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Monday, 9th December 2019

Genetic and functional analysis of the renal filtration diaphragm in health and disease

 

   

grupo2018 LR

 

 

Mar Ruiz Gómez

 

 

  

 

 

 es

Research summary:

 

Our research aims to understand the development, composition and function of the renal filtration system as well as its pathological anomalies. To this end, we make use of two model organisms, the fly Drosophila melanogaster and the zebrafish Danio rerio and apply a variety of genetic, biochemical, molecular and cell biology techniques.

 

 

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Our model organisms: Drosophila melanogaster and zebrafish (Danio rerio).

Drosophila embryonic nephrocytes and zebrafish pronephros are indicated by arrowheads. They are labeled by the expression of specific markers.


 

 

Chronic kidney disease is one of the most prevalent illnesses in modern societies affecting 22% of the aged population and its incidence is increasing. It is a progressive loss of kidney function that if untreated, becomes irreversible and leads to end-stage renal disease requiring renal replacement therapy by dialysis or kidney transplantation. It is now accepted that chronic kidney disease is mainly triggered by insults to the glomerular podocyte, a very specialized kidney cell responsible of the initial stages of urine formation. Most of these insults disrupt slit diaphragm function.

The slit diaphragm is a multiprotein complex formed by two major structural constituents, the Immunoglobulin family members, nephrin and Neph1 and their associated intracellular partners. It acts as a molecular sieve during the process of blood ultrafiltration in the glomerulus, limiting the passage of molecules according to their size and charge. Insults to this structure induce dramatic morphological changes (foot process effacement) and lead to proteinuria.

 

 

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Scheme of vertebrate slit diaphragm, indicating its main protein components.

 

 

Our recent work demonstrated a striking similarity between the vertebrate podocyte slit diaphragm and the filtration diaphragm of the fly nephrocytes that spans the functional, structural and molecular levels (1). These similarities allow us exploit the power of Drosophila as a model organism to study the composition and assembly of the filtration diaphragm as well as its regulation and alterations under pathological conditions.

 

 

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Scanning electron micrograph of Drosophila nephrocytes (left).

The ultrastructure of mammalian slit diaphragm and Drosophila filtration diaphragm are extremely similar, as revelaed by transmission electron microscopy (right).

 

 

We look for novel genes involved in the formation and function of the filtration diaphragm. To this end, we apply the powerful genetic techniques available in Drosophila. Some of the genes we have found and are currently under study are involved in processes such as cell endocytosis or the regulation of the plasma membrane phospholipid composition.

 

 

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Drosophila nephrocytes in wild type animals (A) and in two different mutants isolated in our lab that affect cellular endocytosis (B) or the phospholipid composition of the plasma membrane (C). The filtration diaphragm proteins Duf and Pyd are labeled in green and red respectively. Nuclei in blue.

 

 


Additionally, we examine the dynamism of the filtration diaphragm molecular components and their regulation, focusing on their biochemistry and the filtration diaphragm structure revealed by confocal and electron microscopy. Thus, we have found that the stability of the fly filtration diaphragm is regulated by phosphorylation of Duf, the orthologue of Neph1, by the Src kinase Src64B. Hyperactivation of Src64B induces hyperphosphorylation of Duf and diaphragm damage. In addition, Duf phosphorylation status impinges on the constellation of its intracellular interactors (3).


Our overarching goal is to translate to vertebrates the knowledge acquired in our experimental model, to help understand and treat chronic kidney disease.

 


 

Selected publications:

 

  1. H. Weavers*, S. Prieto-Sánchez*, F. Grawe, A. García-López, R. Artero, M. Wilsch-Braeuninger, M. Ruiz-Gómez, H. Skaer and B. Denholm (2009). The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm. Nature 457: 323-326.
  2. A.S. Tutor and M. Ruiz-Gómez (2013)."Desarrollo embrionario del riñón", en Nefrología Clínica. Ed Médica Panamericana.
  3. A.S. Tutor, S. Prieto-Sánchez and M. Ruiz-Gómez (2014). Src64B phosphorylates Dumbfounded and regulates slit diaphragm dynamics: Drosophila as a model to study nephropathies. Development 141: 367-376.
  4. M. Carrasco-Rando, A. Atienza-Manuel, AS. Tutor and M. Ruiz-Gómez (2015). "Modelling renal development and disease in Drosophila". eLS. John Wiley & Sons, Ltd: Chichester. DOI 10.1002/9780470015902.a0025981

 


 

Doctoral theses:

 

  • Marta Carrasco Rando. "musculus morbidus una E3 ubiquitín ligasa de Drosophila, genera Artrina y mantiene la integridad del sarcómero". Universidad Autónoma de Madrid. 2 de Febrero de 2006. Sobresaliente "cum laude".
  • Silvia Prieto Sánchez. "Función de Dumbfounded, Sticks and stones y Polychaetoid en los nefrocitos en guirnalda de Drosophila melanogaster".Universidad Autónoma de Madrid. 15 de June de 2009. Sobresaliente "cum laude"

 


 

Awards:

 

  • 2009 PINP award to the best Ph.D. thesis to Dr. Silvia Prieto Sánchez.
  • 2010 Award for basic investigation in kidney research from Fundación Renal Iñigo Álvarez de Toledo (FRIAT) to Silvia Prieto Sánchez and Mar Ruiz Gómez.

 

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