Hyperglycemia in the absence of cilia accelerates cystogenesis and induces renal damage.

In polycystic kidney disease (PKD), the rate of cyst formation and disease progression is highly variable. The lack of predictability in disease progression may be due to additional environmental factors or pathophysiological processes called "third hits." Diabetes is a growing epidemic, and recent studies suggest that PKD patients may be at an increased risk for this disease. We sought to determine if hyperglycemia enhances the initiation and rate of cystogenesis. Tamoxifen was administered to adult Ift88 conditional floxed allele mice to induce cilia loss in the presence of Cre. Subsequent administration of streptozotocin resulted in equivalent hyperglycemia in cilia(+) and cilia(-) mice. Hyperglycemia with loss of cilia increased the rate of cyst formation and cell proliferation. Structural and functional alterations in the kidney, including focal glomerular foot process effacement, interstitial inflammation, formation of primitive renal tubules, polyuria, and increased proteinuria, were also observed in hyperglycemic cilia(-) mice. Gene array analysis indicated enhanced Wnt and epithelial-to-mesenchymal transition signaling in the kidney of hyperglycemic cilia(-) mice. These data show that hyperglycemia, in the absence of cilia, results in renal structural and functional damage and accelerates cystogenesis, suggesting that diabetes is a risk factor in the progression of PKD.

Deletion of airway cilia results in noninflammatory bronchiectasis and hyperreactive airways.

The mechanisms for the development of bronchiectasis and airway hyperreactivity have not been fully elucidated. Although genetic, acquired diseases and environmental influences may play a role, it is also possible that motile cilia can influence this disease process. We hypothesized that deletion of a key intraflagellar transport molecule, IFT88, in mature mice causes loss of cilia, resulting in airway remodeling. Airway cilia were deleted by knockout of IFT88, and airway remodeling and pulmonary function were evaluated. In IFT88(-) mice there was a substantial loss of airway cilia on respiratory epithelium. Three months after the deletion of cilia, there was clear evidence for bronchial remodeling that was not associated with inflammation or apparent defects in mucus clearance. There was evidence for airway epithelial cell hypertrophy and hyperplasia. IFT88(-) mice exhibited increased airway reactivity to a methacholine challenge and decreased ciliary beat frequency in the few remaining cells that possessed cilia. With deletion of respiratory cilia there was a marked increase in the number of club cells as seen by scanning electron microscopy. We suggest that airway remodeling may be exacerbated by the presence of club cells, since these cells are involved in airway repair. Club cells may be prevented from differentiating into respiratory epithelial cells because of a lack of IFT88 protein that is necessary to form a single nonmotile cilium. This monocilium is a prerequisite for these progenitor cells to transition into respiratory epithelial cells. In conclusion, motile cilia may play an important role in controlling airway structure and function.

N-methyl-D-aspartate receptor subunit NR3a expression and function in principal cells of the collecting duct.

N-methyl-D-aspartate receptors (NMDARs) are Ca(2+)-permeable, ligand-gated, nonselective cation channels that function as neuronal synaptic receptors but which are also expressed in multiple peripheral tissues. Here, we show for the first time that NMDAR subunits NR3a and NR3b are highly expressed in the neonatal kidney and that there is continued expression of NR3a in the renal medulla and papilla of the adult mouse. NR3a was also expressed in mIMCD-3 cells, where it was found that hypoxia and hypertonicity upregulated NR3a expression. Using short-hairpin (sh) RNA-based knockdown, a stable inner medullary collecting duct (IMCD) cell line was established that had ∼80% decrease in NR3a. Knockdown cells exhibited an increased basal intracellular calcium concentration, reduced cell proliferation, and increased cell death. In addition, NR3a knockdown cells exhibited reduced water transport in response to the addition of vasopressin, suggesting an alteration in aquaporin-2 (AQP2) expression/function. Consistent with this notion, we demonstrate decreased surface expression of glycosylated AQP2 in IMCD cells transfected with NR3a shRNA. To determine whether this also occurred in vivo, we compared AQP2 levels in wild-type vs. in NR3a(-/-) mice. Total AQP2 protein levels in the outer and inner medulla were significantly reduced in knockout mice compared with control mice. Finally, NR3a(-/-) mice showed a significant delay in their ability to increase urine osmolality during water restriction. Thus NR3a may play a renoprotective role in collecting duct cells. Therefore, under conditions that are associated with high vasopressin levels, NR3a, by maintaining low intracellular calcium levels, protects the function of the principal cells to reabsorb water and thereby increase medullary osmolality.

Telomerase immortalization of principal cells from mouse collecting duct.

Recently, the use of overexpression of telomerase reverse transcriptase (TERT) has led to the generation of immortalized human cell lines. However, this cell immortalization approach has not been reported in well-differentiated mouse cells, such as renal epithelial cells. We sought to establish and then characterize a mouse collecting duct cell line, using ectopic expression of mTERT. Isolated primary cortical collecting duct (CCD) cell lines were transduced with mouse (m)TERT, using a lentiviral vector. mTERT-negative cells did not survive blasticidin selection, whereas mTERT-immortalized cells proliferated in selection media for over 40 subpassages. mTERT messenger RNA and telomerase activity was elevated in these cells, compared with an SV40-immortalized cell line. Flow cytometry with Dolichos biflorus agglutinin was used to select the CCD principal cells, and we designated this cell line mTERT-CCD. Cells were well differentiated and exhibited morphological characteristics typically found in renal epithelial cells, such as tight junction formation, microvilli, and primary cilia. Further characterization using standard immunofluorescence revealed abundant expression of aquaporin-2 and the vasopressin type 2 receptor. mTERT-CCD cells exhibited cAMP-stimulated/benzamil-inhibited whole cell currents. Whole cell patch-clamp currents were also enhanced after a 6-day treatment with aldosterone. In conclusion, we have successfully used mTERT to immortalize mouse collecting duct cells that retain the basic in vivo phenotypic characteristics of collecting duct cells. This technique should be valuable in generating cell lines from genetically engineered mouse models.

Reproductive disruption in wild longear sunfish (Lepomis megalotis) exposed to kraft mill effluent.

Worldwide, wild fish living in rivers receiving municipal and industrial discharges may experience endocrine disruption as a result of exposure to anthropogenic pollutants. The purpose of this study was to evaluate the hormonal status of wild fish in a U.S. river receiving unbleached kraft and recycled pulp mill effluent (Pearl River at Bogalusa, LA). We evaluated two alternative hypotheses: the effluent contained constituents that suppressed male and female reproduction, or it contained an androgenic substance that masculinized females. To evaluate the likelihood of fish exposure to effluent, we marked 697 longear sunfish (Lepomis megalotis) over a 2-year period; 83% of recaptured fish were found at the site of initial capture, and only one fish migrated from an effluent-receiving site to a reference site. We can reasonably assume that fish captured from an effluent-receiving site are residents, not transitory migrants. To diagnose endocrine disruption, we measured sex steroid hormone [17beta-estradiol (E2), testosterone (T), and 11-ketotestosterone (11KT)] and vitellogenin (VTG) concentrations in male and female longear sunfish captured at two sites upstream and two sites downstream of the effluent outfall. Kraft pulp mill effluent did not affect male reproductive physiology but did suppress female T and VTG levels when effluent constituted>or=1% of river flow. Masculinization was not observed. Longear sunfish in the Pearl River experience moderate reproductive suppression in response to unbleached kraft and recycled pulp mill effluent.

Activation of the intrarenal renin-angiotensin-system in murine polycystic kidney disease.

The mechanism for early hypertension in polycystic kidney disease (PKD) has not been elucidated. One potential pathway that may contribute to the elevation in blood pressure in PKD is the activation of the intrarenal renin-angiotensin-system (RAS). For example, it has been shown that kidney cyst and cystic fluid contains renin, angiotensin II (AngII), and angiotensinogen (Agt). Numerous studies suggest that ciliary dysfunction plays an important role in PKD pathogenesis. However, it is unknown whether the primary cilium affects the intrarenal RAS in PKD. The purpose of this study was to determine whether loss of cilia or polycystin 1 (PC1) increases intrarenal RAS in mouse model of PKD. Adult Ift88 and Pkd1 conditional floxed allele mice with or without cre were administered tamoxifen to induce global knockout of the gene. Three months after tamoxifen injection, kidney tissues were examined by histology, immunofluorescence, western blot, and mRNA to assess intrarenal RAS components. SV40 immortalized collecting duct cell lines from hypomorphic Ift88 mouse were used to assess intrarenal RAS components in collecting duct cells. Mice without cilia and PC1 demonstrated increased kidney cyst formation, systolic blood pressure, prorenin, and kidney and urinary angiotensinogen levels. Interestingly immunofluorescence study of the kidney revealed that the prorenin receptor was localized to the basolateral membrane of principal cells in cilia (-) but not in cilia (+) kidneys. Collecting duct cAMP responses to AngII administration was greater in cilia (-) vs. cilia (+) cells indicating enhanced intrarenal RAS activity in the absence of cilia. These data suggest that in the absence of cilia or PC1, there is an upregulation of intrarenal RAS components and activity, which may contribute to elevated blood pressure in PKD.