Kidney Week

Abstract: TH-PO698

Actin and Intermediate Filament Protein LAD1 (Ladinin1) Is Dysregulated in ADPKD Mouse Model

Session Information

• ADPKD: Genetic and Model Studies
  October 25, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidney

• 1001 Genetic Diseases of the Kidney: Cystic

Authors

• Kurashige, Mahiro, NIDDK, National Institute of Health, Bethesda, Maryland, United States

Mahiro Kurashige,

• Wu, Tsung-Jui, NIDDK, National Institute of Health, Bethesda, United States

Tsung-Jui Wu,

• Lin, Chengchao, NIDDK, National Institute of Health, Bethesda, Maryland, United States

Chengchao Lin,

• Zhou, Fang, NIDDK, National Institute of Health, Bethesda, Maryland, United States

Fang Zhou,

• Menezes, Luis F., NIDDK, National Institute of Health, Bethesda, Maryland, United States

Luis F. Menezes,

• Germino, Gregory G., NIDDK, National Institute of Health, Bethesda, Maryland, United States

Gregory G. Germino,

Background

There have been reports that Polycystin1 (PC1) co-localizes with actin/intermediate filament and could have functional roles in its organization, but much remains poorly understood. We have previously reported the transcriptomics of an orthologous ADPKD mouse model using 80 kidneys from Pkd1cko;Tg(Cre/Esr1) 102-210 days-old mice, in which Pkd1 knockout was induced at P40. We found Lad1 (Ladinin-1), a poorly characterized intermediate-filament protein, was one of the top differentially expressed genes. Here we focus on characterizing Lad1 expression and function as a candidate regulator of cytoskeleton dynamics in ADPKD.

Methods

16 kidneys (8 WT, 8 KO) were used for qPCR and 12 (6 WT, 6 KO) for immunoblotting. Lad1 mRNA and protein expression levels were evaluated by qPCR and immunoblot in a pair of DBA and LTL cell lines derived from Pkd1^CKO/CKO mouse, with corresponding mutant derived after Cre-expression. Mouse and humand Lad1 were cloned with various epitope tags to ease detection. A human keratinocyte cell line with high levels of endogenous LAD1, HaCat, and an MDCK cell line expressing recombinant, HA-tagged human PC1 were used for characterizing LAD1 and actin dynamics.

Results

Lad1 mRNA and protein levels were 58.8% (exon1-2; 50.0% exon4-5) and 67% lower, respectively, in Pkd1mutant kidneys (p<0.01 for each analysis). In the cell lines, Lad1 mRNA and protein levels were 46.0% (n=3, p=0.16) and 67.7% (n=3, p<0.01) lower in mutants compared to the paired WT cells. In characterizing Lad1 protein, we found that both endogenous and recombinant Lad1 migrates at ~72Kda, not at the size of 59kDa predicted by both its MW and companies that sell Lad1 antibodies. Using the HaCat cell line to better characterize LAD1 function, we found that LAD1 levels increased with cell confluency. Confocal studies determined that LAD1 generally co-localizes with actin in stress fibers and in a cortical “rim” pattern at the cell edge, which is decreased at cell-cell junctions. This pattern was also seen in the MDCK and DBA renal epithelial cell lines.

Conclusion

LAD1 is a poorly understood cytoskeletal protein whose expression increases with cell density but whose level is down-regulated in cystic kidney. Further study will be required to determine whether the downregulation observed in cystic kidney reflects altered cell-cell sensing.

Funding

• NIDDK Support