A short carboxy-terminal domain of polycystin-1 reorganizes the microtubular network and the endoplasmic reticulum.

Mutations of PKD1 cause autosomal dominant polycystic kidney disease (ADPKD), a syndrome characterized by kidney cysts and progressive renal failure. Polycystin-1, the protein encoded by PKD1, is a large integral membrane protein with a short carboxy-terminal cytoplasmic domain that appears to initiate multiple cellular programs. We report now that this polycystin-1 domain contains a novel motif responsible for rearrangements of intermediate filaments, microtubules and the endoplasmic reticulum (ER). This motif reveals homology to CLIMP-63, a microtubule-binding protein that rearranges the ER. Our findings suggest that polycystin-1 influences the shape and localization of both the microtubular network and the ER.

The subcellular localization of TRPP2 modulates its function.

TRPP2, also known as polycystin-2, is a calcium permeable nonselective cation channel that is mutated in autosomal dominant polycystic kidney disease but has also been implicated in the regulation of cardiac development, renal tubular differentiation, and left-to-right (L-R) axis determination. For obtaining further insight into how TRPP2 exerts tissue-specific functions, this study took advantage of PACS-dependent trafficking of TRPP2 in zebrafish larvae. PACS proteins recognize an acidic cluster within the carboxy-terminal domain of TRPP2 that undergoes phosphorylation and mediate retrieval of TRPP2 to the Golgi and endoplasmic reticulum (ER). The interaction of human TRPP2 with PACS proteins can be inhibited by a Ser812Ala mutation (TRPP2(S812A)), thereby allowing TRPP2 to reach other subcellular compartments, and enhanced by a Ser812Asp mutation (TRPP2(S812D)), thereby trapping TRPP2 in the ER. It was found that the TRPP2(S812A) mutant rescued cyst formation of TRPP2-deficient zebrafish larvae to the same degree as wild-type TRPP2, whereas the TRPP2(S812D) mutant was significantly more effective in normalizing the distorted body axis of TRPP2-deficient fish. Surprisingly, the TRPP2(S812D) mutant rescued the abnormalities of L-R asymmetry more effectively than either wild-type or TRPP2(S812A), suggesting that the ER localization of TRPP2 plays an important role in the development of normal L-R asymmetry. Taken together, these findings support the hypothesis that TRPP2 assumes distinct subcellular localizations to exert tissue-specific functions.

Kidney injury molecule 1 (Kim1) is a novel ciliary molecule and interactor of polycystin 2.

Inherited mutations in genes encoding for ciliary proteins lead to a broad spectrum of human diseases, such as polycystic kidney disease (PKD), situs inversus and retinitis pigmentosa. In the human kidney, autosomal dominant PKD (ADPKD) is caused by mutations in PKD1 (PC1), or PKD2 (TRPP2). Both are necessary for ciliary mechanotransduction, whereby bending of the cilium elicits a calcium response in the cell. We have previously shown that overexpression of mutated forms of the chemosensor kidney injury molecule 1 (Kim1) abolishes the flow response in ciliated MDCK cells. Here we identify Kim1 as an endogenous ciliary protein. Kim1 co-precipitates with TRPP2. Mutational analysis reveals that the interaction between Kim1 and TRPP2 requires the ciliary sorting motif in the N-terminus of TRPP2, and the presence of a highly conserved tyrosine in the intracellular tail of Kim1, which has previously been shown to play a role in ciliary flow sensing. These data support the notion that TRPP2 functionally interacts with ciliary chemosensors.

Polycystin-2 immunolocalization and function in zebrafish.

Polycystin-2 functions as a cation-permeable transient receptor potential ion channel in kidney epithelial cells and when mutated results in human autosomal dominant polycystic kidney disease. For further exploration of the in vivo functions of Polycystin-2, this study examined its expression and function during zebrafish embryogenesis. pkd2 mRNA is ubiquitously expressed, and its presence in the larval kidney could be confirmed by reverse transcription-PCR on isolated pronephroi. Immunostaining with anti-zebrafish Polycystin-2 antibody revealed protein expression in motile kidney epithelial cell cilia and intracellular cell membranes. Intracellular localization was segment specific; in the proximal nephron segment, Polycystin-2 was localized to basolateral cell membranes, whereas in the caudal pronephric segment, Polycystin-2 was concentrated in subapical cytoplasmic vesicles. Polycystin-2 also was expressed in muscle cells and in a variety of sensory cells that are associated with mechanotransduction, including cells of the ear, the lateral line organ, and the olfactory placodes. Disruption of Polycystin-2 mRNA expression resulted in pronephric kidney cysts, body axis curvature, organ laterality defects, and hydrocephalus-defects that could be rescued by expression of a human PKD2 mRNA. In-frame deletions in the first extracellular loop and C-terminal phosphofurin acidic cluster sorting protein-1 (PACS-1) binding sites in the cytoplasmic tail caused Polycystin-2 mislocalization to the apical cell surface. Unlike zebrafish intraflagellar transport protein (IFT) mutants, cyst formation was not associated with cilia defects and instead correlated with reduced kidney fluid output, expansion of caudal duct apical cell membranes, and occlusion of the caudal pronephric nephron segment.

Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes.

Podocytes are specialized cells of the kidney that form the blood filtration barrier in the kidney glomerulus. The barrier function of podocytes depends upon the development of specialized cell-cell adhesion complexes called slit-diaphragms that form between podocyte foot processes surrounding glomerular blood vessels. Failure of the slit-diaphragm to form results in leakage of high molecular weight proteins into the blood filtrate and urine, a condition called proteinuria. In this work, we test whether the zebrafish pronephros can be used as an assay system for the development of glomerular function with the goal of identifying novel components of the slit-diaphragm. We first characterized the function of the zebrafish homolog of Nephrin, the disease gene associated with the congenital nephritic syndrome of the Finnish type, and Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome. Zebrafish nephrin and podocin were specifically expressed in pronephric podocytes and required for the development of pronephric podocyte cell structure. Ultrastructurally, disruption of nephrin or podocin expression resulted in a loss of slit-diaphragms at 72 and 96 h post-fertilization and failure to form normal podocyte foot processes. We also find that expression of the band 4.1/FERM domain gene mosaic eyes in podocytes is required for proper formation of slit-diaphragm cell-cell junctions. A functional assay of glomerular filtration barrier revealed that absence of normal nephrin, podocin or mosaic eyes expression results in loss of glomerular filtration discrimination and aberrant passage of high molecular weight substances into the glomerular filtrate.

Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis.

Cilia, as motile and sensory organelles, have been implicated in normal development, as well as diseases including cystic kidney disease, hydrocephalus and situs inversus. In kidney epithelia, cilia are proposed to be non-motile sensory organelles, while in the mouse node, two cilia populations, motile and non-motile have been proposed to regulate situs. We show that cilia in the zebrafish larval kidney, the spinal cord and Kupffer's vesicle are motile, suggesting that fluid flow is a common feature of each of these organs. Disruption of cilia structure or motility resulted in pronephric cyst formation, hydrocephalus and left-right asymmetry defects. The data show that loss of fluid flow leads to fluid accumulation, which can account for organ distension pathologies in the kidney and brain. In Kupffer's vesicle, loss of flow is associated with loss of left-right patterning, indicating that the 'nodal flow' mechanism of generating situs is conserved in non-mammalian vertebrates.