Nephrotic syndrome (NS) is a set of symptoms defined by heavy proteinuria and associated with a host of kidney diseases that cause injury to the glomerulus, the filtration apparatus of the kidney. Primary podocytopathies (Px), a group of diseases including minimal change disease (MCD), primary focal segmental sclerosis (pFSGS), and lupus podocytopathy (LP), are the principal cause of idiopathic NS in both children and adults. The hallmark feature of Px is the ultrastructural finding of podocyte foot process effacement (FPE), so current differential diagnosis of Px relies on technically exhaustive electron microscopy (EM) analysis. During routine immunofluorescence (IF) microscopy of many Px cases, we have observed punctate IgG (P-IgG) immunoreactivity in the glomerulus. P-IgG may represent a disease-specific reactivity that could not only provide clues for understanding Px etiology but could also serve as a diagnostic tool. However, we have found that P-IgG may be misinterpreted as protein reabsorption granules (PRGs), a morphological feature seen in proteinuric conditions. We sought to definitively characterize the key characteristics of PRGs and P-IgG in order to differentiate these features. To accomplish this, we reviewed prior IF immunostaining of MCD biopsies to evaluate anti-human IgG and anti-human albumin staining. We conducted additional IF staining on archived MCD biopsies using antibodies against IgG and against the markers of PRGs: albumin, megalin, and cubilin. We found that the P-IgG demonstrates a diffuse, global distribution pattern that is specific to glomerular epithelium and is fine and scattered. Conversely, the PRGs are coarse, clustered, and frequently demonstrate a focal, segmental pattern in the glomeruli and tubules. Co-staining with albumin and megalin revealed that the P-IgG and the PRGs do not colocalize in the tissue. While the cubilin antibody positively stained the tubular epithelium, it did not stain glomeruli. Our results showed that punctate IgG and protein reabsorption granules are morphologically and constitutionally distinct and do not colocalize with each other, indicating that P-IgG is highly likely to represent a distinct process from epithelial protein reabsorption.

Glomerular derangement is the major feature of a diverse array of kidney disorders that lead to end-stage kidney disease (ESKD). Podocyte dysfunction is central to the underlying pathophysiology of many common glomerular diseases, including diabetic nephropathy (DN), focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), and genetic forms of nephrotic syndrome, and is associated with heavy proteinuria. Loss of podocyte foot process structure, or effacement, is the key feature of podocyte injury in these proteinuric glomerular disorders, also called "podocytopathies". However, the degree of effacement can vary: For instance, it is very disseminated (diffuse) in minimal change disease, but more variable (segmental) in focal segmental glomerulosclerosis. Recent work has shown that nephrin, ROBO2, and SLIT2 are proteins implicated in these podocyte foot process effacement and podocytopathy. We sought to evaluate changes in the expression of these proteins using immunofluorescence microscopy, and the degree of foot process effacement in podocytopathies using ultrastructural morphometry. In podocytopathies, we saw increased expression of ROBO2 and decreased expression of nephrin indicating that, upregulation of ROBO2 may lead to podocyte injury and podocyte injury may result in loss of nephrin. In addition, SLIT2, which binds ROBO2, was found in tubules and in glomeruli. A higher degree of geometric mean foot process width was vii observed in podocytopathies as compared to normal kidney. These findings can be used in the clinical setting to diagnose and monitor disease treatment. / 2024-02-07T00:00:00Z

Introdução: A podocitopenia e a podocitúria são marcadores de injúria glomerular em podocitopatias (POD) e glomerulonefrites proliferativas (GNsP), e mesmo em fases iniciais destas doenças mecanismos pró-fibróticos indutores de glomeruloesclerose e fibrose renal progressiva estão ativados. Objetivo: Avaliar pacientes portadores de glomerulopatias biopsiados em diferentes tempos de evolução clínica, correlacionando lesões morfológicas dos compartimentos glomerular e túbulo-intersticial com a expressão dos RNAm de proteínas associadas ao podócito e de fatores pró-fibróticos no tecido renal. Materiais e Métodos: Foram incluídos no estudo oitenta e quatro pacientes adultos portadores de glomerulopatias de diferentes etiologias submetidos à biópsia renal por indicação clínica. As lesões histológicas foram individualizadas e a porcentagem de fibrose intersticial e atrofia tubular foi quantificada na coloração de Tricrômio de Masson. Foram mensurados no tecido renal o log 10 do RNAm pela reação em cadeia da polimerase em tempo real das proteínas associadas ao podócito alfa actinina-4, podocina e podocalixina e dos fatores pró-fibróticos fator de crescimento transformador ₁ (TGF₁), fator de crescimento do tecido conectivo (CTGF) e fator de crescimento derivado do endotélio A (VEGF-A). A secção livre de neoplasia de rins removidos por câncer renal foram usados como controles da expressão dos RNAm. Resultados: No grupo POD, os diagnósticos histopatológicos foram: Glomeruloesclerose segmentar e focal (n=20), GN membranosa (n=12), Nefropatia diabética (n=9) e Lesões mínimas (n=7); no grupo GNsP foram Nefropatia por IgA (n=15), GN membranoproliferativa (n=5), Nefrite lúpica (n=5) e GN proliferativa mesangial (n=4), e outros diagnósticos (n=7). O RNAm do tecido renal nos pacientes com POD e GNsP foi significativamente menor comparado ao dos controles para todos os genes estudados. A presença de crescentes, independente do estágio evolutivo, foi associada à maior expressão do RNAm de alfa actinina-4 (p=0,04), podocina (p=0,01) e podocalixina (p=0,038). O RNAm dos genes pró-fibróticos também estava inibido comparado a sua expressão no rim normal. Nas GNsP, o VEGF-A (p<0,001) e o CTGF (p<0,001) foram os genes com menor nível de expressão comparado aos controles. Em relação às biópsias com lesões crescênticas, tanto o RNAm do TGFβ1 (p=0,001) como do CTGF (p=0,041) tiveram maior expressão comparado ao RNAm das biópsias sem crescentes. Nas biópsias com fibrose intersticial superior a 30%, a expressão do RNAm de TGFβ1, (p=0,038) e do VEGF-A (p=0,040) foi maior do que nas biópsias com fibrose leve. O maior tempo entre o início da doença clínica e a realização da biópsia renal não teve influência detectável na expressão tecidual do RNAm dos biomarcadores estudados. Conclusões: Pacientes com podocitopatias ou glomerulonefrites proliferativas apresentaram inibição da expressão do RNAm de proteínas associadas ao podócito e de fatores indutores de fibrose renal, achados compatíveis com injúria podocitária e podocitopenia. Nas biópsias renais com maior grau de fibrose intersticial e atrofia tubular, assim também como naquelas com lesões crescênticas, a expressão do RNAm de fatores fibrogênicos como TGF-β1 e CTGF foi significativamente aumentada, o que pode sugerir supra-regulação de moléculas associadas a mecanismos de fibrose renal e patologia glomerular. / Introduction: Both podocitopenia and podocyturia are markers of glomerular injury in podocytopathies (POD) and proliferative glomerulonephritis (PGNs), and even in the early stages of these diseases pro-fibrotic mechanisms leading to glomerulosclerosis and progressive renal fibrosis are running. Objective: This study evaluated patients with glomerulopathies who were biopsied at different times of clinical evolution, correlating morphological lesions of the glomerular and tubulointerstitial compartments with renal messenger RNA (mRNA) expression of podocyteassociated proteins and pro-fibrotic factors. Materials and Methods: The study included eighty-four adult patients with glomerulopathies of different etiologies undergoing kidney biopsy as clinically indicated. The histological lesions were individualized and the percentage of interstitial fibrosis and tubular atrophy was quantified on Trichrome Masson staining. Tissue log 10 mRNA of the podocyte proteins alpha-actinin-4, podocin and podocalyxin and of the pro-fibrotic factors transforming growth factor β₁ (TGFβ₁), connective tissue growth factor (CTGF) and vascular endothelium growth factor A (VEGF-A) was measured by real time polymerase chain reaction. The sections free of neoplasia of kidneys removed for renal cancer were used as controls for the mRNA tissue expression. Results: Results: In the POD group, the histopathological diagnoses were: focal segmental glomerulosclerosis (n=20), membranous (n=12), diabetic nephropathy (n=9) and minimal changes (n=7); in PGNs group were IgA nephropathy (n=15), membranoproliferative (n=5), lupus nephritis (n=5) and mesangial proliferative (n=4), and other diagnoses (n=7). Messenger RNA expression of POD and PGNs groups was significantly lower compared to controls for all the studied genes. The presence of crescents, regardless of their evolutive stage, was associated with higher mRNA expression of alpha-actinin-4 (p=0.04), podocin (p=0.01) and podocalyxin (p=0.038). The mRNA of pro-fibrotic genes was also inhibited compared to their expression in normal kidneys. In PGNs, VEGF-A (p<0.001) and CTGF (p<0.001) were the genes with lowest mRNA levels compared to controls. Regarding the biopsies with crescentic lesions, both the mRNA of TGFβ1 (p=0.001) and CTGF (p=0.041) were highly expressed as compared to those of biopsies without crescents. In biopsies with moderate to severe interstitial fibrosis (more than 30%), the mRNA expression of TGFβ1 (p=0.038) and VEGF-A (p=0.040) was highly expressed compared to biopsies with mild fibrosis. A longer interval between the clinical disease and the performance of kidney biopsy did not have a detectable influence on tissue mRNA expression of the studied biomarkers. Conclusions: Patients with POD or PGNs presented inhibition of the mRNA expression of podocyte-associated proteins and pro-fibrotic factors, findings that are consistent with podocyte injury and podocitopenia. In renal biopsies with a higher degree of interstitial fibrosis and tubular atrophy, as well as those with crescentic lesions, the mRNA expression of fibrogenic factors such as TGF-β1 and CTGF was significantly increased, which may suggest upregulation of molecules associated with mechanisms of renal fibrosis and glomerular pathology.

Introdução: A podocitopenia e a podocitúria são marcadores de injúria glomerular em podocitopatias (POD) e glomerulonefrites proliferativas (GNsP), e mesmo em fases iniciais destas doenças mecanismos pró-fibróticos indutores de glomeruloesclerose e fibrose renal progressiva estão ativados. Objetivo: Avaliar pacientes portadores de glomerulopatias biopsiados em diferentes tempos de evolução clínica, correlacionando lesões morfológicas dos compartimentos glomerular e túbulo-intersticial com a expressão dos RNAm de proteínas associadas ao podócito e de fatores pró-fibróticos no tecido renal. Materiais e Métodos: Foram incluídos no estudo oitenta e quatro pacientes adultos portadores de glomerulopatias de diferentes etiologias submetidos à biópsia renal por indicação clínica. As lesões histológicas foram individualizadas e a porcentagem de fibrose intersticial e atrofia tubular foi quantificada na coloração de Tricrômio de Masson. Foram mensurados no tecido renal o log 10 do RNAm pela reação em cadeia da polimerase em tempo real das proteínas associadas ao podócito alfa actinina-4, podocina e podocalixina e dos fatores pró-fibróticos fator de crescimento transformador ₁ (TGF₁), fator de crescimento do tecido conectivo (CTGF) e fator de crescimento derivado do endotélio A (VEGF-A). A secção livre de neoplasia de rins removidos por câncer renal foram usados como controles da expressão dos RNAm. Resultados: No grupo POD, os diagnósticos histopatológicos foram: Glomeruloesclerose segmentar e focal (n=20), GN membranosa (n=12), Nefropatia diabética (n=9) e Lesões mínimas (n=7); no grupo GNsP foram Nefropatia por IgA (n=15), GN membranoproliferativa (n=5), Nefrite lúpica (n=5) e GN proliferativa mesangial (n=4), e outros diagnósticos (n=7). O RNAm do tecido renal nos pacientes com POD e GNsP foi significativamente menor comparado ao dos controles para todos os genes estudados. A presença de crescentes, independente do estágio evolutivo, foi associada à maior expressão do RNAm de alfa actinina-4 (p=0,04), podocina (p=0,01) e podocalixina (p=0,038). O RNAm dos genes pró-fibróticos também estava inibido comparado a sua expressão no rim normal. Nas GNsP, o VEGF-A (p<0,001) e o CTGF (p<0,001) foram os genes com menor nível de expressão comparado aos controles. Em relação às biópsias com lesões crescênticas, tanto o RNAm do TGFβ1 (p=0,001) como do CTGF (p=0,041) tiveram maior expressão comparado ao RNAm das biópsias sem crescentes. Nas biópsias com fibrose intersticial superior a 30%, a expressão do RNAm de TGFβ1, (p=0,038) e do VEGF-A (p=0,040) foi maior do que nas biópsias com fibrose leve. O maior tempo entre o início da doença clínica e a realização da biópsia renal não teve influência detectável na expressão tecidual do RNAm dos biomarcadores estudados. Conclusões: Pacientes com podocitopatias ou glomerulonefrites proliferativas apresentaram inibição da expressão do RNAm de proteínas associadas ao podócito e de fatores indutores de fibrose renal, achados compatíveis com injúria podocitária e podocitopenia. Nas biópsias renais com maior grau de fibrose intersticial e atrofia tubular, assim também como naquelas com lesões crescênticas, a expressão do RNAm de fatores fibrogênicos como TGF-β1 e CTGF foi significativamente aumentada, o que pode sugerir supra-regulação de moléculas associadas a mecanismos de fibrose renal e patologia glomerular. / Introduction: Both podocitopenia and podocyturia are markers of glomerular injury in podocytopathies (POD) and proliferative glomerulonephritis (PGNs), and even in the early stages of these diseases pro-fibrotic mechanisms leading to glomerulosclerosis and progressive renal fibrosis are running. Objective: This study evaluated patients with glomerulopathies who were biopsied at different times of clinical evolution, correlating morphological lesions of the glomerular and tubulointerstitial compartments with renal messenger RNA (mRNA) expression of podocyteassociated proteins and pro-fibrotic factors. Materials and Methods: The study included eighty-four adult patients with glomerulopathies of different etiologies undergoing kidney biopsy as clinically indicated. The histological lesions were individualized and the percentage of interstitial fibrosis and tubular atrophy was quantified on Trichrome Masson staining. Tissue log 10 mRNA of the podocyte proteins alpha-actinin-4, podocin and podocalyxin and of the pro-fibrotic factors transforming growth factor β₁ (TGFβ₁), connective tissue growth factor (CTGF) and vascular endothelium growth factor A (VEGF-A) was measured by real time polymerase chain reaction. The sections free of neoplasia of kidneys removed for renal cancer were used as controls for the mRNA tissue expression. Results: Results: In the POD group, the histopathological diagnoses were: focal segmental glomerulosclerosis (n=20), membranous (n=12), diabetic nephropathy (n=9) and minimal changes (n=7); in PGNs group were IgA nephropathy (n=15), membranoproliferative (n=5), lupus nephritis (n=5) and mesangial proliferative (n=4), and other diagnoses (n=7). Messenger RNA expression of POD and PGNs groups was significantly lower compared to controls for all the studied genes. The presence of crescents, regardless of their evolutive stage, was associated with higher mRNA expression of alpha-actinin-4 (p=0.04), podocin (p=0.01) and podocalyxin (p=0.038). The mRNA of pro-fibrotic genes was also inhibited compared to their expression in normal kidneys. In PGNs, VEGF-A (p<0.001) and CTGF (p<0.001) were the genes with lowest mRNA levels compared to controls. Regarding the biopsies with crescentic lesions, both the mRNA of TGFβ1 (p=0.001) and CTGF (p=0.041) were highly expressed as compared to those of biopsies without crescents. In biopsies with moderate to severe interstitial fibrosis (more than 30%), the mRNA expression of TGFβ1 (p=0.038) and VEGF-A (p=0.040) was highly expressed compared to biopsies with mild fibrosis. A longer interval between the clinical disease and the performance of kidney biopsy did not have a detectable influence on tissue mRNA expression of the studied biomarkers. Conclusions: Patients with POD or PGNs presented inhibition of the mRNA expression of podocyte-associated proteins and pro-fibrotic factors, findings that are consistent with podocyte injury and podocitopenia. In renal biopsies with a higher degree of interstitial fibrosis and tubular atrophy, as well as those with crescentic lesions, the mRNA expression of fibrogenic factors such as TGF-β1 and CTGF was significantly increased, which may suggest upregulation of molecules associated with mechanisms of renal fibrosis and glomerular pathology.

Introdução: A podocitopenia e a podocitúria são marcadores de injúria glomerular em podocitopatias (POD) e glomerulonefrites proliferativas (GNsP), e mesmo em fases iniciais destas doenças mecanismos pró-fibróticos indutores de glomeruloesclerose e fibrose renal progressiva estão ativados. Objetivo: Avaliar pacientes portadores de glomerulopatias biopsiados em diferentes tempos de evolução clínica, correlacionando lesões morfológicas dos compartimentos glomerular e túbulo-intersticial com a expressão dos RNAm de proteínas associadas ao podócito e de fatores pró-fibróticos no tecido renal. Materiais e Métodos: Foram incluídos no estudo oitenta e quatro pacientes adultos portadores de glomerulopatias de diferentes etiologias submetidos à biópsia renal por indicação clínica. As lesões histológicas foram individualizadas e a porcentagem de fibrose intersticial e atrofia tubular foi quantificada na coloração de Tricrômio de Masson. Foram mensurados no tecido renal o log 10 do RNAm pela reação em cadeia da polimerase em tempo real das proteínas associadas ao podócito alfa actinina-4, podocina e podocalixina e dos fatores pró-fibróticos fator de crescimento transformador ₁ (TGF₁), fator de crescimento do tecido conectivo (CTGF) e fator de crescimento derivado do endotélio A (VEGF-A). A secção livre de neoplasia de rins removidos por câncer renal foram usados como controles da expressão dos RNAm. Resultados: No grupo POD, os diagnósticos histopatológicos foram: Glomeruloesclerose segmentar e focal (n=20), GN membranosa (n=12), Nefropatia diabética (n=9) e Lesões mínimas (n=7); no grupo GNsP foram Nefropatia por IgA (n=15), GN membranoproliferativa (n=5), Nefrite lúpica (n=5) e GN proliferativa mesangial (n=4), e outros diagnósticos (n=7). O RNAm do tecido renal nos pacientes com POD e GNsP foi significativamente menor comparado ao dos controles para todos os genes estudados. A presença de crescentes, independente do estágio evolutivo, foi associada à maior expressão do RNAm de alfa actinina-4 (p=0,04), podocina (p=0,01) e podocalixina (p=0,038). O RNAm dos genes pró-fibróticos também estava inibido comparado a sua expressão no rim normal. Nas GNsP, o VEGF-A (p<0,001) e o CTGF (p<0,001) foram os genes com menor nível de expressão comparado aos controles. Em relação às biópsias com lesões crescênticas, tanto o RNAm do TGFβ1 (p=0,001) como do CTGF (p=0,041) tiveram maior expressão comparado ao RNAm das biópsias sem crescentes. Nas biópsias com fibrose intersticial superior a 30%, a expressão do RNAm de TGFβ1, (p=0,038) e do VEGF-A (p=0,040) foi maior do que nas biópsias com fibrose leve. O maior tempo entre o início da doença clínica e a realização da biópsia renal não teve influência detectável na expressão tecidual do RNAm dos biomarcadores estudados. Conclusões: Pacientes com podocitopatias ou glomerulonefrites proliferativas apresentaram inibição da expressão do RNAm de proteínas associadas ao podócito e de fatores indutores de fibrose renal, achados compatíveis com injúria podocitária e podocitopenia. Nas biópsias renais com maior grau de fibrose intersticial e atrofia tubular, assim também como naquelas com lesões crescênticas, a expressão do RNAm de fatores fibrogênicos como TGF-β1 e CTGF foi significativamente aumentada, o que pode sugerir supra-regulação de moléculas associadas a mecanismos de fibrose renal e patologia glomerular. / Introduction: Both podocitopenia and podocyturia are markers of glomerular injury in podocytopathies (POD) and proliferative glomerulonephritis (PGNs), and even in the early stages of these diseases pro-fibrotic mechanisms leading to glomerulosclerosis and progressive renal fibrosis are running. Objective: This study evaluated patients with glomerulopathies who were biopsied at different times of clinical evolution, correlating morphological lesions of the glomerular and tubulointerstitial compartments with renal messenger RNA (mRNA) expression of podocyteassociated proteins and pro-fibrotic factors. Materials and Methods: The study included eighty-four adult patients with glomerulopathies of different etiologies undergoing kidney biopsy as clinically indicated. The histological lesions were individualized and the percentage of interstitial fibrosis and tubular atrophy was quantified on Trichrome Masson staining. Tissue log 10 mRNA of the podocyte proteins alpha-actinin-4, podocin and podocalyxin and of the pro-fibrotic factors transforming growth factor β₁ (TGFβ₁), connective tissue growth factor (CTGF) and vascular endothelium growth factor A (VEGF-A) was measured by real time polymerase chain reaction. The sections free of neoplasia of kidneys removed for renal cancer were used as controls for the mRNA tissue expression. Results: Results: In the POD group, the histopathological diagnoses were: focal segmental glomerulosclerosis (n=20), membranous (n=12), diabetic nephropathy (n=9) and minimal changes (n=7); in PGNs group were IgA nephropathy (n=15), membranoproliferative (n=5), lupus nephritis (n=5) and mesangial proliferative (n=4), and other diagnoses (n=7). Messenger RNA expression of POD and PGNs groups was significantly lower compared to controls for all the studied genes. The presence of crescents, regardless of their evolutive stage, was associated with higher mRNA expression of alpha-actinin-4 (p=0.04), podocin (p=0.01) and podocalyxin (p=0.038). The mRNA of pro-fibrotic genes was also inhibited compared to their expression in normal kidneys. In PGNs, VEGF-A (p<0.001) and CTGF (p<0.001) were the genes with lowest mRNA levels compared to controls. Regarding the biopsies with crescentic lesions, both the mRNA of TGFβ1 (p=0.001) and CTGF (p=0.041) were highly expressed as compared to those of biopsies without crescents. In biopsies with moderate to severe interstitial fibrosis (more than 30%), the mRNA expression of TGFβ1 (p=0.038) and VEGF-A (p=0.040) was highly expressed compared to biopsies with mild fibrosis. A longer interval between the clinical disease and the performance of kidney biopsy did not have a detectable influence on tissue mRNA expression of the studied biomarkers. Conclusions: Patients with POD or PGNs presented inhibition of the mRNA expression of podocyte-associated proteins and pro-fibrotic factors, findings that are consistent with podocyte injury and podocitopenia. In renal biopsies with a higher degree of interstitial fibrosis and tubular atrophy, as well as those with crescentic lesions, the mRNA expression of fibrogenic factors such as TGF-β1 and CTGF was significantly increased, which may suggest upregulation of molecules associated with mechanisms of renal fibrosis and glomerular pathology.