Intitulé du sujet: To study the role of Emilin1 in diabetic kidney disease progression

Sujet

Codirection:

Nombre de mois: 36 mois

Ecole Doctorale: ED 562 - Bio Sorbonne Paris Cité

Unité de recherche et équipe:

INSERM U970, Paris Cardiovascular Center

Team Kidney, Vessels, immunity and Metabolism

Coordonnées de l’équipe:

56 rue Leblanc, Lab 219

75015 Paris

Secteur: Sciences de la vie / Life Sciences

Langue attendue: Anglais

Niveau de langue attendu: C2

Description

Description du sujet:

Diabetic kidney disease (DKD) is the main microvascular complication of diabetes, affecting approximately one-third of diabetic patients. If microvascular damage were prevented, the life expectancy and the quality of life of patients with diabetes would be most comparable to non-diabetic people. Diabetic kidney disease develops as a consequence of complex interactions between the glomerulus, tubule, interstitium, and vascular components of the kidney. It is therefore important to study the alterations in the diabetic kidney in order to identify mechanisms of protection and thus to identify new therapeutic targets. Tubulointerstitial injury is a key aspect of diabetic nephropathy and an important predictor of renal dysfunction. Diabetes associates increased production of glucose and AGEs that may lead to tubulointerstitial damage by the activation of various pathways. We recently identified Emilin1, a component of the extracellular matrix, as a putative protein involved in fibrosis in diabetic kidney disease. Emilin1 overexpression in kidney fibrotic regions of diabetic patients with diabetic kidney disease support that Emilin1 is secreted by extracellular matrix-producing cells, most probably fibroblast; but its role as (anti-?)fibrotic protein is not known in this context. The main objective of the project is thus to determine the potential roles of Emilin1 in diabetic kidney disease. For that purpose we will use a combination of in vitro and ex vivo approaches with validation on human samples. The project has three main aims: Aim 1: To study Emilin1 expression in kidney cells in DKD We will use archived renal biopsies of patients with DKD and kidneys from diabetic mice with mild fibrosis, the BTBR ob/ob mouse, to study Emilin1 expression in DKD and to analyze whether its expression correlates with TGFβ signaling changes and fibrosis. Aim 2: To study how Emilin1 is involved in fibrogenesis in DKD We will generate kidney cells (fibroblasts and parietal epithelial cells (PECs)) with Emilin1 deficiency to study its role in DKD progression and fibrosis. Aim 3: To modulate Emilin1 expression using miRNA If the aims 1 and 2 confirm the expected role of Emilin1 as anti-fibrotic in DKD, then we will try to sustain its expression in DKD. For that we will study its regulation by miRNAs and we will test antagomir to sustain Emilin1 expression in renal cells Our project could led to the identification of a novel pathway important for fibrogenesis in DKD. Fibrosis has been clearly associated to renal function decline in patients with DKD and TGFβ is involved in that process. Several strategies have been tested in animals with success to block fibrogenesis in DKD by acting on TGFβ. But blocking TGFβ appears risky in human due to its role on inflammation. One approach would be then to block its action only were it is deleterious. If we demonstrate that Emiin1 exerts antifibrotic effects in kidneys via its action on TGFβ activation, then sustaining Emilin1 expression in kidneys, using miRNAs antagomir for example, could represent a novel approach to limit fibrogenesis in DKD.

Compétences requises:

Softwares: GraphPad Prism, Image J, Zotero, R code and Python language would be a plus.

Animal experiment: Handling of mice, injection, Gavage, Organ and sample collection

Cell culture: manipulation of cells according to CSL2 regulation, transfection, lentiviral transduction

Molecular biology: Western blot, PCR, qPCR, Immunohistochemistry, Plasmid cloning

English C2 or C1

Références bibliographiques:

1. Kramer, C. K., Rodrigues, T. C., Canani, L. H., Gross, J. L. & Azevedo, M. J. Diabetic
retinopathy predicts all-cause mortality and cardiovascular events in both type 1 and 2
diabetes: meta-analysis of observational studies. Diabetes Care 34, 1238–1244 (2011).
2. Sawaf, H. et al. Therapeutic Advances in Diabetic Nephropathy. J Clin Med 11, 378 (2022).
3. Mahtal, N., Lenoir, O. & Tharaux, P.-L. Glomerular Endothelial Cell Crosstalk With
Podocytes in Diabetic Kidney Disease. Front Med (Lausanne) 8, 659013 (2021).
4. Van Buren, P. N. & Toto, R. Current update in the management of diabetic nephropathy.
Curr Diabetes Rev 9, 62–77 (2013).
5. Kida, Y. Peritubular Capillary Rarefaction: An Underappreciated Regulator of CKD
Progression. Int J Mol Sci 21, E8255 (2020).
6. Eleftheriadis, T., Antoniadi, G., Pissas, G., Liakopoulos, V. & Stefanidis, I. The renal
endothelium in diabetic nephropathy. Ren Fail 35, 592–599 (2013).
7. Lenoir, O., Gaillard, F., Lazareth, H., Robin, B. & Tharaux, P.-L. Hmox1 Deficiency
Sensitizes Mice to Peroxynitrite Formation and Diabetic Glomerular Microvascular Injuries.
J Diabetes Res 2017, 9603924 (2017).
8. Lenoir, O. et al. Endothelial cell and podocyte autophagy synergistically protect from
diabetes-induced glomerulosclerosis. Autophagy 11, 1130–1145 (2015).
9. Lenoir, O. et al. Direct action of endothelin-1 on podocytes promotes diabetic
glomerulosclerosis. J Am Soc Nephrol 25, 1050–1062 (2014).
10. Garsen, M. et al. Endothelin-1 Induces Proteinuria by Heparanase-Mediated Disruption of
the Glomerular Glycocalyx. J Am Soc Nephrol 27, 3545–3551 (2016).
11. Ohse, T. et al. A new function for parietal epithelial cells: a second glomerular barrier. Am J
Physiol Renal Physiol 297, F1566-1574 (2009).
12. Shirato, I. et al. The development of focal segmental glomerulosclerosis in masugi nephritis
is based on progressive podocyte damage. Virchows Arch 429, 255–273 (1996).
13. Bariéty, J. et al. Posttransplantation relapse of FSGS is characterized by glomerular
epithelial cell transdifferentiation. J Am Soc Nephrol 12, 261–274 (2001).
14. Lazareth, H., Lenoir, O. & Tharaux, P.-L. Parietal epithelial cells role in repair versus
scarring after glomerular injury. Curr Opin Nephrol Hypertens 29, 293–301 (2020).
15. Kawaguchi, T. et al. Diabetic condition induces hypertrophy and vacuolization in glomerular
parietal epithelial cells. Sci Rep 11, 1515 (2021).
16. Holderied, A. et al. Glomerular parietal epithelial cell activation induces collagen secretion
and thickening of Bowman’s capsule in diabetes. Lab Invest 95, 273–282 (2015).
17. Chan, G. C. et al. Differential expression of parietal epithelial cell and podocyte
extracellular matrix proteins in focal segmental glomerulosclerosis and diabetic
nephropathy. Am J Physiol Renal Physiol 317, F1680–F1694 (2019).
18. Zhang, Y., George, J., Li, Y., Olufade, R. & Zhao, X. Matrix Metalloproteinase-9 Expression
Is Enhanced in Renal Parietal Epithelial Cells of Zucker Diabetic Fatty Rats and Is Induced
by Albumin in In Vitro Primary Parietal Cell Culture. PLoS ONE 10, e0123276 (2015).
19. Kietzmann, L. et al. MicroRNA-193a Regulates the Transdifferentiation of Human Parietal
Epithelial Cells toward a Podocyte Phenotype. J Am Soc Nephrol 26, 1389–1401 (2015).

20. Saleem, M. A. et al. A conditionally immortalized human podocyte cell line demonstrating
nephrin and podocin expression. J Am Soc Nephrol 13, 630–638 (2002).
21. Singh, A. et al. High glucose causes dysfunction of the human glomerular endothelial
glycocalyx. Am J Physiol Renal Physiol 300, F40-48 (2011).
22. Luque, Y. et al. Endothelial Epas1 Deficiency Is Sufficient To Promote Parietal Epithelial
Cell Activation and FSGS in Experimental Hypertension. J Am Soc Nephrol 28, 3563–3578
(2017).
23. Lazareth, H. et al. The tetraspanin CD9 controls migration and proliferation of parietal
epithelial cells and glomerular disease progression. Nat Commun 10, 3303 (2019).
24. Bensaada, I. et al. Calpastatin prevents Angiotensin II-mediated podocyte injury through
maintenance of autophagy. Kidney Int 100, 90–106 (2021).
25. Mahtal, N., Lenoir, O. & Tharaux, P.-L. Glomerular Endothelial Cell Crosstalk With
Podocytes in Diabetic Kidney Disease. Front. Med. 8, 659013 (2021).
26. Zanetti, M. et al. EMILIN-1 deficiency induces elastogenesis and vascular cell defects. Mol
Cell Biol 24, 638–650 (2004).
27. Zacchigna, L. et al. Emilin1 Links TGF-β Maturation to Blood Pressure Homeostasis. Cell
124, 929–942 (2006).
28. Louzao-Martinez, L. et al. A proteome comparison between human fetal and mature renal
extracellular matrix identifies EMILIN1 as a regulator of renal epithelial cell adhesion.
Matrix Biology Plus 4, 100011 (2019).
29. Randles, M. J. et al. Identification of an Altered Matrix Signature in Kidney Aging and
Disease. J Am Soc Nephrol 32, 1713–1732 (2021).
30. Qi, Y. et al. TSPAN9 and EMILIN1 synergistically inhibit the migration and invasion of
gastric cancer cells by increasing TSPAN9 expression. BMC Cancer 19, 630 (2019).
31. Braga Gomes, K., Fontana Rodrigues, K. & Fernandes, A. P. The Role of Transforming
Growth Factor-Beta in Diabetic Nephropathy. International Journal of Medical Genetics
2014, 1–6 (2014).
32. Azevedo, R. et al. CD44 glycoprotein in cancer: a molecular conundrum hampering clinical
applications. Clin Proteomics 15, 22 (2018).
33. Eymael, J. et al. CD44 is required for the pathogenesis of experimental crescentic
glomerulonephritis and collapsing focal segmental glomerulosclerosis. Kidney Int 93, 626–
642 (2018).
34. Appel, D. et al. Recruitment of podocytes from glomerular parietal epithelial cells. J Am Soc
Nephrol 20, 333–343 (2009)