INTRODUÇÃO: A Glomerulosclerose Segmentar e Focal (GESF) é a glomerulopatia primária mais prevalente no Brasil e sua incidência vem aumentando no mundo inteiro. Na sua forma primária, caracteriza-se clinicamente por acometer pessoas jovens e causar proteinúria acentuada, geralmente acompanhada de síndrome nefrótica. O mecanismo patogênico tem como evento principal a lesão ao podócito, desencadeado por fatores de natureza variada: vírus, drogas/medicamentos, imunológicos, etc. Em 2004, foi publicada a classificação de Columbia, propondo 5 variantes morfológicas distintas na GESF: colapsante (COL), usual (NOS), apical ou tip lesion (TIP), perihilar (PHI) e variante celular (CEL). Diversos estudos comprovam alterações moleculares em podócitos na GESF. Essas alterações são observadas em diversos sítios podocitários: em moléculas envolvidas na fenda de filtração (slit diaphragm), por exemplo, nefrina, podocina e CD2AP; em moléculas do citoesqueleto podocitário, como a -actinina-4 e sinaptopodina; em moléculas marcadoras de diferenciação dos podócitos, como CD10 e WT-1; e ainda em marcadores de divisão celular como Ki-67 e PCNA. Os objetivos desse estudo foram: 1-) classificar as lesões morfológicas de GESF em biópsia renais nas 5 variantes da GESF propostas na Classificação de Columbia; e 2-) analisar a ocorrência de alterações moleculares podocitárias nestes casos. MÉTODOS: Foram selecionados 131 casos de biópsias renais com diagnóstico de GESF primária no período de 1996 a 2006. Os casos foram classificados de acordo com os critérios de Columbia e posteriormente submetidos a reações imuno-histoquímicas para os marcadores CD10, WT-1, vimentina, sinaptopodina, -actinina-4, GLEPP-1, citoqueratina 8/18, citoqueratina 19 e Ki-67. Os resultados foram submetidos à análise estatística através do teste qui-quadrado. RESULTADOS: A classificação das variantes da GESF se distribuiu da seguinte forma: 38,2% de variante NOS, 36,6% de variante COL, 14,5% de variante TIP, 6,9% de variante PHI e 3,8% de variante CEL. Os casos da variante COL se destacaram das demais variantes pela perda de expressão de marcadores de diferenciação celular, como o CD10 e o WT-1 (p<0,01), perda da molécula do citoesqueleto -actinina-4 (p<0,01) e neo-expressão de citoqueratinas 8-18 (p<0,05) e 19 (p<0,01). Adicionalmente, os casos das variantes COL e CEL se destacam das outras variantes pela expressão do marcador de divisão celular Ki-67 (p<0,05). CONCLUSÃO: a variante COL destacou-se das demais em relação às alterações moleculares observadas na análise imuno-histoquímica. O diagnóstico diferencial desta forma de GESF tem importância clínica por ela estar associada a pior evolução e prognóstico em relação às demais variantes. A integração destes marcadores na rotina diagnóstica pode auxiliar no diagnóstico diferencial da GESF COL / INTRODUCTION: Focal segmental glomerulosclerosis (FSGS) is the most prevalent primary glomerulopathy in Brazil and its incidence is increasing worldwide. Primary FSGS is characterized clinically by affecting young people and causing severe proteinuria, often accompanied by nephrotic syndrome. The pathogenesis is related to podocyte injury, which may be due to several factors: viruses, drugs, immunological, etc. In 2004, the Columbia classification of FSGS identified five histological variants of the disease: collapsing (COL), usual (NOS), tip lesion (TIP), perihilar (PHI) and cellular variant (CEL). Several studies have demonstrated molecular changes in podocytes of FSGS patients, which were observed in molecules involved in the filtering function of these cells (nephrin, podocina and CD2AP), in podocyte cytoskeleton molecules (-actinin-4, and synaptopodin), as well as in molecular markers of podocyte differentiation (CD10 and WT-1) and of cell division (Ki-67 and PCNA). The aim of this study was to classify the FSGS biopsies according to the Columbia classification and to analyze the occurrence of molecular changes in the five morphological variants. METHODS: 131 cases of renal biopsies with a diagnosis of primary FSGS in the period 1996 to 2006 were classified according to the criteria of Columbia and then submitted to immunohistochemical reactions with the following antibodies: CD10, WT-1, Vimentin, Synaptopodin, -actinin-4, GLEPP-1, cytokeratin 8-18, cytokeratin 19, and Ki-67. RESULTS: FSGS cases were classified into five variants as follows: 38.2% of NOS variant, 36.6% COL, 14.5% TIP, 6.9% PHI and 3.8% CEL. The COL variant cases distinguished themselves among the other for having lost the expression of CD10 and WT-1 (p <0.01), and also of -actinin-4 (p <0, 01). Furthermore, they gained expression of the cytokeratin 8-18 (p <0.05) and 19 (p <0.01). The group of CEL and COL variants together differed from the other variants regarding the expression of cell division marker Ki-67 (p <0.05). CONCLUSION: COL variant of FSGS presents molecular changes that differs from others and can be demonstrated by immunohistochemistry. The differential diagnosis of this variant is important because of the worse clinical outcome and prognosis it presents in comparison with other variants. The identification of these markers by immunohistochemical on the routine practice may be useful in the diagnosis of COL FSGS

The increased prevalence of chronic kidney disease (CKD) has become a major global health burden. This increase in CKD burden parallels the increase in hypertension prevalence. In addition, increasing evidence suggest that genetics play a strong role in the susceptibility for renal disease. Inbred mouse strains C57BL/6 and 129S6SvEv differ in their susceptibility to kidney disease when subjected to hypertension using the DOCA/salt uninephrectomy model of hypertension. Similar to others, we found the 129S6SvEv mice to be susceptible to develop severe glomerulosclerosis, whereas the C57BL/6 mice are comparatively resistant. To identify new candidate genes that are involved in the pathogenesis of glomerular disease, we used microarray technology to compare the glomerular transcriptome of both strains and determine changes in glomerular gene expression when subjected to the DOCA/salt uninephrectomy model of systemic hypertension. This approach was accompanied with ultrastructural analysis and glomerular stiffness measurements to identify corresponding structural changes. Here, we have identified novel genes associated with strain differences and hypertension, and we used immunohistochemistry to validate their expression in podocytes and glomerular arterioles in murine and human kidneys. The increased understanding of the molecular mechanisms underlying hypertension-associated podocyte injury and glomerular damage which will result from these studies, will ultimately lead to identification of novel pharmacologic targets or therapeutic strategies for patients with hypertension and renal disease. / 2017-11-02T00:00:00Z

Background: Focal segmental glomerulosclerosis (FSGS) is a component of childhood nephrotic syndrome occurring in 10%-20% of all cases. Over time, 25%-50% of children with FSGS develop kidney failure disease. We followed a cohort of children with FSGS in order to delineate the risk profile of progression to kidney failure (KF). Methods: We evaluated patient data collected from 1977 to 2002 at a regional mid-Atlantic nephrology center in the United States. KF was defined primarily for those patients whose serum creatinine (SCr) value doubled compared with the SCr value from a previous visit. Patients who received dialysis or a kidney transplant were also defined as having KF. We analyzed patient data for those who had at least two visits with SCr values recorded. Various baseline characteristics of patients who had developed KF and those with no kidney failure (NKF) were compared. Hazard ratios and correlation were used to further investigate potential risk factors of the kidney failure. We also compared the inverse SCr trend for KF and NKF patients using weighted linear regression. Results: Thirty-four of 43 FSGS patients had adequate follow-up data. About 60% of the patients developed KF over the study period. The average age of the KF patients at diagnosis of FSGS was 9 years, and that of NKF patients 12 years (P=0.05). FSGS patients with KF had a significantly higher mean diastolic blood pressure (DBP) at baseline, compared to those with NKF (P<0.0001). Other baseline characteristics including race, body mass index (BMI), systolic blood pressure, total cholesterol, urinary protein/creatinine ratio and calculated glomerular filtration rate (cGFR) were not significantly different. Baseline DBP was a significant risk factor in progression to KF (HR: 1.03; 95%CI: 1.01-1.06). Inverse SCr values were significantly decreased over time in KF patients (P=0.01). Conclusions: The data of this study indicate that children diagnosed with FSGS who are younger than 10 years and have elevated baseline DBP are more likely to develop kidney failure. The non-significant hazard ratios for other baseline characteristics including gender, race, and BMI are not instrumental risk factors. These results may help understand what may affect progression towards kidney failure in children with FSGS.

Nephrotic syndrome (NS) is the second leading cause of chronic kidney disease (CKD) presenting under the age of 30. NS presents in children with edema and severe proteinuria, caused by the effacement of podocyte foot processes within the glomerular filtration barrier. Patients with steroid-resistant NS (SRNS) frequently develop end-stage renal disease (ESRD). Additionally, renal biopsies from these patients often reveal focal segmental glomerulosclerosis (FSGS). Pathogenic mutations in known monogenic disease genes have been found in 11-45% children with FSGS/SRNS. Notably, most Mendelian etiologies exhibit recessive inheritance, while dominant vertical inheritance with incomplete penetrance is observed in the remainder. The role of de novo variants (DNVs) in NS necessitates further investigation. Tripartite motif containing 8, TRIM8, is an E3 ubiquitin ligase. De novo TRIM8 variants were previously implicated in a syndromic disease consisting of neurodevelopmental delay, epilepsy, cerebral atrophy, and nephrotic syndrome. In this study, we recapitulate the patient-specific mutations in inducible overexpression cell lines and in CRISPR/Cas9-generated mouse models. N-terminal MYC or GFP-tagged TRIM8 inducible cell lines were generated and characterized using the pInducer21 system. Western blot and immunofluorescence data show that MYC- and GFP-TRIM8 were induced by doxycycline in immortalized podocyte cell lines. Candidate interactors for TRIM8 from the literature and stratified using kidney single cell mRNA sequencing expression were cloned into mammalian expression vectors. Finally, a Trim8 knockout allele (c. 56_162del; p.H20Qfs*124 and c.367_463+304delins46) was generated and bred to yield an allelic series of wildtype, heterozygous and homozygous animals. These mice exhibited normal survival and did not demonstrate proteinuria through three to four months of life. Overall, further studies are ongoing with regards to the continued monitoring of proteinuria and kidney dysfunction, as well as the potential interactor cloning and cell line characterization. / 2025-02-06T00:00:00Z

L’implication d’un facteur circulant et des dysfonctions du système immunitaire entrainant les altérations de la barrière de filtration glomérulaire a été suggérée dans la pathogénèse de la hyalinose segmentaire et focal récidivante. Nous avons identifié par spectrométrie de masse la présence de la protéine CASK dans des sérums de patients après immunoadsoroption sur une colonne de protéine A. CASK recombinante est capable d'induire des modifications de l’architecture des podocytes in vitro, tels qu’une redistribution de la protéine de diaphragme de fente ZO-1 et de la protéine régulatrice d’actine synaptopodine, et une perte de fibres de stress d’actine. Ces podocytes acquièrent ainsi un phénotype motile et une perméabilité accrue à l’albumine en présence de CASK recombinante in vitro. L’injection de CASK chez des souris entraine une protéinurie et l’effacement des pédicelles de podocytes. L’interaction entre CASK et son récepteur CD98 dans les podocytes a été mise en évidence par l’expérience de pontage covalent et co-immunoprécipitation. L’inhibition de l’expression de CD98 par ARNi a permis de préserver l’architecture des podocytes en présence de CASK. Nous avons remarqué la surexpression de CASK dans les monocytes chez les patients atteints de la HSF récidivante par rapport aux témoins. In vitro, CASK est surexprimée dans les macrophages ayant une polarité M2 et est retrouvée dans le surnageant de la culture de ces cellules. La sécrétion de CASK est associée aux exosomes qui sont des microvésicules d’origine endosomale. Dans les cellules, CASK est partiellement co-distribuée avec ALIX, un marqueur exosomal, et leur interaction a été mise en évidence par co-immunoprécipitation. CASK est fortement exprimée dans les exosomes de patients atteints de HSF récidivante comparé aux donneurs sains. Le traitement des podocytes par des exosomes issus des macrophages de type M2 induit des altérations du cytosquelette et augmente la motilité des podocytes comme cela avait été observé en présence de CASK recombinante. Pour conclure, nous avons identifié CASK comme nouveau facteur soluble qui pourrait jouer un rôle au cours de la HSF récidivante après transplantation rénale. Ces découvertes ouvrent de nouvelles orientations pour le traitement des malades atteints de SNI récidivant. / Focal segmental glomerulosclerosis (FSGS) is often associated with a high rate of progression to end-stage renal disease. The idiopathic form has a high recurrence rate (rFSGS) after transplantation suggesting the presence of a systemic circulating factor that causes the glomerular permeability. This factor can be removed by plasmapheresis or immunoadsorption using protein-A columns. We used mass spectrometry to analyze the proteins eluted from protein-A columns, taken from patients with rFSGS after immunoadsorption. A serum form of calcium/calmodulin-dependent serine/threonine kinase (CASK) was identified in rFSGS patients but not in controls. In cultured podocytes, recombinant CASK induced reorganization of the actin cytoskeleton. We also demonstrated the interaction of CASK with CD98 at the cell surface. Injection of recombinant CASK in mice induced proteinuria and foot process effacement on podocytes. We identified that CASK is produced by monocytes in patients with rFSGS. CASK is also expressed and secreted by M2 polarized macrophages but not by M1 subset. CASK was associated with exosomes produced by these cells. CASK has a partial codistribution with ALIX, an exosomal component involved in their development. We’ve also demonstrated that CASK interacts with ALIX in M2 macrophages. Moreover exosomes derived from M2 macrophages cause podocytes cytoskeleton alterations and increase of podocyte motility as observed previously with recombinant CASK. In conclusion, a serum form of CASK secreted by macrophages acts as a permeability factor in patients with rFSGS suggesting its involvement in the physiopathology of rFSGS.

Loss of the renal microvasculature is a common feature of almost all forms of chronic kidney disease (CKD). Here we explored the role of the angiogenic chemokine stromal cell-derived factor-1-alpha (SDF-1) and its cognate receptor CXCR4 in experimental and human CKD. CXCR4 was present on endothelial cells and podocytes, while SDF-1 was detectable on podocytes, arteriolar smooth muscle cells, interstitial fibroblasts and occasional endothelial cells. CXCR4 mRNA was elevated in the kidneys of rats with CKD and chronic antagonism of CXCR4 accelerated renal decline and capillary loss. Acute SDF-1 infusion activated glomerular endothelial nitric oxide synthase (eNOS) in vivo, while functional response to SDF-1 was impaired in glomerular endothelial cells derived from eNOS-/- mice. Finally, CXCR4 mRNA was also found to be increased in biopsies of patients with secondary focal segmental glomerulosclerosis. These observations indicate that local eNOS-dependent SDF-1/CXCR4 signaling exerts a compensatory reno-protective effect in the setting of CKD.

Loss of the renal microvasculature is a common feature of almost all forms of chronic kidney disease (CKD). Here we explored the role of the angiogenic chemokine stromal cell-derived factor-1-alpha (SDF-1) and its cognate receptor CXCR4 in experimental and human CKD. CXCR4 was present on endothelial cells and podocytes, while SDF-1 was detectable on podocytes, arteriolar smooth muscle cells, interstitial fibroblasts and occasional endothelial cells. CXCR4 mRNA was elevated in the kidneys of rats with CKD and chronic antagonism of CXCR4 accelerated renal decline and capillary loss. Acute SDF-1 infusion activated glomerular endothelial nitric oxide synthase (eNOS) in vivo, while functional response to SDF-1 was impaired in glomerular endothelial cells derived from eNOS-/- mice. Finally, CXCR4 mRNA was also found to be increased in biopsies of patients with secondary focal segmental glomerulosclerosis. These observations indicate that local eNOS-dependent SDF-1/CXCR4 signaling exerts a compensatory reno-protective effect in the setting of CKD.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22124号 / 工博第4654号 / 新制||工||1726(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 跡見 晴幸, 教授 浜地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Background and objective: By reviewing our current understanding of oxidative injury as a cause of focal segmental glomerulosclerosis (FSGS), we hope to advance the use of antioxidants as a promising treatment in addition to the other therapeutic modalities to slow the rate of progression. Methods: Key references from the past concerning oxidative injury and FSGS were analyzed, together with those from a PubMed search of the literature from 1997 to 2007, to form the basis of this commentary. Results: In animal studies in FSGS produced by subtotal nephrectomy or puromycin injections, evidence of oxidant injury provided the rationale for disease reversal with an antioxidant such as high dose vitamin E. Clinical trial in children with FSGS using vitamin E resulted in significant reduction in proteinuria. Other treatment modalities in children with FSGS over the past four decades were reviewed. These consisted of one or more of the following medications: oral prednisone, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and intravenous methylprednisolone with and without cyclophosphamide or cyclosporine. The prognosis with these recent therapeutic interventions improved the outcome of children with FSGS compared to no treatment as advocated earlier. However, when the current regimen of combined treatment was compared with the regimen of prednisone plus cyclophosphamide, there was no difference in Kaplan-Meier kidney survival rate at a mean follow-up of 12 to 16 years. Conclusion: In reviewing current concepts of oxidant injury and other mechanisms of injury in the development of FSGS and the available modalities of treatment, we call into question, whether the cost and side effects of intravenous methylprednisolone is justifiable on the basis of unchanged kidney survival rates with continuing this particular mode of intervention.

No description available.