Robert J. Kolb

Ciliary Polycystin-2 Is a Mechanosensitive Calcium Channel Involved in Nitric Oxide Signaling Cascades

Cardiovascular complications such as hypertension are a continuous concern in patients with autosomal dominant polycystic kidney disease (ADPKD). The PKD2 encoding for polycystin-2 is mutated in ≈15% of ADPKD patients. Here, we show that polycystin-2 is localized to the cilia of mouse and human vascular endothelial cells. We demonstrate that the normal expression level and localization of polycystin-2 to cilia is required for the endothelial cilia to sense fluid shear stress through a complex biochemical cascade, involving calcium, calmodulin, Akt/PKB, and protein kinase C. In response to fluid shear stress, mouse endothelial cells with knockdown or knockout of Pkd2 lose the ability to generate nitric oxide (NO). Consistent with mouse data, endothelial cells generated from ADPKD patients do not show polycystin-2 in the cilia and are unable to sense fluid flow. In the isolated artery, we further show that ciliary polycystin-2 responds specifically to shear stress and not to mechanical stretch, a pressurized biomechanical force that involves purinergic receptor activation. We propose a new role for polycystin-2 in transmitting extracellular shear stress to intracellular NO biosynthesis. Thus, aberrant expression or localization of polycystin-2 to cilia could promote high blood pressure because of inability to synthesize NO in response to an increase in shear stress (blood flow).

TAZ Promotes PC2 Degradation through a SCFβ-Trcp E3 Ligase Complex

ABSTRACT Studies of a TAZ knockout mouse reveal a novel function of the transcriptional regulator TAZ, that is, as a binding partner of the F-box protein β-Trcp. TAZ−/− mice develop polycystic kidney disease (PKD) and emphysema. The calcium-permeable cation channel protein polycystin 2 (PC2) is overexpressed in kidneys of TAZ−/− mice as a result of decreased degradation via an SCFβ-Trcp E3 ubiquitin ligase pathway. Replacements of serines in a phosphodegron motif in TAZ prevent β-Trcp binding and PC2 degradation. Coexpression of a cytoplasmic fragment of polycystin 1 blocks the PC2-TAZ interaction and prevents TAZ-mediated degradation of PC2. Depletion of TAZ in zebrafish also results in a cystic kidney accompanied by overexpression of PC2. These results establish a common role of TAZ across vertebrate species in a protein degradation pathway regulated by phosphorylation and implicate deficiencies in this pathway in the development of PKD.

α-Actinin-4 Is Required for Normal Podocyte Adhesion

Mutations in the α-actinin-4 gene ACTN4 cause an autosomal dominant human kidney disease. Mice deficient in α-actinin-4 develop a recessive phenotype characterized by kidney failure, proteinuria, glomerulosclerosis, and retraction of glomerular podocyte foot processes. However, the mechanism by which α-actinin-4 deficiency leads to glomerular disease has not been defined. Here, we examined the effect of α-actinin-4 deficiency on the adhesive properties of podocytes in vivo and in a cell culture system. In α-actinin-4-deficient mice, we observed a decrease in the number of podocytes per glomerulus compared with wild-type mice as well as the presence of podocyte markers in the urine. Podocyte cell lines generated from α-actinin-4-deficient mice were less adherent than wild-type cells to glomerular basement membrane (GBM) components collagen IV and laminin 10 and 11. We also observed markedly reduced adhesion of α-actinin-4-deficient podocytes under increasing shear stresses. This adhesion deficit was restored by transfecting cells with α-actinin-4-GFP. We tested the strength of the integrin receptor-mediated linkages to the cytoskeleton by applying force to microbeads bound to integrin using magnetic pulling cytometry. Beads bound toα-actinin-4-deficient podocytes showed greater displacement in response to an applied force than those bound to wild-type cells. Consistent with integrin-dependent α-actinin-4-mediated adhesion, phosphorylation of β1-integrins on α-actinin-4-deficient podocytes is reduced. We rescued the phosphorylation deficit by transfecting α-actinin-4 into α-actinin-4-deficient podocytes. These results suggest that α-actinin-4 interacts with integrins and strengthens the podocyte-GBM interaction thereby stabilizing glomerular architecture and preventing disease.

Collecting duct cells that lack normal cilia have mislocalized vasopressin-2 receptors

Polycystic kidney disease (PKD) is a ciliopathy characterized by renal cysts and hypertension. These changes are presumably due to altered fluid and electrolyte transport in the collecting duct (CD). This is the site where vasopressin (AVP) stimulates vasopressin-2 receptor (V2R)-mediated aquaporin-2 (AQP2) insertion into the apical membrane. Since cysts frequently occur in the CD, we studied V2R and AQP2 trafficking and function in CD cell lines with stunted and normal cilia [cilia (-), cilia (+)] derived from the orpk mouse (hypomorph of the Tg737/Ift88 gene). Interestingly, only cilia (-) cells grown on culture dishes formed domes after apical AVP treatment. This observation led to our hypothesis that V2R mislocalizes to the apical membrane in the absence of a full-length cilium. Immunofluorescence indicated that AQP2 localizes to cilia and in a subapical compartment in cilia (+) cells, but AQP2 levels were elevated in both apical and basolateral membranes in cilia (-) cells after apical AVP treatment. Western blot analysis revealed V2R and glycosylated AQP2 in biotinylated apical membranes of cilia (-) but not in cilia (+) cells. In addition, apical V2R was functional upon apical desmopressin (DDAVP) treatment by demonstrating increased cAMP, water transport, and benzamil-sensitive equivalent short-circuit current (I(sc)) in cilia (-) cells but not in cilia (+) cells. Moreover, pretreatment with a PKA inhibitor abolished DDAVP stimulation of I(sc) in cilia (-) cells. Thus we propose that structural or functional loss of cilia leads to abnormal trafficking of AQP2/V2R leading to enhanced salt and water absorption. Whether such apical localization contributes to enhanced fluid retention and hypertension in PKD remains to be determined.

STRATEGY FOR THE DEVELOPMENT OF A MATCHED SET OF TRANSPORT-COMPETENT, ANGIOTENSIN RECEPTOR–DEFICIENT PROXIMAL TUBULE CELL LINES

SummaryIn the proximal convoluted tubule (PCT) angiotensin II (Ang II) modulates fluid and electrolyte transport through at least two pharmacologically distinct receptor subtypes: AT1 and AT2. Development of cell lines that lack these receptors are potentially useful models to probe the complex cellular details of Ang II regulation. To this end, angiotensin receptor-deficient mice were bred with an Immortomouse®, which harbors a thermolabile SV40 large-T antigen (Tag). S1 PCT segments from kidneys of F2 mice were microdissected, placed in culture, and maintained under conditions that enhanced cell growth, i.e., promoted Tag expression and thermostability. Three different types of angiotensin receptor-deficient cell lines, (AT1A [−/−], Tag [+/−]), (AT1B[−/−], Tag [+/−]),and (AT1B[−/−], Tag [+/+]), as well as wild type cell lines were generated. Screening and characterization, which were conducted under culture conditions that promoted cellular differentiation, included: measurements of transepithelial transport, such as basal monolayer short-circuit current (Isc; −3 to 3 μA/cm2), basal monolayer conductance (G, 2 to 10 mS/cm2), Nain3+-phosphate cotransport (ΔIsc of 2 to 3 μA/cm2 at 1 mM), and Nain3+-succinate contransport (ΔIsc of 1 to 9 μA/cm2 at 2 mM). Morphology of cell monolayers showed an extensive brush border, well-defined tight junctions, and primary cilia. Receptor functionality was assessed by Ang II-stimulated \-arrestin 2 translocation and showed an Ang II-mediated response in wild type but not (AT1A [−/ −], AT1B [−/−]) cells. Cell line were amplified, yielding a virtually unlimited supply of highly differentiated, transportcompetent, angiotensin receptor-deficient PCT cell lines.