In situ assessment of Mindin as a biomarker of podocyte lesions in diabetic nephropathy.

Diabetic nephropathy (DN) is the leading cause of chronic kidney disease and end-stage renal failure worldwide. Several mechanisms are involved in the pathogenesis of this disease, which culminate in morphological changes such as podocyte injury. Despite the complex diagnosis and pathogenesis, limited attempts have been made to establish new biomarkers for DN. The higher concentration of Mindin protein in the urine of patients with type 2 diabetes mellitus suggests that it plays a role in DN. Therefore, this study investigated whether in situ protein expression of Mindin can be considered a potential DN biomarker. Fifty renal biopsies from patients diagnosed with DN, 57 with nondiabetic glomerular diseases, including 17 with focal segmental glomerulosclerosis (FSGS), 14 with minimal lesion disease (MLD) and 27 with immunoglobulin A nephropathy (IgAN), and 23 adult kidney samples from autopsies (control group) were evaluated for Mindin expression by immunohistochemistry. Podocyte density was inferred by Wilms' tumor 1 (WT1) immunostaining, while foot process effacement was assessed by transmission electron microscopy. Receiver operative characteristic (ROC) analysis was performed to determine the biomarker sensitivity/specificity. Low podocyte density and increased Mindin expression were observed in all cases of DN, regardless of their class. In the DN group, Mindin expression was significantly higher than that in the FSGS, MCD, IgAN and control groups. Higher Mindin expression was significantly positively correlated with foot process effacement only in class III DN cases. Furthermore, Mindin protein presented high specificity in the biopsies of patients with DN (p < 0.0001). Our data suggest that Mindin may play a role in DN pathogenesis and is a promising biomarker of podocyte lesions.

In situ evaluation of podocytes in patients with focal segmental glomerulosclerosis and minimal change disease.

Podocyte injury in focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) results from the imbalance between adaptive responses that maintain homeostasis and cellular dysfunction that can culminate in cell death. Therefore, an in situ analysis was performed to detect morphological changes related to cell death and autophagy in renal biopsies from adult patients with podocytopathies. Forty-nine renal biopsies from patients with FSGS (n = 22) and MCD (n = 27) were selected. In situ expression of Wilms Tumor 1 protein (WT1), light chain microtubule 1-associated protein (LC3) and caspase-3 protein were evaluated by immunohistochemistry. The foot process effacement and morphological alterations related to podocyte cell death and autophagy were analyzed with transmission electronic microscopy. Reduction in the density of WT1-labeled podocytes was observed for FSGS and MCD cases as compared to controls. Foot process width (FPW) in control group was lower than in cases of podocytopathies. In FSGS group, FPW was significantly higher than in MCD group and correlated with proteinuria. A density of LC3-labeled podocytes and the number of autophagosomes in podocytes/ pedicels were higher in the MCD group than in the FSGS group. The number of autophagosomes correlated positively with the estimated glomerular filtration rate in cases of MCD. The density of caspase-3-labeled podocytes in FSGS and MCD was higher than control group, and a higher number of podocytes with an evidence of necrosis was detected in FSGS cases than in MCD and control cases. Podocytes from patients diagnosed with FSGS showed more morphological and functional alterations resulting from a larger number of lesions and reduced cell adaptation.

Renal expression of cytokines and chemokines in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of end-stage renal disease worldwide. Inflammatory mediators have been implicated in the pathogenesis of DN, thus considered an inflammatory disease. However, further studies are required to assess the renal damage caused by the action of these molecules. Therefore, the objective of this study was to analyze the expression of cytokines and chemokines in renal biopsies from patients with DN and to correlate it with interstitial inflammation and decreased renal function. METHODS: Forty-four native renal biopsies from patients with DN and 23 control cases were selected. In situ expression of eotaxin, MIP-1α (macrophage inflammatory protein-1α), IL-8 (interleukin-8), IL-4, IL-10, TNF-α (tumor necrosis factor-α), TNFR1 (tumor necrosis factor receptor-1), IL-1ß, and IL-6 were evaluated by immunohistochemistry. RESULTS: The DN group showed a significant increase in IL-6 (p < 0.0001), IL-1ß (p < 0.0001), IL-4 (p < 0.0001) and eotaxin (p = 0.0012) expression, and a decrease in TNFR1 (p = 0.0107) and IL-8 (p = 0.0262) expression compared to the control group. However, there were no significant differences in IL-10 (p = 0.4951), TNF-α (p = 0.7534), and MIP-1α (p = 0.3816) expression among groups. Regarding interstitial inflammation, there was a significant increase in IL-6 in scores 0 and 1 compared to score 2 (p = 0.0035), in IL-10 in score 2 compared to score 0 (p = 0.0479), and in eotaxin in score 2 compared to scores 0 and 1 (p < 0.0001), whereas IL-8 (p = 0.0513) and MIP-1α (p = 0.1801) showed no significant differences. There was a tendency for negative correlation between eotaxin and estimated glomerular filtration rate (eGFR) (p = 0.0566). CONCLUSIONS: Our results indicated an increased in situ production of cytokines and chemokines in DN, including IL-6, IL-1ß, IL-4, and eotaxin. It was observed that, possibly, eotaxin may have an important role in the progression of interstitial inflammation in DN and in eGFR decrease of these patients.

Analysis of serum inflammatory mediators in type 2 diabetic patients and their influence on renal function.

AIM: To evaluate the serum concentrations of inflammatory mediators in patients with type 2 diabetes mellitus (T2DM) with or without renal alteration (RA) function. METHODS: Serum samples from 76 patients with T2DM and 24 healthy individuals were selected. Patients with T2DM were divided into two groups according to eGFR (> or < 60mL/min/1.73m2). Cytokines, chemokines and adipokines levels were evaluated using the Multiplex immunoassay and ELISA. RESULTS: TNFR1 and leptin were higher in the T2DM group with RA than in the T2DM group without RA and control group. All patients with T2DM showed increased resistin, IL-8, and MIP-1α compared to the control group. Adiponectin were higher and IL-4 decreased in the T2DM group with RA compared to the control group. eGFR positively correlated with IL-4 and negatively with TNFR1, TNFR2, and leptin in patients with T2DM. In the T2DM group with RA, eGFR was negatively correlated with TNFR1 and resistin. TNFR1 was positively correlated with resistin and leptin, as well as resistin with IL-8 and leptin. CONCLUSION: Increased levels of TNFR1, adipokines, chemokines and decrease of IL-4 play important role in the inflammatory process developed in T2DM and decreased renal function. We also suggest that TNFR1 is a strong predictor of renal dysfunction in patients with T2DM.

Focal and Segmental Glomerulosclerosis and Membranous Nephropathy overlapping in a patient with Nephrotic Syndrome: a case report / Glomeruloesclerose Segmentar e Focal e Nefropatia Membranosa sobrepostas em paciente com Síndrome Nefrótica: relato de caso

Abstract Introduction: Some cases of membranous nephropathy (MGN) present focal segmental glomerulosclerosis (FSGS) typically associated with disease progression. However, we report a case of a patient who seemed to have MGN and FSGS, both primary. Case presentation: A 17-year-old female, Caucasian, presenting lower extremity edema associated with episodes of foamy urine and high blood pressure, had physical and laboratorial exams indicating nephrotic syndrome. A renal biopsy was performed and focal and segmental glomerulosclerosis were observed under light microscopy in some glomeruli presented as tip lesion, and in others it was accompanied by podocyte hypertrophy and podocyte detachment in urinary space, compatible with podocytopathy FSGS. Besides, there were thickened capillary loops with basement membrane irregularities due to "spikes" compatible with MGN stage II. Immunofluorescence showed finely granular IgG, IgG4, and PLA2R deposits in capillary loops and, in electron microscopy, subepithelial deposits and foot process effacement. These morphological findings are compatible with FSGS and MGN stage II. Conclusions: In the present case, clinical and morphological characteristics showed a possible overlap of primary FSGS and MGN as focal and segmental glomerulosclerosis does not seem to be related with MGN progression but with the podocytopathy FSGS.

Resumo Introdução: Alguns casos de nefropatia membranosa (NM) apresentam glomeruloesclerose segmentar e focal (GESF) tipicamente associada a progressão da doença. Contudo, relatamos o caso de uma paciente que parece ter NM e GESF, ambas primárias. Apresentação do caso: Uma jovem branca de 17 anos de idade com edema de membros inferiores associado a episódios de urina espumosa e hipertensão apresentou-se com achados físicos e laboratoriais sugestivos de síndrome nefrótica. Foi realizada biópsia renal. GESF foi observada por microscopia de luz em alguns glomérulos que apresentavam lesões de ponta, enquanto em outros o achado era acompanhado por hipertrofia podocitária e descolamento de podócitos no espaço urinário, compatíveis com podocitopatia GESF. Além disso, as alças capilares estavam espessadas com irregularidades na membrana basal devido a "espículas" compatíveis com NM estágio II. Imunofluorescência revelou depósitos finamente granulares de IgG, IgG4 e PLA2R nas alças capilares. Microscopia eletrônica exibiu depósitos subepiteliais e apagamento de pedicelos. Tais achados morfológicos são compatíveis com GESF e NM estágio II. Conclusões: No presente caso, as características clínicas e morfológicas revelaram uma possível sobreposição de GESF primária e NM, uma vez que a glomeruloesclerose segmentar e focal não parece estar relacionada com a progressão da NM, mas com a podocitopatia GESF.

Focal and Segmental Glomerulosclerosis and Membranous Nephropathy overlapping in a patient with Nephrotic Syndrome: a case report.

INTRODUCTION: Some cases of membranous nephropathy (MGN) present focal segmental glomerulosclerosis (FSGS) typically associated with disease progression. However, we report a case of a patient who seemed to have MGN and FSGS, both primary. CASE PRESENTATION: A 17-year-old female, Caucasian, presenting lower extremity edema associated with episodes of foamy urine and high blood pressure, had physical and laboratorial exams indicating nephrotic syndrome. A renal biopsy was performed and focal and segmental glomerulosclerosis were observed under light microscopy in some glomeruli presented as tip lesion, and in others it was accompanied by podocyte hypertrophy and podocyte detachment in urinary space, compatible with podocytopathy FSGS. Besides, there were thickened capillary loops with basement membrane irregularities due to "spikes" compatible with MGN stage II. Immunofluorescence showed finely granular IgG, IgG4, and PLA2R deposits in capillary loops and, in electron microscopy, subepithelial deposits and foot process effacement. These morphological findings are compatible with FSGS and MGN stage II. CONCLUSIONS: In the present case, clinical and morphological characteristics showed a possible overlap of primary FSGS and MGN as focal and segmental glomerulosclerosis does not seem to be related with MGN progression but with the podocytopathy FSGS.

A novel single amino acid deletion impairs fibronectin function and causes familial glomerulopathy with fibronectin deposits: case report of a family.

BACKGROUND: Glomerulopathy with fibronectin deposits is an autosomal dominant disease associated with proteinuria, hematuria, hypertension and renal function decline. Forty percent of the cases are caused by mutations in FN1, the gene that encodes fibronectin. CASE PRESENTATION: This report describes two cases of Glomerulopathy with fibronectin deposits, involving a 47-year-old father and a 14-year-old son. The renal biopsies showed glomeruli with endocapillary hypercellularity and large amounts of mesangial and subendothelial eosinophilic deposits. Immunohistochemistry for fibronectin was markedly positive. Whole exome sequencing identified a novel FN1 mutation that leads to an amino-acid deletion in both patients (Ile1988del), a variant that required primary amino-acid sequence analysis for assessment of pathogenicity. Our primary sequence analyses revealed that Ile1988 is very highly conserved among relative sequences and is positioned in a C-terminal FN3 domain containing heparin- and fibulin-1-binding sites. This mutation was predicted as deleterious and molecular mechanics simulations support that it can change the tertiary structure and affect the complex folding and its molecular functionality. CONCLUSION: The current report not only documents the occurrence of two GFND cases in an affected family and deeply characterizes its anatomopathological features but also identifies a novel pathogenic mutation in FN1, analyzes its structural and functional implications, and supports its pathogenicity.

Is it possible to predict parameters of the Oxford classification of primary IgA Nephropathy from clinical laboratory data? Focus on the role of segmental glomerulosclerosis subtypes.

INTRODUCTION: IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world and has a broad range of histological and clinical manifestations, ranging from morphologically normal to globally sclerotic glomeruli with clinical manifestations varying from isolated hematuria to end stage renal disease. This study aims to assess sensitivity, specificity and accuracy of clinical data at the time of biopsy in predicting 2017 updated Oxford classification parameters and to investigate if subtypes of segmental sclerosis (FSGS) influence clinical presentation. MATERIAL AND METHODS: Renal biopsies from 103 patients with IgAN were analyzed. Oxford classification was updated and FSGS lesions were subclassified. ROC curves, univariate and multivariate logistic regression were used. RESULTS: In Oxford classification, the majority of patients had mesangial hypercellularity in less than a half of glomeruli (M0), did not have endocapillary hypercellularity (E0), had segmental glomerulosclerosis (S1), had interstitial fibrosis and tubular atrophy in more than a half of the sample (T2) and had no crescents (C0). Hypertension increases the chance of M1 in 2.54x (p = 0.02). For each unit of increased creatinine, 2.6x more chances of E1 (p = 0.001). S1 is predicted by proteinuria with 75% sensitivity and 90.9% specificity (p < 0.0001). For each unit of increase in GFR, there is a reduction of 6% in the chance of T2 in relation to T0 (p = 0.0001). If hypertension, there is 5x more chances of T2 than T0 (p = 0.01). For each unit of increase in creatinine, there are 2.8x more chances of crescents- C (p = 0.003). Creatinine also showed 75.8% sensitivity and 75% specificity for prediction of C (p = 0.002). Inversely, for each unit of GFR, the chance of C is reduced by 4% (p = 0.007). Other clinical data related with C are hypertension (p = 0.03) and proteinuria (p = 0.02). To determine the role of FSGS subtypes in clinical presentation, we divided patients in S0 and S1 groups. Proteinuria was the only clinical parameter with significative difference, respectively, 0.3 (0-2.1) and 1.6 (0.02-16.2) g/24 h (p < 0.0001). FSGS subtypes related to proteinuria were cellular (p = 0.03) and peri-hilar (p = 0.02). Subtypes classically related to podocytopathies showed no correlation with clinical data. CONCLUSION: In the future, with noninvasive methods for diagnosis of IgAN, it will be essential to predict Oxford classification parameters using clinical laboratory data for establishment of prognosis and therapeutics. We showed that Oxford classification parameters correspond to some clinical laboratory data, making this approach possible. FSGS lesions not specifically related to podocytopathies may also influence clinical parameters that affect renal disease progression.

Podocin and uPAR are good biomarkers in cases of Focal and segmental glomerulosclerosis in pediatric renal biopsies.

There are controversies whether Minimal Change Disease (MCD) and Focal and Segmental Glomerulosclerosis (FSGS) are distinct glomerular lesions or different manifestations within the same spectrum of diseases. The uPAR (urokinase-type plasminogen activator receptor) and some slit diaphragm proteins may be altered in FSGS glomeruli and may function as biomarkers of the disease in renal biopsies. Thus, this study aims to evaluate the diagnostic potential of uPAR and glomerular proteins for differentiation between MCD and FSGS in renal pediatric biopsy. Renal biopsies from 50 children between 2 and 18 years old were selected, with diagnosis of MCD (n = 29) and FSGS (n = 21). Control group consisted of pediatric autopsies (n = 15) from patients younger than 18 years old, with no evidences of renal dysfunction. In situ expressions of WT1, nephrin, podocin and uPAR were evaluated by immunoperoxidase technique. Renal biopsy of patients with MCD and FSGS expressed fewer WT1 (p≤0.0001, F = 19.35) and nephrin (p<0.0001; H = 21.54) than patients in the control group. FSGS patients expressed fewer podocin than control (p<0.0359, H = 6.655). FSGS cases expressed more uPAR than each of control and MCD (p = 0.0019; H = 12.57) and there was a positive and significant correlation between nephrin and podocin (p = 0.0026, rS = 0.6502) in these cases. Podocin had sensitivity of 73.3% and specificity of 86.7% (p = 0.0068) and uPAR had sensitivity of 78.9% and specificity of 73.3% (p = 0.0040) for diagnosis of FSGS patients. The main limitation of the study is the limited number of cases due to the difficulty in performing biopsy in pediatric patients. Podocin and uPAR are good markers for FSGS and differentiate these cases from MCD, reinforcing the theory of distinct glomerular diseases. These findings suggest that podocin and uPAR can be used as biomarkers in the routine analysis of renal biopsies in cases of podocytopathies when the lesion (sclerosis) is not sampled.

Evaluation of the Diagnostic Potential of uPAR as a Biomarker in Renal Biopsies of Patients with FSGS.

Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are primary glomerulopathies leading to proteinuria, known as podocytopathies, which share syndromic and morphological similarities. Morphological similarity occurs in cases of FSGS in which the sclerotic lesion was not sampled in renal biopsy, due to the focal nature of the disease. Differentiating these entities is very important, especially in cases of suspected FSGS but with sclerotic lesion not sampled, as they are diseases that apparently have different pathogenic mechanisms and prognosis. The difference in uPAR expression in situ among these two entities may be related to a distinct molecular mechanism involved in pathogenesis. Thus, finding biomarkers involved in the pathogenesis and that can also help in differential diagnosis is very relevant. The aim of this work was to evaluate the potential of urokinase-type plasminogen activator receptor (uPAR) as a biomarker in renal biopsies of patients with podocytopathies (n = 38). Immunohistochemistry showed that FSGS (n = 22) had increased uPAR expression in podocytes compared with both the MCD group (n = 16; p = 0.0368) and control group (n = 21; p = 0.0076). ROC curve (p = 0.008) showed that this biomarker has 80.95% of specificity in biopsies of patients with FSGS. Therefore, uPAR presented a high specificity in cases of podocytopathies associated with sclerosis and it can be considered a potential biomarker for FSGS.

Nodular glomerulosclerosis in a non-diabetic hypertensive, dyslipidemic, smoker patient: a case report.

INTRODUCTION: This is a case report of a patient with idiopathic nodular glomerulosclerosis whose pathogenesis and morphology are similar to diabetic nephropathy. CASE PRESENTATION: A 64-year-old Brazilian man, leukoderma, dyslipidemic, obese with chronic obstructive pulmonary disease secondary to tobacco smoking, known to be hypertensive for five years and he had no history of diabetes. He was admitted with sudden anasarca, rapid loss of renal function and needed to start hemodialysis immediately. Renal biopsy was performed, and the sections were examined by light microscopy, immunofluorescence and electron microscopy. Morphological and ultrastructural findings showed that the profile of the disease studied herein strongly resembles diabetic nephropathy. However, the absence of diabetes mellitus, the presence of arteriolar hyalinosis in renal arterioles, tobacco smoking, and other clinical factors observed can play a significant role in nodular formation. CONCLUSION: The clinical features of the patient, and most importantly, the fact that he is a smoker, favor the diagnosis of "nodular glomerulosclerosis associated with smoking", a nomenclature proposed by some authors as an alternative to the term idiopathic nodular glomerulosclerosis. This clinical case report highlights idiopathic nodular glomerulosclerosis as a rare disease of little known etiopathogenesis; thus, further studies are necessary in order to elucidate the causes of this disease.

Nodular glomerulosclerosis in a non-diabetic hypertensive, dyslipidemic, smoker patient: a case report / Glomeruloesclerose nodular em paciente fumante não diabético, hipertenso e dislipidêmico: relato de caso

Abstract Introduction: This is a case report of a patient with idiopathic nodular glomerulosclerosis whose pathogenesis and morphology are similar to diabetic nephropathy. Case presentation: A 64-year-old Brazilian man, leukoderma, dyslipidemic, obese with chronic obstructive pulmonary disease secondary to tobacco smoking, known to be hypertensive for five years and he had no history of diabetes. He was admitted with sudden anasarca, rapid loss of renal function and needed to start hemodialysis immediately. Renal biopsy was performed, and the sections were examined by light microscopy, immunofluorescence and electron microscopy. Morphological and ultrastructural findings showed that the profile of the disease studied herein strongly resembles diabetic nephropathy. However, the absence of diabetes mellitus, the presence of arteriolar hyalinosis in renal arterioles, tobacco smoking, and other clinical factors observed can play a significant role in nodular formation. Conclusion: The clinical features of the patient, and most importantly, the fact that he is a smoker, favor the diagnosis of "nodular glomerulosclerosis associated with smoking", a nomenclature proposed by some authors as an alternative to the term idiopathic nodular glomerulosclerosis. This clinical case report highlights idiopathic nodular glomerulosclerosis as a rare disease of little known etiopathogenesis; thus, further studies are necessary in order to elucidate the causes of this disease.

Resumo Introdução: Este é um relato de caso de um paciente com glomeruloesclerose nodular idiopática, cuja patogênese e morfologia são semelhantes à nefropatia diabética. Apresentação do caso: Homem, 64 anos de idade, leucodermo, com dislipidemia, obesidade, doença pulmonar obstrutiva crônica secundária ao tabagismo, hipertenso há cinco anos e sem história de diabetes mellitus. Ele foi internado com anasarca súbita, perda rápida da função renal com necessidade de hemodiálise imediata. A biópsia renal foi realizada, e as seções foram examinadas por microscopia luz, imunofluorescência e microscopia eletrônica. Achados morfológicos e ultraestruturais mostraram que o perfil da doença estudado fortemente se assemelha à nefropatia diabética. No entanto, a ausência de diabetes mellitus, a presença de hialinose arteriolar em arteríolas renais, o fumo do tabaco, e outros fatores clínicos observados podem desempenhar um papel significativo na formação nodular. Conclusão: As características clínicas do paciente e, o mais importante, o fato de que ele é fumante favorecem o diagnóstico de "glomeruloesclerose nodular associada ao tabagismo", uma nomenclatura proposta por alguns autores como uma alternativa para o termo glomeruloesclerose nodular idiopática. Este relato de caso clínico realça glomeruloesclerose nodular idiopática como uma doença rara, de etiopatogenia pouco conhecida. Desse modo, mais estudos são necessários para elucidar as causas desta doença.