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| Author(s): |
Mychel Raony Paiva Teixeira Morais
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2019-12-05 |
| Examining board members: |
Telma Maria Tenorio Zorn;
Vanessa Morais Freitas;
Graziella Eliza Ronsein;
Karina Thieme
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| Advisor: | Telma Maria Tenorio Zorn; Sebastian Andres San Martin Henriquez |
| Abstract | |
Kidney development in diabetic pregnancies has been widely studied, however, little is known about the alterations on the metanephric extracellular matrix (ECM) in this gestational condition. The ECM plays an essential role during kidney development, therefore, changes in its composition, organization and physicochemical properties must have a negative effect on this organ. We hypothesize that the maternal diabetes mellitus (DM) restricts kidney development and alters metanephric ECM composition and turnover. Thus, this study aimed to investigate the effects of the maternal DM on the kidney structure and urinary production rate of pre-term fetuses, as well as to globally characterize the composition, organization and alterations on the metanephric ECM, using a model of pregnancy complicated by type 1 diabetes induced by a dose of alloxan (40 mg/kg) in mice, without insulin therapy. In this model, normoglycemic (control) and diabetic females were mated with normoglycemic males from the 30th to the 50th day post-induction, and fetuses of normoglycemic and diabetic mothers (FNM and FDM, respectively) were collected on the 19th embryonic day (E19). Compared to FNM, we observed that FDM were severely hyperglycemic and asymmetrically growth restricted characterized by retarded body growth in relation to head development. Growth-restricted FDM had kidneys with reduced mass and volume compared to normal FMN. Amniotic fluid analysis revealed polyhydramnios due to fetal polyuria and glycosuria in the diabetic group. Stereological analysis showed reduced volumetric proportion of the renal cortex and pelvis dilation in FDM. We observed reduced nephron endowment associated to increased corpuscular volume and dilation of convoluted tubules in FMD compared to FNM. Ultrastructural analysis of the glomerular filtration barrier by transmission electron microscopy revealed segmental thickening of the glomerular basement membrane (GBM) and morphological alterations of the foot processes of podocytes in mature glomeruli of FDM. The metanephric matrisome in mice was investigated for the first time by tandem mass spectrometry using samples enriched for ECM proteins. We identified 86 structural (collagens, adhesive glycoproteins and proteoglycans) and 75 ECM-associated proteins (regulatory, affiliated and secreted factors), including known markers of GMB maturity. The proteomic analysis showed increased levels of EMILIN-3 and COL6A6 in FDM kidneys, which was further validated by immunoblot. Real-time PCR and immunostaining analyses showed accumulation of collagen IV in FDM kidneys, particularly of COL4A1 in mature glomeruli. We also found a discrete increase of panlaminin and reduction of perlecan in mature glomeruli, besides accumulation of agrin on the apical surface of convoluted tubules of FDM by immunohistochemistry. In gel zymography and immunoblot revealed reduced activation of MMP-2 and MT1- MMP, respectively, in FDM kidneys. We also verified increased levels of TIMP-2 by real-time PCR, immunoblot and immunohistochemistry. These analyses allowed us to conclude that the maternal DM restricted intrauterine kidney development in mice and promoted ECM accumulation in the fetal kidney, particularly of collagen, due to a misbalanced expression and turnover of its proteins. (AU) | |
| FAPESP's process: | 15/03525-2 - Effects of the type 1 diabetes on the fetal reprogramming with focus on the glomerular extracellular matrix in mice model |
| Grantee: | Mychel Raony Paiva Teixeira Morais |
| Support Opportunities: | Scholarships in Brazil - Doctorate |