Inhibition of pannexin-1 does not restore electrolyte balance in precystic Pkd1 knockout mice.

Mutations in PKD1 and PKD2 cause autosomal dominant polycystic kidney disease (ADPKD), which is characterized by the formation of fluid-filled cysts in the kidney. In a subset of ADPKD patients, reduced blood calcium (Ca2+) and magnesium (Mg2+) concentrations are observed. As cystic fluid contains increased ATP concentrations and purinergic signaling reduces electrolyte reabsorption, we hypothesized that inhibiting ATP release could normalize blood Ca2+ and Mg2+ levels in ADPKD. Inducible kidney-specific Pkd1 knockout mice (iKsp-Pkd1-/-) exhibit hypocalcemia and hypomagnesemia in a precystic stage and show increased expression of the ATP-release channel pannexin-1. Therefore, we administered the pannexin-1 inhibitor brilliant blue-FCF (BB-FCF) every other day from Day 3 to 28 post-induction of Pkd1 gene inactivation. On Day 29, both serum Ca2+ and Mg2+ concentrations were reduced in iKsp-Pkd1-/- mice, while urinary Ca2+ and Mg2+ excretion was similar between the genotypes. However, serum and urinary levels of Ca2+ and Mg2+ were unaltered by BB-FCF treatment, regardless of genotype. BB-FCF did significantly decrease gene expression of the ion channels Trpm6 and Trpv5 in both control and iKsp-Pkd1-/- mice. Finally, no renoprotective effects of BB-FCF treatment were observed in iKsp-Pkd1-/- mice. Thus, administration of BB-FCF failed to normalize serum Ca2+ and Mg2+ levels.

PKD2/polycystin-2 inhibits LPS-induced acute lung injury in vitro and in vivo by activating autophagy.

Polycystin-2 (PC2), which is a transmembrane protein encoded by the PKD2 gene, plays an important role in kidney disease, but its role in lipopolysaccharide (LPS)-induced acute lung injury (ALI) is unclear. We overexpressed PKD2 in lung epithelial cells in vitro and in vivo and examined the role of PKD2 in the inflammatory response induced by LPS in vitro and in vivo. Overexpression of PKD2 significantly decreased production of the inflammatory factors TNF-α, IL-1ß, and IL-6 in LPS-treated lung epithelial cells. Moreover, pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, reversed the inhibitory effect of PKD2 overexpression on the secretion of inflammatory factors in LPS-treated lung epithelial cells. We further demonstrated that overexpression of PKD2 could inhibit LPS-induced downregulation of the LC3BII protein levels and upregulation of SQSTM1/P62 protein levels in lung epithelial cells. Moreover, we found that LPS-induced changes in the lung wet/dry (W/D) weight ratio and levels of the inflammatory cytokines TNF-α, IL-6 and IL-1ß in the lung tissue were significantly decreased in mice whose alveolar epithelial cells overexpressed PKD2. However, the protective effects of PKD2 overexpression against LPS-induced ALI were reversed by 3-MA pretreatment. Our study suggests that overexpression of PKD2 in the epithelium may alleviate LPS-induced ALI by activating autophagy.

Probenecid slows disease progression in a murine model of autosomal dominant polycystic kidney disease.

Development of autosomal dominant polycystic kidney disease (ADPKD) involves renal epithelial cell abnormalities. Cystic fluid contains a high level of ATP that, among other effects, leads to a reduced reabsorption of electrolytes in cyst-lining cells, and thus results in cystic fluid accumulation. Earlier, we demonstrated that Pkd1RC/RC mice, a hypomorphic model of ADPKD, exhibit increased expression of pannexin-1, a membrane channel capable of ATP release. In the current study, we found that human ADPKD cystic epithelia have higher pannexin-1 abundance than normal collecting ducts. We hypothesized that inhibition of pannexin-1 function with probenecid can be used to attenuate ADPKD development. Renal function in male and female Pkd1RC/RC and control mice was monitored between 9 and 20 months of age. To test the therapeutic effects of probenecid (a uricosuric agent and a pannexin-1 blocker), osmotic minipumps were implanted in male and female Pkd1RC/RC mice, and probenecid or vehicle was administered for 42 days until 1 year of age. Probenecid treatment improved glomerular filtration rates and slowed renal cyst formation in male mice (as shown in histopathology). The mechanistic effects of probenecid on sodium reabsorption and fluid transport were tested on polarized mpkCCDcl4 cells subjected to short-circuit current measurements, and in 3D cysts grown in Matrigel. In the mpkCCDcl4 epithelial cell line, probenecid elicited higher ENaC currents and attenuated in vitro cyst formation, indicating lower sodium and less fluid retention in the cysts. Our studies open new avenues of research into targeting pannexin-1 in ADPKD pathology.

The inhibition of MDM2 slows cell proliferation and activates apoptosis in ADPKD cell lines.

INTRODUCTION: Autosomal dominant polycystic kidney disease (ADPKD) is characterised by progressive cysts formation and renal enlargement that in most of cases leads to end stage of renal disease (ESRD). This pathology is caused by mutations of either PKD1 or PKD2 genes that encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. These proteins function as receptor-channel complex able to regulate calcium homeostasis. PKD1/2 loss of function impairs different signalling pathways including cAMP and mTOR that are considered therapeutic targets for this disease. In fact, Tolvaptan, a vasopressin-2 antagonist that reduces cAMP levels, is the only drug approved for ADPKD treatment. Nevertheless, some ADPKD patients developed side effects in response to Tolvaptan including liver damage. Conversely, mTOR inhibitors that induced disease regression in ADPKD animal models failed the clinical trials. RESULTS: Here, we show that the inhibition of mTOR causes the activation of autophagy in ADPKD cells that could reduce therapy effectiveness by drug degradation through the autophagic vesicles. Consistently, the combined treatment with rapamycin and chloroquine, an autophagy inhibitor, potentiates the decrease of cell proliferation induced by rapamycin. To overcome the dangerous activation of autophagy by mTOR inhibition, we targeted MDM2 (a downstream effector of mTOR signalling) that is involved in TP53 degradation by using RG7112, a small-molecule MDM2 inhibitor used for the treatment of haematologic malignancies. The inhibition of MDM2 by RG7112 prevents TP53 degradation and increases p21 expression leading to the decrease of cell proliferation and the activation of apoptosis. CONCLUSION: The targeting of MDM2 by RG7112 might represent a new therapeutic option for the treatment of ADPKD.

Effects of polycystin­1 N­terminal fragment fusion protein on the proliferation and apoptosis of rat mesangial cells.

Mesangial proliferative glomerulonephritis (MsPGN) is characterized by widespread mesangial cell proliferation and an accumulation of extracellular matrix (ECM) in the mesangial area. In a previous study we developed a polycystin­1 N­terminal fragment (PC­1 NF) fusion protein that inhibits the proliferation of cyst­lining epithelial cells in autosomal dominant polycystic kidney disease. In addition, the PC­1 NF fusion protein arrests the cell cycle of cancer cells at the G0/G1 phase, inhibiting their proliferation. In the present study, the effect of the PC­1 NF fusion protein on MsPGN was investigated. It was found that the PC­1 NF fusion protein inhibited the proliferation of rat mesangial cells and induced G0/G1 phase arrest and apoptosis in vitro. PC­1 NF fusion protein treatment also resulted in a decrease in mRNA expression levels of proliferating cell nuclear antigen, cyclin D1 and B­cell lymphoma­2 (Bcl­2) and an increase in mRNA expression levels of Bcl­2­associated X protein (Bax) and p21Waf1. Furthermore, a decrease in Bcl­2, c­fos, c­jun and protein kinase C­α protein levels was observed, whereas Bax protein levels increased. Additionally, PC­1 NF fusion protein induced ECM degradation and inhibited ECM expansion. The results also demonstrated that PC­1 NF fusion protein treatment resulted in a decrease in type IV collagen and tissue inhibitor of metalloproteinase mRNA levels but an increase in matrix metalloproteinase 2 mRNA levels. In combination, these results suggest that the PC­1 NF fusion protein inhibits proliferation, promotes apoptosis and induces ECM degradation in MsPGN rats. This study offers novel perspectives for the treatment of MsPGN.

Distribution and function of polycystin-2 in mouse retinal ganglion cells.

The polycystin family of transient receptor potential (TRP) channels form Ca(2+) regulated cation channels with distinct subcellullar localizations and functions. As part of heteromultimeric channels and multi-protein complexes, polycystins control intracellular Ca(2+) signals and more generally the translation of extracellular signals and stimuli to intracellular responses. Polycystin-2 channels have been cloned from retina, but their distribution and function in retinal ganglion cells (RGCs) have not yet been established. In the present study, we determined cellular and subcellular localization as well as functional properties of polycystin-2 channels in RGCs. Polycystin-2 expression and distribution in RGCs was assessed by immunohistochemistry on vertical cryostat section of mouse retina as well as primary cultured mouse RGCs, using fluorescence microscopy. Biophysical and pharmacological properties of polycystin-2 channels isolated from primary cultured RGCs were determined using planar lipid bilayer electrophysiology. We detected polycystin-2 immunoreactivity both in the ganglion cell layer as well as in primary cultured RGCs. Subcellular analysis revealed strong cytosolic localization pattern of polycystin-2. Polycystin-2 channel current was Ca(2+) activated, had a maximum slope conductance of 114 pS, and could be blocked in a dose-dependent manner by increasing concentrations of Mg(2+). The cytosolic localization of polycystin-2 in RGCs is in accordance with its function as intracellular Ca(2+) release channel. We conclude that polycystin-2 forms functional channels in RGCs, of which biophysical and pharmacological properties are similar to polycystin-2 channels reported for other tissues and organisms. Our data suggest a potential role for polycystin-2 in RGC Ca(2+) signaling.

Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1.

BACKGROUND: When challenged with extracellular fluid shear stress, vascular endothelial cells are known to release nitric oxide, an important vasodilator. Here, we show that the ability of cultured endothelial cells to sense a low range of fluid shear depends on apical membrane organelles, called cilia, and that cilia are compartments required for proper localization and function of the mechanosensitive polycystin-1 molecule. METHODS AND RESULTS: Cells with the Pkd1(null/null) or Tg737(orpk/orpk) mutation encoded for polycystin-1 or polaris, respectively, are unable to transmit extracellular shear stress into intracellular calcium signaling and biochemical nitric oxide synthesis. Cytosolic calcium and nitric oxide recordings further show that fluid shear sensing is a cilia-specific mechanism because other mechanical or pharmacological stimulation does not abolish calcium and nitric oxide signaling in polycystin-1 and polaris mutant endothelial cells. Polycystin-1 localized in the basal body of Tg737(orpk/orpk) endothelial cells is insufficient for a fluid shear stress response. Furthermore, the optimal shear stress to which the cells respond best does not alter the apical cilia structure but modifies the responsiveness of cells to higher shear stresses through proteolytic modification of polycystin-1. CONCLUSIONS: We demonstrate for the first time that polycystin-1 (required for cilia function) and polaris (required for cilia structure) are crucial mechanosensitive molecules in endothelial cells. We propose that a distinctive communication with the extracellular microenvironment depends on the proper localization and function of polycystin-1 in cilia.