Characterizing Glomerular Barrier Dysfunction with Patient-Derived Serum in Glomerulus-on-a-Chip Models: Unveiling New Insights into Glomerulonephritis.

Glomerulonephritis (GN) is characterized by podocyte injury or glomerular filtration dysfunction, which results in proteinuria and eventual loss of kidney function. Progress in studying the mechanism of GN, and developing an effective therapy, has been limited by the absence of suitable in vitro models that can closely recapitulate human physiological responses. We developed a microfluidic glomerulus-on-a-chip device that can recapitulate the physiological environment to construct a functional filtration barrier, with which we investigated biological changes in podocytes and dynamic alterations in the permeability of the glomerular filtration barrier (GFB) on a chip. We also evaluated the potential of GN-mimicking devices as a model for predicting responses to human GN. Glomerular endothelial cells and podocytes successfully formed intact monolayers on opposite sides of the membrane in our chip device. Permselectivity analysis confirmed that the chip was constituted by a functional GFB that could accurately perform differential clearance of albumin and dextran. Reduction in cell viability resulting from damage was observed in all serum-induced GN models. The expression of podocyte-specific marker WT1 was also decreased. Albumin permeability was increased in most models of serum-induced IgA nephropathy (IgAN) and membranous nephropathy (MN). However, sera from patients with minimal change disease (MCD) or lupus nephritis (LN) did not induce a loss of permeability. This glomerulus-on-a-chip system may provide a platform of glomerular cell culture for in vitro GFB in formation of a functional three-dimensional glomerular structure. Establishing a disease model of GN on a chip could accelerate our understanding of pathophysiological mechanisms of glomerulopathy.

Caracterización de enfermedades glomerulares: análisis de 22 años de biopsias renales / Frequency of glomerular diseases in an analysis of 550 kidney biopsies

BACKGROUND: The frequency of glomerular diseases is dynamic and varies according to geographic area. AIM: To evaluate the frequency of primary and secondary glomerulopathies, their demographic profile and main clinical characteristics. MATERIAL AND METHODS: Renal biopsies from native kidneys performed between 1999 and 2020 were retrospectively reviewed. Demographic characteristics, clinical presentation, most relevant laboratory tests, frequency of primary and secondary glomerulopathies were analyzed. RESULTS: We analyzed 550 kidney biopsies from patients with a median age of 48 years (64% females). Nephrotic syndrome was the main indication for renal biopsy. Primary and secondary glomerulopathies occurred with similar frequency. Within the primary glomerulopathies, membranous nephropathy (34.1%) was the most common, followed by IgA nephropathy (31.1%) and focal segmental glomerulosclerosis (14.1%). Among the secondary glomerulopathies, lupus nephropathy was the most common (41.7%), followed by pauciimmune glomerulonephritis (27.1%) and diabetic nephropathy (6.4%). When comparing the results with other regions, significant differences were observed with reported frequencies in United States, Europe, Asia and the rest of Latin America. CONCLUSIONS: The most common primary glomerulopathies were membranous nephropathy and IgA nephropathy. Among the secondary glomerulopathies lupus nephropathy and pauci-immune glomerulonephritis were the most common. Compared to international registries, we observed a high proportion of membranous nephropathy and pauci-immune glomerulonephritis.

Epidemiology of biopsy-proven glomerular diseases in Chinese children: A scoping review.

Background: Glomerular disease is the leading cause of chronic kidney disease globally. No scoping review reports have focused on China's spectrum of glomerular diseases in children. This study aimed to systematically identify and describe retrospective studies on pediatric glomerular disease based on available data on sex, age, study period, and region. Methods: Six databases were systematically searched for relevant studies from initiation to December 2021 in PubMed, Embase, Web of Science, Global Health Library, Wangfang Database, and CNKI. Results: Thirty-four studies were identified in the scoping review, including 40,430 patients with biopsy-proven diagnoses. The proportion of boys was significantly higher than that of girls. In this study, 28,280 (70%) cases were primary glomerular disease, 10,547 (26.1%) cases were diagnosed as secondary glomerular disease, and 1146 (2.8%) cases were hereditary glomerular disease. Minimal change disease is the most common glomerular disease among children in China, followed by mesangial proliferative glomerulonephritis, IgA nephropathy, and purpura nephritis. We observed increments in glomerular diseases in periods 2 (2001-2010) and 3 (2011-2021). The proportion of major glomerular diseases varies significantly in the different regions of China. Conclusion: The spectrum of pediatric glomerular diseases varied across sex, age groups, study periods, and regions, and has changed considerably over the past 30 years.

COVID-19 in Patients with Glomerular Disease: Follow-Up Results from the IRoc-GN International Registry.

Background: The acute and long-term effects of severe acute respiratory syndrome coronavirus 2 infection in individuals with GN are still unclear. To address this relevant issue, we created the International Registry of COVID-19 infection in GN. Methods: We collected serial information on kidney-related and -unrelated outcomes from 125 GN patients (63 hospitalized and 62 outpatients) and 83 non-GN hospitalized patients with coronavirus disease 2019 (COVID-19) and a median follow-up period of 6.4 (interquartile range 2.3-9.6) months after diagnosis. We used logistic regression for the analyses of clinical outcomes and linear mixed models for the longitudinal analyses of eGFR. All multiple regression models were adjusted for age, sex, ethnicity, and renin-angiotensin-aldosterone system inhibitor use. Results: After adjustment for pre-COVID-19 eGFR and other confounders, mortality and AKI did not differ between GN patients and controls (adjusted odds ratio for AKI=1.28; 95% confidence interval [CI], 0.46 to 3.60; P=0.64). The main predictor of AKI was pre-COVID-19 eGFR (adjusted odds ratio per 1 SD unit decrease in eGFR=3.04; 95% CI, 1.76 to 5.28; P<0.001). GN patients developing AKI were less likely to recover pre-COVID-19 eGFR compared with controls (adjusted 6-month post-COVID-19 eGFR=0.41; 95% CI, 0.25 to 0.56; times pre-COVID-19 eGFR). Shorter duration of GN diagnosis, higher pre-COVID-19 proteinuria, and diagnosis of focal segmental glomerulosclerosis or minimal change disease were associated with a lower post-COVID-19 eGFR. Conclusions: Pre-COVID-19 eGFR is the main risk factor for AKI regardless of GN diagnosis. However, GN patients are at higher risk of impaired eGFR recovery after COVID-19-associated AKI. These patients (especially those with high baseline proteinuria or a diagnosis of focal segmental glomerulosclerosis or minimal change disease) should be closely monitored not only during the acute phases of COVID-19 but also after its resolution.

Establishment of a diagnostic model for glomerular micro thrombosis in patients with lupus nephritis based on machine learning / 中华风湿病学杂志

Objective:To establish a diagnostic model for glomerular micro thrombosis (GMT) in lupus nephritis through clinical indicators.Methods:A continuous collection of patients diagnosed with lupus nephritis (LN) by renal biopsy in the Department of Nephrology, Shenzhen Second People's Hospital, from January 2010 to March 2021. All patients were admitted and discharged through the inclusion and exclusion criteria. Demographic data, clinical characteristics, biochemical indicators, and immune indicators were collected. A GMT diagnosis model was established from the most important variables among the abovementioned variables through machine learning and Logistic stepwise regression analysis. The model was presented through a nomogram. The receiver operating characteristic curve (ROC), the clinical decision curve and the calibration curve were used to evaluate the model discrimination, clinical use and accuracy, respectively. The internal verification of the model was carried out by repeated sampling 500 times by the Bootstrap method.Results:There were a total of 129 patients with lupus nephritis including the study, including 117 females (90.7%); the average age was (34±11) years. There were 39 patients with GMT (30.2%). Using machine learning to screen out the top 10 important variables from 47 candidate variables, then through logistic stepwise regression analysis, five variables were further screened to establish the diagnostic model of GMT, namely hemoglobin [ OR(95% CI)=0.966(0.943, 0.990), P=0.005], serum C3 [ OR(95% CI)=0.133(0.022, 0.819), P=0.030], systolic blood pressure [ OR(95% CI)=1.027(1.005, 1.049), P=0.017], lymphocyte count [ OR(95% CI)=0.462(0.213, 0.999), P=0.049], and TT [ OR(95% CI)=1.260(0.993, 1.597), P=0.057]. Draw up the equation of the GMT diagnosis model of lupus nephritis and establish a nomogram to present the model. The area under curve (AUC) of the diagnostic model was 0.823, 95% CI(0.753, 0.893), indicating that the model had a reasonable degree of discrimin-ation. The Hosmer-Lemeshow test showed a perfect fit between the predicted GMT risk and the observed GMT risk ( χ2= 14.62, P=0.067). The clinical decision curve and clinical impact curve reflect that the model had a good clinical application value, especially when the threshold probability is between 0.4 and 0.6, the application value is more significant. In addition, after 500 repeated samplings by the Bootstrap method, the average AUC was 0.825, 95% CI(0.753, 0.893), which is basically the same as the AUC obtained by the original model. Conclusion:We established a diagnostic model of GMT inLN based on clinical indicators through machine learning and logistic stepwise regression analysis. It is used for early diagnosis of the risk of GMT before the renal biopsy.

Ultrasound-guided percutaneous renal biopsy at a university hospital: retrospective analysis of success and complication rates.

OBJECTIVE: To evaluate the success and complication rates of ultrasound-guided renal biopsy at a tertiary care hospital. MATERIALS AND METHODS: This was a retrospective analysis of 97 ultrasound-guided renal biopsies, all performed by the same radiologist, between 1 March, 2017 and 31 October, 2019. RESULTS: Of the 97 biopsies evaluated, 87 had a definitive pathological diagnosis. In five cases (5.4%), the biopsy results were inconclusive and a second procedure was required. In seven procedures (7.6%), there were complications, all of which were properly resolved. CONCLUSION: Ultrasound-guided renal biopsy has proven to be a safe, effective method for the diagnosis of nephropathies, with high success rates.

OBJETIVO: Avaliar taxas de sucesso e de complicações de biópsias renais guiadas por ultrassonografia em um hospital terciário. MATERIAIS E MÉTODOS: Análise retrospectiva de 97 biópsias renais realizadas entre 1º de março de 2017 e 31 de outubro de 2019, guiadas por ultrassonografia e executadas por um único médico radiologista. RESULTADOS: Oitenta e sete biópsias apresentaram diagnóstico anatomopatológico definitivo. Cinco biópsias foram inconclusivas (5,4%) e precisaram de um segundo procedimento. Houve complicações em sete procedimentos (7,6%), todas devidamente solucionadas. CONCLUSÃO: A biópsia renal guiada por ultrassonografia demonstrou ser um método efetivo e seguro para o diagnóstico de nefropatias, com elevada taxa de sucesso em sua execução.

Ultrasound-guided percutaneous renal biopsy at a university hospital: retrospective analysis of success and complication rates / Biópsia renal percutânea guiada por ultrassonografia: análise retrospectiva de sucesso e complicações em um hospital universitário

Abstract Objective: To evaluate the success and complication rates of ultrasound-guided renal biopsy at a tertiary care hospital. Materials and Methods: This was a retrospective analysis of 97 ultrasound-guided renal biopsies, all performed by the same radiologist, between 1 March, 2017 and 31 October, 2019. Results: Of the 97 biopsies evaluated, 87 had a definitive pathological diagnosis. In five cases (5.4%), the biopsy results were inconclusive and a second procedure was required. In seven procedures (7.6%), there were complications, all of which were properly resolved. Conclusion: Ultrasound-guided renal biopsy has proven to be a safe, effective method for the diagnosis of nephropathies, with high success rates.

Resumo Objetivo: Avaliar taxas de sucesso e de complicações de biópsias renais guiadas por ultrassonografia em um hospital terciário. Materiais e Métodos: Análise retrospectiva de 97 biópsias renais realizadas entre 1º de março de 2017 e 31 de outubro de 2019, guiadas por ultrassonografia e executadas por um único médico radiologista. Resultados: Oitenta e sete biópsias apresentaram diagnóstico anatomopatológico definitivo. Cinco biópsias foram inconclusivas (5,4%) e precisaram de um segundo procedimento. Houve complicações em sete procedimentos (7,6%), todas devidamente solucionadas. Conclusão: A biópsia renal guiada por ultrassonografia demonstrou ser um método efetivo e seguro para o diagnóstico de nefropatias, com elevada taxa de sucesso em sua execução.

A Personalized Glomerulus Chip Engineered from Stem Cell-Derived Epithelium and Vascular Endothelium.

Progress in understanding kidney disease mechanisms and the development of targeted therapeutics have been limited by the lack of functional in vitro models that can closely recapitulate human physiological responses. Organ Chip (or organ-on-a-chip) microfluidic devices provide unique opportunities to overcome some of these challenges given their ability to model the structure and function of tissues and organs in vitro. Previously established organ chip models typically consist of heterogenous cell populations sourced from multiple donors, limiting their applications in patient-specific disease modeling and personalized medicine. In this study, we engineered a personalized glomerulus chip system reconstituted from human induced pluripotent stem (iPS) cell-derived vascular endothelial cells (ECs) and podocytes from a single patient. Our stem cell-derived kidney glomerulus chip successfully mimics the structure and some essential functions of the glomerular filtration barrier. We further modeled glomerular injury in our tissue chips by administering a clinically relevant dose of the chemotherapy drug Adriamycin. The drug disrupts the structural integrity of the endothelium and the podocyte tissue layers, leading to significant albuminuria as observed in patients with glomerulopathies. We anticipate that the personalized glomerulus chip model established in this report could help advance future studies of kidney disease mechanisms and the discovery of personalized therapies. Given the remarkable ability of human iPS cells to differentiate into almost any cell type, this work also provides a blueprint for the establishment of more personalized organ chip and 'body-on-a-chip' models in the future.

Morphofunctional study on the effects of passive smoking in kidneys of rats / Estudo morfofuncional sobre os efeitos do tabagismo passivo em rins de ratos

ABSTRACT Objective To analyze whether passive inhalation of cigarette smoke causes morphological, structural, and functional changes in kidneys of rats. Methods Wistar rats, aged eight weeks, weighing on average 260g, were divided into Control Group and Smoking Group. Each group was subdivided into four groups of ten animals for morphofunctional analysis, in a period of seven and 28 days. The Smoking Group was exposed to smoke of 40 cigarettes per day, at certain times and in automated equipment for cigarette burning, called smoking machine (SM-MC-01). After the exposure period, urine and blood samples were collected for the functional analyses, and the kidneys were dissected and submitted to histological procedures for morphoquantitative analyses. Results After exposure of animals of the Smoking Group, the following were observed: lower weight gain; lower water and feed intake; decreased renal weight, diameter, and volume; reduction in cortical thickness and glomerular volume density; decrease in glomerular and capsular diameter; increase in mesangial density; decreased urine volume; increased levels of glucose, serum creatinine and microalbuminuria; decreased urinary creatinine levels and creatinine clearance rate. Conclusion Passive smoking negatively influences renal morphology and glomerular filtration rate, with effects similar to those described in the literature regarding active smoking.

RESUMO Objetivo Analisar se a inalação passiva da fumaça do cigarro proporciona alterações morfológicas, estruturais e funcionais nos rins de ratos. Métodos Ratos Wistar, com oito semanas de idade, pesando, em média, 260g, foram divididos em Grupo Controle e Grupo Tabagista. Cada grupo foi subdividido em quatro grupos de dez animais para análise morfofuncional, em um período de sete e 28 dias. O Grupo Tabagista foi exposto à fumaça de 40 cigarros por dia, em horários determinados e equipamento automatizado de queima de cigarros, denominado smoking machine (SM-MC-01). Após o período de exposição, foram coletadas amostras de urina e sangue para as análises funcionais, e os rins foram dissecados e submetidos a procedimentos histológicos para análises morfoquantitativas. Resultados Após a exposição dos animais do Grupo Tabagista, observou-se menor ganho de peso; menor consumo de água e ração; menor peso, diâmetro e volume renal; redução em espessura cortical e densidade de volume glomerular; diminuição no diâmetro glomerular e capsular; aumento na densidade mesangial; volume urinário diminuído; níveis aumentados de glicose, creatinina sérica e microalbuminúria; níveis reduzidos de creatinina urinária e redução da taxa de depuração da creatinina. Conclusão O tabagismo passivo influencia negativamente na morfologia renal e na taxa de filtração glomerular, com efeitos semelhantes aos descritos na literatura em relação ao tabagismo ativo.

Glomerular Endothelial Cells as Instigators of Glomerular Sclerotic Diseases.

Glomerular endothelial cell (GEnC) dysfunction is important in the pathogenesis of glomerular sclerotic diseases, including Focal Segmental Glomerulosclerosis (FSGS) and overt diabetic nephropathy (DN). GEnCs form the first cellular barrier in direct contact with cells and factors circulating in the blood. Disturbances in these circulating factors can induce GEnC dysfunction. GEnC dysfunction occurs in early stages of FSGS and DN, and is characterized by a compromised endothelial glycocalyx, an inflammatory phenotype, mitochondrial damage and oxidative stress, aberrant cell signaling, and endothelial-to-mesenchymal transition (EndMT). GEnCs are in an interdependent relationship with podocytes and mesangial cells, which involves bidirectional cross-talk via intercellular signaling. Given that GEnC behavior directly influences podocyte function, it is conceivable that GEnC dysfunction may culminate in podocyte damage, proteinuria, subsequent mesangial activation, and ultimately glomerulosclerosis. Indeed, GEnC dysfunction is sufficient to cause podocyte injury, proteinuria and activation of mesangial cells. Aberrant gene expression patterns largely contribute to GEnC dysfunction and epigenetic changes seem to be involved in causing aberrant transcription. This review summarizes literature that uncovers the importance of cross-talk between GEnCs and podocytes, and GEnCs and mesangial cells in the context of the development of FSGS and DN, and the potential use of GEnCs as efficacious cellular target to pharmacologically halt development and progression of DN and FSGS.

Renal intravascular large B cell lymphoma: the first case report in Korea and a review of the literature.

Herein, we describe the first case of renal intravascular large B cell lymphoma in Korea occurring in a 66-year-old female. She presented with mild fever and dyspnea. On physical and laboratory evaluations, hemophagocytic lymphohistiocytosis was suspected, but the bone marrow biopsy results were unremarkable. During the work-up, massive proteinuria developed, which led to a renal biopsy. The renal architecture was relatively well-preserved, but the glomeruli were hypercellular with the infiltration of atypical, large lymphoid cells with increased nucleus-cytoplasm ratio and clumped chromatin. Similar cells were also present in the peritubular capillaries. The tumor cells exhibited membranous staining for CD20 and CD79a. After the diagnosis of intravascular large B cell lymphoma, the patient received rituximab-based chemotherapy under close follow-up.

Characterization of the renal cortical transcriptome following Roux-en-Y gastric bypass surgery in experimental diabetic kidney disease.

INTRODUCTION: Roux-en-Y gastric bypass surgery (RYGB) reduces albuminuria and the long-term incidence of end-stage renal disease in patients with obesity and diabetes. Preclinical modeling in experimental diabetic kidney disease demonstrates that improvements in glomerular structure likely underpin these findings. RESEARCH DESIGN AND METHODS: In adult male Zucker diabetic fatty (ZDF) rats, we profiled the effect of RYGB on weight and metabolic control as well biochemical, structural and ultrastructural indices of diabetic renal injury. Furthermore, we sequenced the renal cortical transcriptome in these rats and used bioinformatic pathway analyses to characterize the transcriptional alterations governing the renal reparative response to RYGB. RESULTS: In parallel with improvements in weight and metabolic control, RYGB reduced albuminuria, glomerulomegaly, podocyte stress and podocyte foot process effacement. Pathway analysis of RYGB-induced transcriptomic changes in the renal cortex highlighted correction of disease-associated alterations in fibrosis, inflammation and biological oxidation pathways. RYGB reversed disease-associated changes in the expression of transforming growth factor (TGF)-ß superfamily genes that strongly correlated with improvements in structural measures of glomerulopathy. CONCLUSIONS: Improved glomerular structure in ZDF rats following RYGB is underpinned by pathway level changes, including interruption of the TGF-ß-driven early profibrotic programme. Our data provide an important layer of experimental support for clinical evidence demonstrating that RYGB arrests renal damage in patients with obesity and type 2 diabetes.

Activation of Notch1 signaling in podocytes by glucose-derived AGEs contributes to proteinuria.

INTRODUCTION: Advanced glycation end-products (AGEs) are implicated in the pathogenesis of diabetic nephropathy (DN). Previous studies have shown that AGEs contribute to glomerulosclerosis and proteinuria. Podocytes, terminally differentiated epithelial cells of the glomerulus and the critical component of the glomerular filtration barrier, express the receptor for AGEs (RAGE). Podocytes are susceptible to severe injury during DN. In this study, we investigated the mechanism by which AGEs contribute to podocyte injury. RESEARCH DESIGN AND METHODS: Glucose-derived AGEs were prepared in vitro. Reactivation of Notch signaling was examined in AGE-treated human podocytes (in vitro) and glomeruli from AGE-injected mice (in vivo) by quantitative reverse transcription-PCR, western blot analysis, ELISA and immunohistochemical staining. Further, the effects of AGEs on epithelial to mesenchymal transition (EMT) of podocytes and expression of fibrotic markers were evaluated. RESULTS: Using human podocytes and a mouse model, we demonstrated that AGEs activate Notch1 signaling in podocytes and provoke EMT. Inhibition of RAGE and Notch1 by FPS-ZM1 (N-Benzyl-4-chloro-N-cyclohexylbenzamide) and DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester), respectively, abrogates AGE-induced Notch activation and EMT. Inhibition of RAGE and Notch1 prevents AGE-induced glomerular fibrosis, thickening of the glomerular basement membrane, foot process effacement, and proteinuria. Furthermore, kidney biopsy sections from people with DN revealed the accumulation of AGEs in the glomerulus with elevated RAGE expression and activated Notch signaling. CONCLUSION: The data suggest that AGEs activate Notch signaling in the glomerular podocytes. Pharmacological inhibition of Notch signaling by DAPT ameliorates AGE-induced podocytopathy and fibrosis. Our observations suggest that AGE-induced Notch reactivation in mature podocytes could be a novel mechanism in glomerular disease and thus could represent a novel therapeutic target.

Demonstration of Subclinical Early Nephrotoxicity Induced by Occupational Exposure to Silica among Workers in Pottery Industry.

BACKGROUND: For many years, several studies drew attention to the possible nephrotoxic effects of silica and distinct renal dysfunction involving glomerular and renal tubules in workers exposed to silica. OBJECTIVE: To determine the early signs of subclinical nephrotoxic effects among some Egyptian workers exposed to silica in the pottery industry. METHODS: This study was carried out in El-Fawakhir handicraft pottery area, in Greater Cairo, Egypt. The studied population included 29 non-smoking male workers occupationally exposed to silica in addition to 35 non-smoking administrative male subjects who represented the comparison group in the study. Measured urinary parameters were concentrations of total protein (TP), microalbumin (Malb), activities of alkaline phosphatase (ALP), γ-glutamyl transferase (γ-GT), lactate dehydrogenase (LDH), kidney injury molecule-1 (KIM-1), and silicon (Si). RESULTS: Silica-exposed workers showed significantly (p<0.05) increased levels of urinary TP, Malb, ALP, γ-GT, LDH, and KIM-1 compared with the comparison group. Among the silicaexposed group, increased urinary Si levels were positively and significantly correlated (Spearman's ρ>0.60, p<0.001 for all variables) with the elevated urinary proteins (including KIM-1) and enzymes levels. All measured urinary parameters were positively and significantly correlated (ρ>0.75, p<0.001 for all variables) with the duration of work among exposed subjects. No significant correlation was observed between the measured variables and the age of workers. CONCLUSION: There is associated subclinical glomerular and tubular affection among silica exposed workers, which is related to the duration and intensity of exposure.

Identification of Podocyte Cargo Proteins by Proteomic Analysis of Clathrin-Coated Vesicles.

Background: Clathrin-mediated endocytosis (CME) plays a fundamental role in podocyte health. Genetic ablation of genes implicated in CME has been shown to cause severe proteinuria and foot process effacement in mice. However, little is known about the cargo of clathrin-coated vesicles (CCVs) in podocytes. The goal of this study was to isolate CCVs from podocytes and identify their cargo by proteomic analysis. Methods: Glomeruli isolated from Podocin-Cre Rosa-DTRflox mouse kidneys were seeded and treated with diphtheria toxin to obtain pure primary podocyte cultures. CCVs were isolated by differential gradient ultracentrifugation, and enrichment of CCVs was assessed by immunoblotting and electron microscopy (EM). Liquid chromatography-mass spectrometry (LC-MS) was performed for proteomic analysis. Proteins with higher abundance than transferrin receptor protein 1 were evaluated for CCV cargo potential against previously published literature. Immunofluorescence staining of identified cargo proteins and CCVs was performed in podocytes for further verification. Results: Immunoblotting for multiple protein markers of CME revealed enrichment in the CCV fraction. Enrichment of CCVs among other small vesicles was observed via EM. Proteomics yielded a total of >1200 significant proteins. Multiple-step data analysis revealed 36 CCV-associated proteins, of which 10 represent novel, highly abundant cargo proteins in podocytes. Colocalization of cargo proteins and CCVs on immunostaining was observed. Conclusions: Our identification of podocyte CCV cargo proteins helps to elucidate the importance of endocytic trafficking for podocyte health and maintenance of the glomerular environment.

Automated Image Analyses of Glomerular Hypertrophy in a Mouse Model of Diabetic Nephropathy.

Background: Glomerular hypertrophy is a hallmark of kidney injury in metabolically induced renal diseases such as obesity-associated glomerulopathies and diabetic nephropathy (DN). Methods: Using light sheet fluorescent microscopy (LSFM) and 3D image analysis, we tested algorithms for automated and unbiased quantification of total glomerular numbers and individual glomerular volume in the uninephrectomized (UNx) db/db mouse model of DN. Results: At 6 weeks after surgery, db/db and UNx db/db mice showed increased urine albumin-to-creatinine ratio (ACR) compared with db/+ control mice. Before euthanasia, glomeruli were labeled in vivo by injecting tomato lectin. Whole-kidney LSFM 3D image analysis revealed that mean glomerular volume was significantly increased in UNx db/db mice compared with db/+ mice. Moreover, analysis of individual glomerular volume showed a shift in volume distribution toward larger glomeruli and thereby demonstrated additive effects of diabetes and UNx on induction of glomerular hypertrophy. The automatized quantification showed no significant differences in glomerular numbers among db/+, db/db, and UNx db/db mice. These data correlated with glomerular numbers as quantified by subsequent stereologic quantification. Conclusions: Overall, LSFM coupled with automated 3D histomorphometric analysis was demonstrated to be advantageous for unbiased assessment of glomerular volume and numbers in mouse whole-kidney samples. Furthermore, we showed that injection of fluorescently labeled lectin and albumin can be used as markers of nephron segments in the mouse kidneys, thus enabling functional assessment of kidney physiology, pathology, and pharmacology in preclinical rodent models of kidney disease.

Fibrillary Glomerulonephritis and DnaJ Homolog Subfamily B Member 9 (DNAJB9).

Fibrillary GN (FGN) is a rare glomerular disease that is diagnosed based on the presence of fibrils in glomeruli. The fibrils are typically noncongophilic, randomly oriented, and measure 12-24 nm. Traditionally, electron microscopy (EM) has been an important tool to aid in the diagnosis of FGN by identifying the fibrils and to distinguish it from other entities that could mimic FGN. However, recently DnaJ homolog subfamily B member 9 (DNAJB9) has emerged as both a specific and sensitive biomarker in patients with FGN. It allows prompt diagnosis and alleviates reliance on EM. DNAJB9 is a cochaperone of heat shock protein 70 and is involved in endoplasmic reticulum protein-folding pathways. But its role in the pathogenesis of FGN remains elusive. DNAJB9 may act as a putative antigen or alternatively it may secondarily bind to misfolded IgG in the glomeruli. These hypotheses need future studies to elucidate the role of DNAJB9 in the pathogenesis of FGN. The treatment regimen for FGN has been limited due to paucity of studies. Most patients receive combination immunosuppressive regimens. Rituximab has been studied the most in FGN and it may delay disease progression. Prognosis of FGN remains poor and 50% require dialysis within 2 years of diagnosis. Despite its poor prognosis in native kidneys, the rate of recurrence post-transplantation is low (20%) and patient as well as allograft outcomes are similar to patients without FGN.

Assessment of long term outcomes after buccal mucosal graft urethroplasty: the impact of chronic kidney disease.

OBJECTIVES: To compare and assess various outcomes and success of buccal mucosal graft urethroplasty (BMGU) in patients with CKD versus patients having normal renal function. MATERIAL AND METHODS: This was a retrospective, single centre study, during period 2013 to 2017. Patients were grouped into two groups. Group 1 had patients with estimated Glomerular Filtration Rate (eGFR)>60mL/min/1.73m2 while group 2 had patients with eGFR <60mL/min/1.73m2. eGFR was calculated according to the MDRD equation. The two groups were compared with regard to various outcomes like length, location of stricture, technique of graft placement, intra-operative blood loss (haemoglobin drop), duration of hospital stay, post-operative complications and recurrence. RESULTS: A total of 223 patients were included in study with group 1 had 130 patients and group 2 had 93 patients. Mean age of patients with CKD were higher (47.49 years versus 29.13 years). The mean follow-up period was comparable between both groups (23.29 months and 22.54 months respectively). Patients with CKD had more post-operative Clavien Grade 2 or higher complications (p=0.01) and a greater recurrence rates (p<0.001) than in non-CKD patients. On multivariate analysis, age and CKD status was significant predictor of urethroplasty success (p=0.004) (OR= 14.98 (1.952-114.94, 95% CI). CONCLUSIONS: CKD patients are more prone to post-operative complications in terms of wound infection, graft uptake and graft failure and higher recurrence rates following BMGU.