Polycystic kidney disease in Han:SPRD Cy rats is associated with elevated expression and mislocalization of SamCystin

Abstract

Polycystic kidney disease (PKD) in Han:SPRD Cy rats is caused by a missense mutation in Anks6 (also called Pkdr1), leading to an R823W substitution in SamCystin, a protein that contains ankyrin repeats and a sterile alpha motif (SAM). The cellular function of SamCystin and the role of the Cy (R823W) mutation in cyst formation are unknown. In normal SPRD rats, SamCystin was found to be expressed in proximal tubules and glomeruli; protein expression was highest at 7 days of age and declined by ∼50-60% at 45-84 days of age. In Cy/+ and Cy/Cy kidneys, expression of SamCystin was lower than in +/+ kidneys at 3 and 7 days but became elevated at 21 days. Immunohistochemical analysis revealed that SamCystin was distributed on the brush border of proximal tubules in normal rat kidneys. In Cy/+ kidneys, there were robust SamCystin staining in cyst-lining epithelial cells and loss of apical localization, and increased number of PCNA-positive cells in cyst-lining epithelia. Verapamil, an L-type Ca(2+) channel blocker, accelerated PKD progression in this model and caused a further increase in the expression and abnormal distribution of SamCystin. We conclude that aberrant expression and mislocalization of R823W SamCystin lead to increased cell proliferation and renal cyst formation.

Fig. 1.

Tissue distribution and relative SamCystin abundance in kidneys of 21-day-old +/+, Cy/+, and Cy/Cy rats. A: immunoblots for +/+ and Cy/+ kidney lysates probed with rabbit polyclonal antibodies against 2 epitopes of rat SamCystin. Molecular weight markers are shown on the left side of each blot. The antibody detected a 110-kDa band, consistent with the predicted molecular weight of SamCystin. Incubation of the antibody with excess competing peptide eliminated the band, demonstrating antibody specificity. B: SamCystin protein was detected in the brain, kidney, and testis, but not the liver, consistent with SamCystin mRNA expression in a previous report (3).

Fig. 2.

Temporal changes in protein levels for SamCystin in +/+, Cy/+, and Cy/Cy kidneys. A: representative immunoblots for renal SamCystin levels in +/+, Cy/+, and Cy/Cy rats at 3, 7, and 21 days (d) of age, and +/+ and Cy/+ rats at 7, 45, and 84 days of age. Levels of GAPDH were used to verify equal loading of total cell protein. B: SamCystin levels were determined by measurement of band intensity normalized to GAPDH protein levels. For each time point, SamCystin expression (SamCystin/GAPDH) was set to 100% for +/+ kidneys. *P < 0.05 and **P < 0.01 compared with age-matched +/+ kidneys.

Fig. 3.

SamCystin localizes to the luminal aspect of the proximal tubule in normal kidneys. SamCystin was detected in kidney sections of 21-day-old rats using an anti-SamCystin antibody and a secondary antibody conjugated to Alexa 568 (red: A, C, D, E, G, and H). Proximal tubules were stained with PHA-E lectin (green: B, C, and D) and distal tubules were stained with calbindin-D28K (green: F, G, and H). In +/+ kidneys, SamCystin localized to the brush border of proximal tubules but did not appear to be expressed in distal tubules, consistent with a previous report (3).

Fig. 4.

Cellular distribution of SamCystin in +/+, Cy/+, and Cy/Cy kidneys. A: SamCystin (red) localized to brush border of normal proximal tubules stained for PHA-E lectin (green) in 7-day-old +/+ and Cy/+ kidneys. By contrast, there was diffuse SamCystin staining in the cytosol and the basolateral aspect of cells in proximal tubule cysts of Cy/+ and Cy/Cy kidneys. Nuclei were stained with DAPI (blue) to indicate individual cells in the merged images. B: in 21-day-old Cy/+ and Cy/Cy kidneys, SamCystin (red) was diffusely distributed throughout the cystosol and basolateral aspects of cystic epithelial cells. C: as with earlier time points, SamCystin localized to the luminal surface of proximal tubules of kidneys of 84-day-old +/+ rats. Cellular distribution of SamCystin in 84-day-old Cy/+ kidneys was consistent with a loss of specific staining at the brush border. Incubation of the antibody with excess competing peptide eliminated SamCystin staining demonstrating antibody specificity (data not shown).

Fig. 5.

Cysts with aberrant SamCystin expression in Cy/+ kidneys have increased cell proliferation. Representative kidney sections from 84-day-old Cy/+ rats were stained with an antibody to SamCystin (brown color; A, C) or proliferating cell nuclear antigen (PCNA), a proliferation marker (B, D). Immunohistochemical staining for PCNA indicated that cysts with elevated and mislocalized SamCystin have increased number of proliferating cells.

Fig. 6.

Effect of verapamil (VP) on expression and distribution of SamCystin in +/+ and Cy/+ rat kidneys. Rats were treated with vehicle (Control; A, D) or 20 mg/kg VP (B, E) twice daily from 5 to 12 wk of age. Representative kidney sections were stained for SamCystin (brown color) and counterstained with hematoxylin and eosin. Kidney section from 180-day-old untreated +/+ rats (C) and Cy/+ rats (F). SamCystin staining was very weak in +/+ kidneys of 84- and 180-day-old rats (A-C), consistent a reduction in overall SamCystin expression. B: VP had no effect on SamCystin expression in +/+ kidneys. In contrast to the normal kidney, there was persistent expression of SamCystin in the Cy/+ kidney at 84 days of age (D) and VP treatment caused a further increase in SamCystin expression and mislocalization, similar to its staining pattern in kidneys of an 180-day-old Cy/+ rat (F).

Fig. 7.

Effect of VP on SamCystin protein levels in the kidneys of +/+ and Cy/+ rats. Western blot analysis shows that there was increased expression of SamCystin in vehicle (CONT)-treated Cy/+ kidneys at 84 days of age compared with age-matched +/+ kidneys and that VP treatment caused a further increase in its expression. By contrast, VP treatment had no effect of SamCystin expression in +/+ kidneys. **P < 0.001, compared with CONT-treated +/+ kidneys. #P < 0.01, compared with CONT-treated Cy/+ kidneys. †P < 0.001, compared with VP-treated +/+ kidneys.