane thickening in PMN. Augmented infiltration and activation of myeloid leukocytes and B lymphocytes are found, profiling delicate crosstalk of immune cells in PMN kidneys. Overall, these results provide valuable insights into the roles of podocytes and immune cells in PMN, and comprehensive resources toward the complete understanding of PMN pathophysiology.

#234 Clinical spectrum and genetic variants of Alport syndrome and thin basement membrane nephropathy—single center experience

Abstract Background and aims Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are genetically heterogeneous kidney diseases that manifest with kidney involvement, AS also with hearing and visual impairment. Electron microscopic examination shows characteristic ultrastructural changes in the glomerular basement membrane (GBM) in AS and TBMN, but cannot reliably distinguish between the two entities. Therefore, molecular genetic studies and the identification of individual pathogenic variants in the target genes are crucial to confirm the diagnosis. The aim of our study is to determine the significance of the genetic variants associated with the ultrastructural changes of GBM found in the renal biopsy. Method We retrospectively analyzed records of patients from the University Medical Centre Ljubljana in which ultrastructural GBM changes characteristic for AS and TBMN were found in the renal biopsy, including their clinical, laboratory and histological features. Molecular genetic investigations were made using the new generation sequencing method (NGS) to determine germline pathogenic variants in genes directly related to AS and TBMN (COL4A3, COL4A4 and COL4A5) and in genes that are important in syndromes with a related clinical picture (COL4A6, CD151, LMX1B, MYH9, FN1 and MYO1E). The pathogenicity of variants was determined according to the Clinical Molecular Genetic Society best practice guidelines. Results The study included 73 patients with ultrastructurally confirmed thin GBM. Indications for kidney biopsy were chronic nephritis syndrome in 33 patients (45%), acute worsening of renal function in 16 (22%), nephrotic syndrome in 9 (12%), nephrotic proteinuria and chronic kidney disease in 6 (8%), isolated proteinuria in 5 (7%) and suspected vasculitis in 4 patients (6%). At the time of kidney biopsy average age was 50 ± 16 years, average creatinine 112 ± 70 µmol/L and average estimated glomerular filtration rate (eGFR) 65 ± 24 ml/min/1.73 m2, 7 patients had eGFR below 30 ml/min/1.73 m2. Average daily proteinuria was 2.4 ± 3.3 gr and 88% of patients had hematuria. 38% of them have hearing impairment and 43% had positive familiar history of kidney disease. We recorded 33 patients (45%) with genetic variants associated with AS or TBMN: 22 patients (67%) with pathogenic variants, 2 patients (6%) with likely pathogenic and 9 patients (27%) with variants of uncertain significance. The presence of a positive genetic analysis was associated with a higher level of hematuria (p = 0.003), a lower level of proteinuria (p = 0.004) and a positive family history of kidney disease (p &amp;lt; 0.001). It was also associated with hearing loss, but the association was not statistically significant (p = 0.099). Patients with genetic variants had similar eGFR to those with negative genetics (63.9 ml/min/1.73 m2 (58.3-69.4) vs 66.2 ml/min/1.73 m2 (60.0-72.3)), p = 0.698. We also found no differences in GBM thickness in patients without compared to those with identified gene variants (213 nm (160-315) vs. 209 nm (140-410)), p = 0.761. 32 patients (44%) had only focal chronic changes and ultrastructural changes of thin GBM present on kidney biopsy and 41 patients (56%) had also been diagnosed with other renal pathology. When comparing these two groups, patients without associated pathology had lower daily proteinuria (p = 0.048), higher level of hematuria (p = 0.021), frequent hearing impairment (p = 0.008) and also positive genetics analysis (p = 0.016). Conclusion We have shown that in patients with proven ultrastructural alterations in GBM, the presence of so far recognized genetic variants associated with AS and TBMN is 45%, and corresponds to the frequency of other phenotypic characteristics of AS, including hearing impairment and positive family history. In the future, additional clinical tools will be required to accurately determine the role of coexistent thin GBM on the prognosis of patients with other relevant kidney pathology and other genetic tools beyond NGS should be employed to explore other pathologic genetic variants associated with the AS spectrum.

#2984 SLE associated fibrillary glomerulonephritis vs SLE nephritis with fibrils: can a straightforward distinction always be made?

Abstract Background and Aims Fibrillary glomerulonephritis (FGN) is an uncommon but serious kidney disease with unclear aetiology, variable prognosis, and a not well-established treatment. FGN has been associated with autoimmune diseases including SLE. Other aetiologies comprise diabetes, malignancies, HCV and chronic infections. Our goal is to discuss the pathologic and clinical differential diagnosis conundrums that a patient with a suspected SLE presented. Method We present the case of a 53-year-old woman evaluated for proteinuria (1 g/24 h), and glomerular haematuria. Renal function was normal. She referred mild arthralgias for the past two years. Her medical history was unremarkable for diabetes and hypertension. ANA and anti-dsDNA were positive. The rest of the immunological work-up was normal as well as protein immunofixation/free light chains. HCV, HBV, and HIV serology was negative. A thorough evaluation for malignancy was negative. A renal biopsy was performed. Results The renal biopsy showed enlarged glomeruli with mesangial matrix expansion (Congo-red negative), mild mesangial hypercellularity and focal endocapillary hypercellularity without hyaline deposits, fibrinoid necrosis or crescents, without significant chronic lesions (Fig. 1). A probable diagnosis based on light microscope could be that of a lupus nephritis class III with low activity and low chronicity indices. Immunofluorescence demonstrated weak or moderate immunofixation of IgG (2+), IgA(1+), C3(2+), C1q(1+), κ(1+) and λ(1+) in the mesangium and segmentally in the capillary walls with a smudgy appearance. IgM, C4, fibrinogen and albumin were negative. The lack of a full-house pattern and, more significantly, the low C1q intensity raised some questions about the validity of a lupus nephritis diagnosis. Ultrastructural evaluation revealed abundant fibrils that occupied the mesangium and permeated the glomerular basement membranes (GBMs). Fibrils were non branching and randomly oriented with a mean diameter of ∼15 nm (range: 11,87 nm -18,84 nm) (Fig. 2). No electron dense deposits, fingerprint deposits or tubuloreticular inclusions were detected. Immunohistochemical staining for DNA-J heat-shock protein family member B9 (DNAJB9) was then performed showing strong positivity in the mesangial matrix and GBMs. Interestingly, C4d was also strongly positive with the same pattern as DNAJB9. The diagnosis of fibrillary glomerulonephritis was suggested, mostly based on ultrastructural evaluation and immunohistochemical findings. Conclusion It is well known that the characteristic features of lupus nephritis (intense C1q staining, full-house staining, extraglomerular deposits, tubuloreticular inclusions, and combined subendothelial and subepithelial deposits) are, in some cases, neither universally present nor specific for lupus nephritis. Of special interest is the identification of fibrils in the renal biopsy of an SLE patient. The crucial importance of distinguishing between SLE associated FGN and SLE nephritis with fibrils is based on the generally poor renal prognosis of FGN as well as the unsatisfactory performance of immunosuppression. The differential diagnosis can be very challenging especially when DNAJB9 is not available.