Molecular Identification of Canine Podocalyxin-Like Protein 1 as a Renal Tubulogenic Regulator

Abstract

GP135 is an apical membrane protein expressed in polarized MDCK epithelial cells. When cultured in three-dimensional collagen gel, MDCK cells form branching tubules in response to hepatocyte growth factor stimulation in a manner that simulates the embryonic renal development. During this process, GP135 displays transient loss of membranous localization but reappears at the cell surface when nascent lumen emerges from the developing tubules. Despite being used for decades as the canonical hallmark of apical surface, the molecular identity and the significance of the dynamic expression of GP135 during the tubulogenic process remain elusive. For exploring the function of GP135, the full-length cDNA encoding GP135 was obtained. Sequence alignments and features analysis confirm GP135 as a canine homolog of podocalyxin, confirming the finding of an earlier independent study. Immunohistochemical assays on canine kidney sections identified both glomerular and tubular distribution of GP135 along the nephron. Mutant MDCK cells expressing siRNA targeted at two regions of GP135 show defects in hepatocyte growth factor-induced tubulogenesis. Re-expression of full-length and an O-linked glycosylation abbreviated construct of GP135 could recapitulate the tubulogenesis process lacking in siRNA knockdown cells; however, a deletion construct devoid of the cytoplasmic domain failed to rescue the phenotype. In summary, the data identify the MDCK apical domain marker GP135 as a tubular form of podocalyxin and provide evidence for its importance in renal tubulogenesis.

Trafficking of Crumbs3 during Cytokinesis Is Crucial for Lumen Formation

“…Because one of our Crb3 hairpins showed a reduction of GP135 levels (Supplemental Figure S7A), we cannot rule out a role for GP135 in lumen formation. Meder et al (2005) found a requirement of GP135 for lumen formation, although this result could not be reproduced by Cheng et al (2005). Furthermore, the second hairpin we used did not cause a reduction of GP135, compared with the control cell line used, although reproducing the predominant no-lumen phenotype of the first hairpin (Supplemental Figure S7B).…”

The Role of Podocalyxin in Health and Disease

“…33 Similarly, blocking podocalyxin expression prevents tubule formation in a kidney tubulogenesis model. 34 Moreover, podocalyxin is essential for extension of foot processes in kidney podocytes (discussed in the Podocalyxin in the Developing Kidney section). 2 Thus, mounting evidence suggests a role for podocalyxin in cellular morphogenesis.…”

Podocalyxin in the Diagnosis and Treatment of Cancer

“…Since human RCC arise from renal tubule cells, the MDCK model cell line may offer valuable insights into the molecular mechanisms of tumour progression and metastasis in RCC. Although renal tubules in human and rabbit do not express detectable levels of PC protein, MDCK cells and adult canine kidney tubular cells have detectable PC (Cheng et al, 2005;Meder et al, 2005). As mentioned in previous sections, MCF7 and PC3 cells are derived from metastatic breast and prostate tumours, respectively.…”

“…6) in MCF7 result in sorting of these exogenously expressed proteins to the apical membrane domain. Conversely, using a similar series of PC mutants expressed in MDCK cells, two other laboratories show that precise apical targeting of PC requires its intracellular domain (Cheng et al, 2005;Meder et al, 2005). In these studies, although the majority of intracellular-deletion PC mutants were still sorted to the apical domain, only full-length PC with an intact O-linked glycosylation extracellular domain plus an intracellular domain was targeted to apical domains in a way that precisely co-localized with endogenous PC.…”

“…While the parental MDCK line formed organized spheres with a central lumen when suspended in matrix, deletion of PC disrupts this architecture, and MDCK fail to form an organized lumen (Meder et al, 2005). Correspondingly, using siRNA-mediated knockdown of endogenous P C i n M D C K , C h e n g e t a l s h o w t h a t endogenous PC is required for hepatocyte growth factor (HGF)-induced tubulogenesis (Chen et al, 2005). Transfection with siRNA-resistant PC (FL) and mutant constructs reveals that tubule formation of MDCK requires the intracellular domain.…”