Diameters of spherical cysts with distinct lumens were measured using Image-Pro Premier 9.2 64?bit. pathway, whose activity is also regulated at the cilium. Cilia are PD146176 (NSC168807) formed by intraflagellar transport (IFT), the bi-directional transport of protein cargo along the ciliary axoneme by IFT-B and -A complexes. In mice, loss of most IFT-B proteins causes absent or stunted cilia and the inability to respond to the Hh signal10. In contrast, loss of the IFT-A proteins, THM1 (TTC21B) and IFT122, results in accumulation of proteins in bulb-like structures at the distal tip of shortened cilia and enhanced activation of the Hh pathway11,12. Deletion of or genes in the kidney or globally during late embryogenesis causes renal cysts13C15. Hh signaling has been reported to promote renal proliferative diseases, including renal cell carcinoma16,17 and fibrosis18, and several studies suggest Hh signaling may also influence cystogenesis19C22. Cystic kidneys of several mouse models have shown upregulation of (LTL) or agglutinin (DBA) lectins or with antibody against Tamm-Horsfall Protein (THP) to examine the tubular origin of GLI1?+?cells. While cystic cells did not label with LTL, a marker of proximal tubules, DBA or THP staining of cystic cells suggested that this cysts originated from collecting duct or Loop of Henle tubules, respectively, and that GLI1-positive epithelial cells were present in these cysts (Fig.?2; Physique?S4). Open in a separate window Physique 1 GLI1 is usually upregulated in human ADPKD renal tissue. (A) Western blot analysis for GLI1 in normal human kidney (NHK) and ADPKD extracts of the renal cortex. Bars CD14 (mean SEM) are band intensity normalized to -actin, and represented as fold change from NHK, set to 1 1.0. Quantification of GLI1 levels was performed on 6 NHK and 5 ADPKD tissue extracts (Summary Table?S1). Statistical significance was determined by an unpaired t-test. *P? ?0.05 (B) Immunohistochemistry for GLI1 on NHK and ADPKD sections of the renal cortex. Scale bar?=?50?m. Open in a separate windows Physique 2 GLI1-expressing epithelial cells derive from collecting duct and Loop of Henle tubules. GLI1 immunohistochemistry and staining with DBA, LTL and THP on ADPKD sections of the renal cortex. Scale bar?=?100?m. Ciliary trafficking and Hedgehog signaling are intact in ADPKD primary renal epithelial cells In mice, ciliary length appears to affect PKD severity5,6. Further, increased ciliary length has been reported in the mutant mouse, which harbors an ADPKD PD146176 (NSC168807) mutation25, and in and knock-down or expression of a dominant-negative form of Bbs3 in IMCD cells resulted in absence of PC1 in the cilium29, while combined deficiency of and in retinal pigment epithelial (RPE) cells caused ciliary accumulation of PC230. To determine if the BBSome is usually conversely affected in ADPKD, we examined the localization of BBS components, BBS2 and BBS5 (Fig.?3). Similar to the IFT proteins, the BBS proteins localized normally along the ciliary axoneme. Together, these data suggest that polycystin dysfunction does not overtly affect the ciliary trafficking machinery. We examined Hh status in ADPKD primary renal epithelial cells. Using qPCR, we found that and transcript levels were comparable in NHK and ADPKD cells (Fig.?4A). Additionally, we examined SMO localization, which enriches in the cilium upon pathway stimulation8. In the absence of Hh agonist, SMO was mostly undetected in primary cilia of NHK and ADPKD cells, but following treatment with SAG, a SMO agonist, NHK and ADPKD cells showed comparable ciliary enrichment of SMO (Fig.?4B), suggesting comparable Hh signaling levels. These data indicate that ADPKD primary renal epithelial cells have Hh signaling machinery and respond appropriately to Hh modulation. Open in a separate window Physique 4 Human primary renal epithelial cells have Hh signaling machinery. (A) qPCR analysis on NHK and ADPKD primary renal epithelial cells. Bars represent mean SEM of 3 NHK PD146176 (NSC168807) and 3 ADPKD cell lines (Summary Table?S1). (B) Immunofluorescence for SMO (green) and acetylated -tubulin (red) in presence or absence of SAG. Experiments were replicated in 5 NHK and 5 ADPKD cell lines (Summary Table?S1). Scale bar?=?25?m. Hh inhibitors reduce cAMP-induced proliferation and microcyst formation of human primary ADPKD renal cells Since Hh signaling affects proliferation of multiple cell types, we examined proliferation of ADPKD cells in response to Hh modulators. NHK and ADPKD cells were treated with SAG or with SMO or GLI antagonists, Sant2 or Gant61, respectively, alone or in combination with SAG, for 48?hours. Cell counts were then obtained. As control,.
Diameters of spherical cysts with distinct lumens were measured using Image-Pro Premier 9