The major goal of our laboratory is to elucidate the molecular mechanisms of post-filtration protein handling by proximal tubules in health and disease.

Vladimir Marshansky, PhD 
Assistant Professor of Medicine 
185 Cambridge Street, CPZN 8.212
Boston, MA 02114
Phone: (617) 724-9815
Fax: (617) 643-3182
Email: vmarshansky@partners.org

 

Role of kidney proximal tubules in metabolism of proteins:

The major goal of our laboratory is to elucidate the molecular mechanisms of post-filtration protein handling by proximal tubules in health and disease. Recent advances in cell and molecular biology of kidney in combination with proteomic analysis of urine indicate a crucial role of megalin/cubilin-pathway in proteins reabsorption and handling (including albumin, vitamin binding proteins, hormones, cytokines and lgG) by proximal tubule epithelial cell. The physiological (health) and pathophysiological (disease) significance of protein degradation via endosomal-lysosomal pathway as well as potential role of released "protein fragments" in kidney function are unclear. However, "protein-wasting" is associated with renal pathologies such as Fanconi syndrome and diabetic nephropathy. Excessive, non-physiological levels of proteins in urine can adversely affect epithelial cell function, and it is important to define the pathways and mechanisms involved in protein reabsorption and trafficking. Our previous studies [1-6] have examined the protein trafficking by megalin/cubilin-mediated endocytosis in kidney proximal tubules under physiological conditions. We have uncovered the differential expression and targeting of Arf family small GTPases in the function of the early endosomal compartment of proximal tubules [2,5,6] and have discovered fragmentation/degradation of reabsorbed protein(s) (i.e. albumin) in early endosomal compartment of proximal tubules [1,4].

Our current research interests include:

1) Study the expression, processing, targeting and trafficking of megalin and cubilin. Protein reabsorption via receptor-mediated endocytosis is an important function of the kidney proximal tubules (PT) which involves megalin and cubilin receptors. Here [8] we studied the expression and targeting of megalin and cubilin in PT epithelial cells. Polarized MTC (Mouse Tubular Cells) and IRPT (Immortalized Rat Proximal Tubule) cells cultured on filters were used as a model of PT epithelium. Differential expression of megalin and cubilin polarization was assessed by quantitative real-time PCR and by Western blot analysis. Both megalin and cubilin mRNA levels were up-regulated initially followed by down-regulation during increased epithelial polarization. However, megalin transcripts were 10-30 times more abundant than cubilin transcripts. Western blot analysis revealed the presence of intact (600 kD) megalin at all stages of polarization. Intact full-length (460 kD) cubilin was identified only in completely polarized cells, while at early stages of polarization various cubilin fragments (50 - 200 kD) were detected. Megalin and cubilin are considered as tandem/synergistic receptors. Surprisingly, confocal microscopy revealed a lack of co-targeting and co-localization of megalin and cubilin in polarized MTC and IRPT cells. Megalin was targeted apically to and above tight junctions and partially colocalized with ZO1, while cubilin was found in vesicles below tight junctions. Finally, we also studied the expression and targeting of megalin and cubilin in PT in situ. Both receptors were targeted to the apical pole of mouse PT, but were not always colocalized. Some PT cells exclusively expressed megalin while others expressed cubilin. Thus, our studies demonstrate the differential post-translational processing and targeting of megalin and cubilin receptors during polarization [8]. The molecular and cell biological mechanisms governing the function of both receptors are currently under investigation in our laboratory.

2) Elucidate the role of Arf small GTPases in endosomal function of proximal tubules. ADP-ribosylation factor (Arf) small GTPases are critical regulators of vesicular trafficking both in exo- and endocytosis pathways. GDP-bound Arfs ae inactive while GTP activates Arfs and leads to their recruitment onto target membranes. This activation is catalysed by guanine nucleotide exchange factors (GEFs). Inactivation of Arfs (conversion into the Arf-GDP form) is catalyzed by GTPase activating proteins (GAPs) which enhance the very low intrinsic GTPase activity of Arfs and triggers their release from target membranes. Thus, both regulation of specific activities and targeting of Arf isoforms to different intracellular membranes are most likely achieved through selective sets of GEF and GAP proteins. Previously, we reported the expression and distribution of Arf6 and ARNO in the proximal tubule epithelial cell receptor-mediated endocytosis pathway, where they are co-localized with megalin and the V-ATPase in situ as well as in purified early endosomes in vitro [3,4]. In our recent studies, we have also demonstrated the localization of Arf1 in the apical pole of proximal tubule cells and its colocalization  with megalin [7]. We also showed that V-ATPase dependent intra-endosomal acidification stimulates the recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal membranes, implicating this process in endosomal function of megalin/cubilin-mediated pathway in situ [5,6].

3) Uncover the role of V-ATPase and endosomal acidification in function of megalin/cubilin-pathway. Numerous mammalian intracellular organelles and carrier vesicles have an acidic lumen generated by V-ATPase in conjunction with a parallel chloride conductance. Acidification of proximal tubule endosomes is driven by the V-ATPase and also regulated by other proteins of acidification machinery (CLC5 and NHE3) [2, 4-6]. The V-ATPase controls a variety of intracellular processes. Acidification of early endosomes (pH ~5.5) is required for dissociation of some ligand-receptor complexes and a low pH within lysosomes (pH ~4.5 - 5.0) is necessary for the activity of proteases. Previously, we demonstrated that protein (albumin) fragmentation-degradation is already occurring in early endosomes [1,4]. Recently, we showed that acidification of kidney proximal tubule endosomes is required for the recruitment of Arf6 small GTPase and ARNO (its cognate GDP/GTP - exchange factor) to the endosomal membrane, a step that precedes and it required for coat formation on nascent endosomal carrier vesicles [5,6]. Based on this observation, we also proposed that V-ATPase driven acidification-dependent recruitment of ARNO & Arf6 to endosomal membrane is due to interaction with "pH-sensing protein" (PSP). Thus we examined the mechanism of ARNO and Arf6 recruitment to proximal tubule endosomes. For the first time, our studies identify the transmembrane a2-subunit of the V-ATPase as a protein that interacts with the cytosolic endosomal "coat" components ARNO/Arf6. The V-ATPase a2-subunit is, therefore, a candidate PSP that could participate in the pH-dependent recruitment of ARNO/Arf6 to the membrane of acidic endosomes [9-11]. We hypothesize that Arf6 and ARNO could regulate the megalin/cubilin-mediated endocytosis pathway in proximal tubule epithelial cells [8], and that the V-ATPase has a central role in this process.

In summary: The disruption of vesicular trafficking events in different intracellular compartments gives rise to a variety of human disorders, including some forms of cancer and neurological diseases. Disorders related to kidney function include some forms of Fanconi syndrome as well as diabetic nephropathy. The ongoing studies of our laboratory aim to elucidate the cell biological events that could provide potential therapeutic targets and specific diagnostic/prognostic markers of kidney diseases, including diabetic nephropathy: one of the major causes of kidney failure and associated mortality in the United States.

 

References:

  1. Marshansky V, Fleser A, Noel J, Bourgoin S, and Vinay P. Isolation of heavy endosomes from dog proximal tubules in suspension. J Memb Biol. 1996. 153: 59 - 73.
  2. Marshansky V and Vinay P. Proton gradient formation in early endosomes from proximal tubules. Biochim Biophys Acta. 1996. 1284: 171 - 180.
  3. Marshansky V, Bourgoin S, Londono I, Bendayan M, and Vinay P. Identification of ADP-ribosylation factor-6 in brush-border membrane and early endosomes of human kidney proximal tubules. Electrophoresis. 1997. 18: 538-547.
  4. Marshansky V, Bourgoin S, Londono I, Bendayan M, Maranda B, and Vinay P. Receptor-mediated endocytosis in kidney proximal tubules: recent advances and hypothesis. Electrophoresis. 1997. 18: 2661 - 2676
  5. Maranda B, Brown D, Casanova JE, Bourgoin S, Vinay P, Ausiello DA, and Marshansky V. Intraendosomal pH-sensitive recruitment of the Arf-nucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes. J Biol Chem. 2001. 276: 18540 - 18550.
  6. Marshansky V, Ausiello DA, and Brown D. Physiological importance of endosomal acidification: potential role in proximal tubulopathies. Current Opinion in Nephrology and Hypertension. 2002. 11: 527 - 537.
  7. El Annan J, Brown D, Breton S, Bourgoin S, Ausiello DA, and Marshansky V. Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ. Am J Physiol. (Cell Physiol.) 2003. In Press.

 

Selected Recent Conferences and Abstracts:

  1. Bechoua S, Hurtado-Lorenzo A, El-Annan J, Da Silva N, Breton S, Ingelfinger JR, Fyfe JC, Ausiello DA, Brown D, and Marshansky V. Expression and targeting of megalin and cubilin receptors during polarization of proximal tubule epithelial cells. "The American Society for Cell Biology 43rd Annual Meeting." (December 13-17, 2003) San Francisco, CA, USA. Mol Biol Cell. 14: 479a.
  2. Marshansky V. Is the V-ATPase a pH-sensing protein? The a2-subunit localizes in kidney endosomes and interacts with the Arf6 GEF, ARNO. "Gordon Research Conference: Molecular and Cellular Bioenergetics." (June 22-27, 2003), Kimbal Union Academy, Meriden, NH, USA.
  3. Hurtado-Lorenzo A, El Annan J, Bechoua S, Futai M, Moran MF, Ausiello DA, Brown D, and Marshansky V. When small GTPases meet with ATPases: Functional significance of this "rendez-vous" for kidney epithelial cells. "HUPO 2nd Annual & IUBMB XIX World Congress." (October 8 - 11, 2003) Montreal , QC, Canada. Mol & Cell Proteomics. 2(9): 860.
  4. Hurtado-Lorenzo A, El Annan J, Bechoua S, Futai M, Bourgoin S, Casanova J, Brown D, Ausiello DA, and Marshansky V. V-type ATPase as a possible pH-sensing protein: a2-subunit of V-ATPase localizes in endosomes and interacts with ARNO in kidney proximal tubules. "The American Society for Cell Biology 43rd Annual Meeting." (December 13-17, 2003) San Francisco, CA, USA. Mol Biol Cell. 14: 237a-238a.