Ciliopathies are a heterogeneous group of diseases that exhibit broad clinical phenotypes, including renal cysts, retinal degeneration, and cerebellar vermis aplasia. Nephronophthisis (NPHP) is a renal ciliopathy that causes chronic kidney disease and is characterized by kidney cysts at the cortico-medullary border. Among the 10 different disease-causing genes (NPHP1-NPHP10), mutations in NPHP3, NPHP6, or NPHP8 cause the most severe ciliopathy variants of NPHP, Joubert syndrome, and Meckel Syndrome. In this study, we tested the hypothesis that loss of function of these three most severe disease-associated genes leads to morphological defects in a three-dimensional (3D) renal cell culture [murine (m) inner medullary collecting duct (IMCD) 3] model by either lack of cilia formation and/or cell polarity defects. Stable knockdown cell lines were examined in 3D spheroid culture followed by rhodamine-phalloidin staining to assess spheroid architecture. We observed significantly higher percentages of abnormal spheroids for all three stable cell lines compared with control short-hairpin RNA cells. In addition, stable knockdown of Nphp3, Nphp6, and Nphp8 results in reduced cilia numbers and elevated cAMP levels in mIMCD3 cells. We demonstrate that, following gene knockdown of Nphp3, Nphp6, or Nphp8, treatment with the somatostatin agonist octreotide (2 μM) reduces the percentage of abnormal spheroids compared with control. This study reveals that the loss of Nphp3, Nphp6, or Nphp8 leads to cilia abnormalities and cell polarity defects, resulting in spheroid abnormalities, which can be rescued by inhibiting cAMP levels with octreotide treatment.
- polycystic kidney disease
nephronophthisis-related ciliopathies (NPHP-RC) are a clinically and genetically heterogeneous group of diseases that exhibit shared clinical phenotypes, including renal cysts, retinal degeneration, and cerebellar vermis aplasia (6). Among the 10 different NPHP disease-causing genes (NPHP1-NPHP10), mutations in NPHP3, NPHP6, or NPHP8 cause the most severe NPHP-RC, Joubert Syndrome, and Meckel Syndrome (7). Studies into these diseases have revealed that virtually all of the gene products involved in the pathogenesis in humans, mice, or zebrafish are expressed at the primary cilia-centrosome complex (6, 15). Cilia are a key component of a number of signal transduction pathways, including Hedgehog (shh) (2) and Wnt signaling (3, 5). Centrosomal proteins regulate cell polarity and oriented cell division and participate in the mitotic spindle formation during mitosis (3, 13).
Seventeen ciliopathy genes are known to cause NPHP-RC if mutated (for review, see Ref. 6), whereas NPHP3, NPHP6/Cep290, or NPHP8/RPGRIP1L mutations lead to the most severe ciliopathy phenotypes, including NPHP-Joubert syndrome and Meckel syndrome. Because mutation of these genes causes overlapping disease, phenotypes suggest that the mutated disease proteins may interact and function in a common pathway (13). Although the pathogenesis has been studied extensively, no clear pathways have been delineated, and no effective treatment has been developed.
Recent studies have established that cAMP stimulates mural epithelial cell proliferation and secretion of fluid into cysts in patients with polycystic kidney disease (PKD) (16). Elevated cAMP levels are found in the kidney, liver, vascular smooth muscle, and choroid plexus, in various animal models of PKD and NPHP (4, 9, 14, 16). A recent study from our group has demonstrated that Nphp10 (Sdccag8) disappears from cell junctions upon treatment of cells with the cAMP analog 8-bromo-cAMP (11). These studies prompted us to examine whether loss of function of NPHP-RC genes causes cellular abnormalities in the renal cystic disease model of three-dimensional (3D) spheroids (10) and whether the effect can be rescued by modulation of cAMP levels.
Generation of stable knockdown murine inner medullary collecting duct cells: shRNA duplexes were designed and cloned into RNAi-Ready pSIREN-RetroQ vector (Clonetech). Short-hairpin RNA (shRNA) target sequences for Nphp genes were as follows: Nphp3 (5′-GGATTAATCTACCACCGTTTG-3′, 5′-GGATTCCACTCCAGAAGAAGA-3′, and 5′-GGACATACACGATGAGCAAGA-3′), Nphp6 (5′-GGAAGAAATTGTTCAGCAAAG-3′, 5′-GGAAGACCTTCATGTTCTTCA-3′, and 5′-GGAGCAGACAGTAGCAGAACA-3′), and Nphp8 (5′-GGATCAAGCTATTCGACTTTA-3′, 5′-GGATTTGGATCGCTACCTTAA-3′, and 5′-GGGAAATGCAACAAGGGAAAG-3′).
Retrovirus was generated by transfecting 293T cells with the shRNA constructs along with pVSV and p-gag-pol constructs. Murine (m) inner medullary collecting duct (IMCD) 3 cells were infected with the supernatant. IMCD3 cells were then selected with puromycin resistance for 10 days at 10 μg/ml.
Quantitative real-time PCR.
RNA was isolated using the RNeasy Mini Kit (Qiagene). First-strand cDNA was performed using Moloney murine leukemia virus reverse transcriptase (Invitrogen). Accumulation of PCR products was measured in real time by using iTaq SYBR green supermix with the MyiQ Single Color Real-time PCR Detection System (Bio-Rad).
3D spheroid cell culture was performed using Geltrex Reduced Growth Factor Basement Membrane Matrix (Invitrogen) in chamber slides. Spheroids were stained with rhodamine-phalloidin and DAPI. The spheroid architecture was examined under an inverted confocal microscope (Leica).
mIMCD3 cell lines were grown on the filter and serum starved for 48 h. Cells were fixed and stained with acetylated α-tubulin for the cilia and anti-somatostatin receptor antibodies. Anti-mouse Alexa fluor 594 and anti-rabbit Alexa fluor-488 were used as secondary antibodies and DAPI as nuclear stain. The membrane filters were mounted on slides, and the images were taken under an inverted confocal microscope (Leica). A total of 100 cells were counted for each group for cilia quantification.
Measurement of intracellular cAMP.
Enzyme-linked immunosorbent assay was performed on cell lysates in triplicate samples using a Direct Cyclic AMP Enzyme Immunoassay kit (Arbor Assays).
RESULTS AND DISCUSSION
In the present study, we generated mIMCD3 cell lines of stable knockdown of Nphp3, Nphp6, or Nphp8 with retroviral delivery of three shRNA duplexes for each gene. Quantitative RT-PCR analysis revealed that all three shRNA duplexes for each gene reduced mRNA expression of each gene. However, Nphp3.1, Nphp6.1, and Nphp8.1 were more efficient than the other two shRNA duplexes for the same gene (Fig. 1, A–C). To test whether knockdown of Nphp subtype affects other Nphp mRNA expression, we used the cDNAs from these cells and tested in the context of Nphp3, Nphp6, and Nphp8 primer sets in a reciprocal real-time PCR analysis. These results confirm the specificity of the knockdown by shRNA duplexes (Fig. 1D). To test the effects of Nphp knockdown on the cell proliferation, growth assay was performed by plating an equal number of cells and counting them by an automatic counter (TC10; Bio-Rad) at different time intervals. Our data suggested retarded growth of Nphp knockdown cells compared with the sh-neg control cell line (Fig. 2A). To investigate the roles of Nphp3, Nphp6, and Nphp8 gene products in tissue architecture, we examined Nphp knockdown cell lines in a 3D spheroid culture system that reflects the cell biology of kidney collecting duct. Nphp3, Nphp6, and Nphp8 knockdown mIMCD3 cell lines were grown in the 3D culture system for 3–4 days to develop spheroid structure. Spheroids were then stained with rhodamine-phalloidin for F-actin and DAPI (for nuclear staining) to visualize spheroid architecture under an inverted confocal microscopy. Spheroid architecture was examined by focusing at the equator of the spheroids to visualize lumen formation. Spheroids with no lumen and/or misaligned nuclei were considered as abnormal (Fig. 3A).
We found significantly higher percentages of abnormal spheroids for all three stable knockdown cell lines (Nphp3, 73%; Nphp6, 84%; and Nphp8, 56%) compared with control shRNA cells (21%) (Fig. 3B). Spheroid abnormalities from cells with knockdown of Nphp genes by small-interfering RNA have also been reported in a recent study (13).
We investigated the cilia defects by growing sh-neg, Nphp3, Nphp6, and Nphp8 cell lines on membrane filters under conditions of ciliogenesis (see methods). Cilia quantification data (Fig. 2, B and C) suggested that lack of all three Nphp genes under this study resulted in significant reduction in cilia numbers in the stable knockdown cells. Because cilia are sensory organelles for the kidney cells, lack of cilia might perturb signaling events critical for normal polarity maintenance.
Previous studies have established that cAMP plays a central role in the progression of cystic disease in patients with autosomal dominant PKD by stimulating epithelial cell proliferation and secretion of fluid into cysts (12). This has provided a strong rationale for therapies targeting cAMP signaling. Somatostatin analogs acting on Gi protein-coupled receptors provided an alternative path to inhibit cAMP signaling in tubular epithelial cells in patients with PKD. Research studies showed that somatostatin receptor inhibitor octreotide significantly reduces cAMP levels and rates of cysts expansion in freshly isolated bile ducts from PCK rats grown in 3D culture (9).
We treated Nphp3, Nphp6, and Nphp8 knockdown spheroids with the somatostatin agonist octreotide (2 μM) and observed that treatment of knockdown spheroids with octreotide (2 μM) reduced the percentage of abnormal spheroids to 29% (Nphp3), 35% (Nphp6), and 35% (Nphp8), whereas vehicle control abnormal spheroids were 68% (Nphp3), 87% (Nphp6), and 65% (Nphp8) (Fig. 4). Because octreotide was a known ligand for somatostatin receptors (Sstr), we evaluated the mRNA expression level of somatostatin receptors (Sstr1–5) using cDNA from the mIMCD3 cell line by RT-PCR analysis. Our expression data suggested that, except for Sstr4, all other Sstr mRNAs were abundant in mIMCD3 cells (Fig. 5A). In addition, imaging analysis revealed that only Sstr3 was localized to the primary cilium (Fig. 5B). Our data are in agreement with the studies by others where it has been clearly demonstrated that a consensus sequence of third intracellular loop3 of Sstr3 was critical for cilia localization (1). We then analyzed the intracellular cAMP concentrations in octreotide-treated or untreated control, Nphp3-sh, Nphp6-sh, and Nphp8-sh cell lines. Our data suggested that elevated intracellular cAMP levels in Nphp knockdown cells compared with the control sh-neg cells. Octrotide treatment significantly reduces cAMP levels in the Nphp knockdown cells or control cells (Fig. 5C).
This study reveals that lack of Nphp3, Nphp6, or Nphp8 leads to cell polarity defects, resulting in spheroid abnormalities that can be rescued by inhibiting intracellular cAMP levels with octreotide treatment. Recently, in a randomized clinical trial, long-acting octreotide has been reported to slow down disease progression in patients with polycystic liver disease (8). Our study confirms that manipulation of the cAMP pathways could be one of the therapeutic approaches in treating patients with NPHP-RC.
F. Hildebrandt is an Investigator of the Howard Hughes Medical Institute, a Frederick G. L. Huetwell Professor, and a Doris Duke Distinguished Clinical Scientist. This research was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases to F. Hildebrandt (DK-068306 and DK-090917), the March of Dimes (6-F711-241), and the Center for Organogenesis of the University of Michigan.
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: A.K.G., T.W.H., and F.H. conception and design of research; A.K.G. performed experiments; A.K.G. analyzed data; A.K.G. interpreted results of experiments; A.K.G. prepared figures; A.K.G. drafted manuscript; T.W.H. and F.H. edited and revised manuscript; F.H. approved final version of manuscript.
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