BMP4 regulates asymmetric Pkd2 distribution in mouse nodal immotile cilia and ciliary mechanosensing required for left-right determination.

BACKGROUND: Mouse nodal immotile cilia mechanically sense the bending direction for left-right (L-R) determination and activate the left-side-specific signaling cascade, leading to increased Nodal activity. Asymmetric distribution of Pkd2, a crucial channel for L-R determination, on immotile cilia has been reported recently. However, the causal relationship between the asymmetric Pkd2 distribution and direction-dependent flow sensing is not well understood. Furthermore, the underlying molecular mechanism directing this asymmetric Pkd2 distribution remains unclear. RESULTS: The effects of several recombinant proteins and inhibitors on the Pkd2 distribution were analyzed using super-resolution microscopy. Notably, bone morphogenetic protein 4 (BMP4) affected the Pkd2 distribution. Additionally, three-dimensional manipulation of nodal immotile cilia using optical tweezers revealed that excess BMP4 caused defects in the mechanosensing ability of the cilia. CONCLUSIONS: Experimental data together with model calculations suggest that BMP4 regulates the asymmetric distribution of Pkd2 in nodal immotile cilia, thereby affecting the ability of these cilia to sense the bending direction for L-R determination. This study, for the first time, provides insight into the relationship between the asymmetric protein distribution in cilia and their function.

New Variants Identified by Next-Generation Sequencing in Polycystic Kidney Disease Patients.

Polycystic kidney disease (PKD) is a common inherited disease characterized by multiple cysts in kidneys and various extra renal manifestations. Molecular diagnosis plays a crucial role in confirming both the clinical diagnosis and preimplantation genetic diagnosis furthermore, selecting appropriate treatment options. This study aimed to expand the understanding of genetic mutations in patients with polycystic kidney disease and to improve the management of patients. The study included 92 patients with a clinical diagnosis of PKD based on renal ultrasound criteria. Targeted next-generation sequencing was performed using a custom panel kit. Of the 92 patients included in the study, pathogenic/likely pathogenic variants of the PKD1, PKD2 genes were detected in 37 patients (40.2%), while 8 patients (8.6%) had variants with uncertain clinical significance. After the additional assessment of pathogenic/likely pathogenic variants, it was found that 15 of the variants in PKD1 and 2 of the variants in PKD2 have not been reported in the literature previously. Additionally, pathogenic variants, 5 of which were novel, have been identified in different genes in 8 patients. This study presented the largest patient cohort conducted in Turkey. These findings were significant in expanding our understanding of the genetic variations associated with polycystic kidney disease. The study contributed the literature data on polycystic kidney disease by reporting important findings that could pave the way for further investigations in the diagnosis, treatment, and management of the affected patients.

Roles of cerebrospinal fluid-contacting neurons as potential neural stem cells in the repair and regeneration of spinal cord injuries.

Cerebrospinal fluid-contacting neurons (CSF-cNs) represent a distinct group of interneurons characterized by their prominent apical globular protrusions penetrating the spinal cord's central canal and their basal axons extending towards adjacent cells. Identified nearly a century back, the specific roles and attributes of CSF-cNs have just started to emerge due to the historical lack of definitive markers. Recent findings have confirmed that CSF-cNs expressing PKD2L1 possess attributes of neural stem cells, suggesting a critical function in the regeneration processes following spinal cord injuries. This review aims to elucidate the molecular markers of CSF-cNs as potential neural stem cells during spinal cord development and assess their roles post-spinal cord injury, with an emphasis on their potential therapeutic implications for spinal cord repair.

The Mechanosensitive Pkd2 Channel Modulates the Recruitment of Myosin II and Actin to the Cytokinetic Contractile Ring.

Cytokinesis, the last step in cell division, separates daughter cells through mechanical force. This is often through the force produced by an actomyosin contractile ring. In fission yeast cells, the ring helps recruit a mechanosensitive ion channel, Pkd2, to the cleavage furrow, whose activation by membrane tension promotes calcium influx and daughter cell separation. However, it is unclear how the activities of Pkd2 may affect the actomyosin ring. Here, through both microscopic and genetic analyses of a hypomorphic pkd2 mutant, we examined the potential role of this essential gene in assembling the contractile ring. The pkd2-81KD mutation significantly increased the counts of the type II myosin heavy chain Myo2 (+18%), its regulatory light chain Rlc1 (+37%) and actin (+100%) molecules in the ring, compared to the wild type. Consistent with a regulatory role of Pkd2 in the ring assembly, we identified a strong negative genetic interaction between pkd2-81KD and the temperature-sensitive mutant myo2-E1. The pkd2-81KD myo2-E1 cells often failed to assemble a complete contractile ring. We conclude that Pkd2 modulates the recruitment of type II myosin and actin to the contractile ring, suggesting a novel calcium-dependent mechanism regulating the actin cytoskeletal structures during cytokinesis.

Human Genetics of Defects of Situs.

Defects of situs are associated with complex sets of congenital heart defects in which the normal concordance of asymmetric thoracic and abdominal organs is disturbed. The cellular and molecular mechanisms underlying the formation of the embryonic left-right axis have been investigated extensively in the past decade. This has led to the identification of mutations in at least 33 different genes in humans with heterotaxy and situs defects. Those mutations affect a broad range of molecular components, from transcription factors, signaling molecules, and chromatin modifiers to ciliary proteins. A substantial overlap of these genes is observed with genes associated with other congenital heart diseases such as tetralogy of Fallot and double-outlet right ventricle, d-transposition of the great arteries, and atrioventricular septal defects. In this chapter, we present the broad genetic heterogeneity of situs defects including recent human genomics efforts.

Ciliary intrinsic mechanisms regulate dynamic ciliary extracellular vesicle release from sensory neurons.

Extracellular vesicles (EVs) are submicron membranous structures and key mediators of intercellular communication.1,2 Recent research has highlighted roles for cilia-derived EVs in signal transduction, underscoring their importance as bioactive extracellular organelles containing conserved ciliary signaling proteins.3,4 Members of the transient receptor potential (TRP) channel polycystin-2 (PKD-2) family are found in ciliary EVs of the green algae Chlamydomonas and the nematode Caenorhabditis elegans5,6 and in EVs in the mouse embryonic node and isolated from human urine.7,8 In C. elegans, PKD-2 is expressed in male-specific EV-releasing sensory neurons, which extend ciliary tips to ciliary pore and directly release EVs into the environment.6,9 Males release EVs in a mechanically stimulated manner, regulate EV cargo content in response to mating partners, and deposit PKD-2::GFP-labeled EVs on the vulval cuticle of hermaphrodites during mating.9,10 Combined, our findings suggest that ciliary EV release is a dynamic process. Herein, we identify mechanisms controlling dynamic EV shedding using time-lapse imaging. Cilia can sustain the release of PKD-2-labeled EVs for 2 h. This extended release doesn't require neuronal transmission. Instead, ciliary intrinsic mechanisms regulate PKD-2 ciliary membrane replenishment and dynamic EV release. The kinesin-3 motor kinesin-like protein 6 (KLP-6) is necessary for initial and extended EV release, while the transition zone protein NPHP-4 is required only for sustained EV release. The dynamic replenishment of PKD-2 at the ciliary tip is key to sustained EV release. Our study provides a comprehensive portrait of real-time ciliary EV release and mechanisms supporting cilia as proficient EV release platforms.

Evidence for Two Subpopulations of Cerebrospinal Fluid-Contacting Neurons with Opposite GABAergic Signaling in Adult Mouse Spinal Cord.

Spinal cerebrospinal fluid-contacting neurons (CSF-cNs) form an evolutionary conserved bipolar cell population localized around the central canal of all vertebrates. CSF-cNs were shown to express molecular markers of neuronal immaturity into adulthood; however, the impact of their incomplete maturation on the chloride (Cl-) homeostasis as well as GABAergic signaling remains unknown. Using adult mice from both sexes, in situ hybridization revealed that a proportion of spinal CSF-cNs (18.3%) express the Na+-K+-Cl- cotransporter 1 (NKCC1) allowing intracellular Cl- accumulation. However, we did not find expression of the K+-Cl- cotransporter 2 (KCC2) responsible for Cl- efflux in any CSF-cNs. The lack of KCC2 expression results in low Cl- extrusion capacity in CSF-cNs under high Cl- load in whole-cell patch clamp. Using cell-attached patch clamp allowing recordings with intact intracellular Cl- concentration, we found that the activation of ionotropic GABAA receptors (GABAA-Rs) induced both depolarizing and hyperpolarizing responses in CSF-cNs. Moreover, depolarizing GABA responses can drive action potentials as well as intracellular calcium elevations by activating voltage-gated calcium channels. Blocking NKCC1 with bumetanide inhibited the GABA-induced calcium transients in CSF-cNs. Finally, we show that metabotropic GABAB receptors have no hyperpolarizing action on spinal CSF-cNs as their activation with baclofen did not mediate outward K+ currents, presumably due to the lack of expression of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Together, these findings outline subpopulations of spinal CSF-cNs expressing inhibitory or excitatory GABAA-R signaling. Excitatory GABA may promote the maturation and integration of young CSF-cNs into the existing spinal circuit.

Genotype-phenotype of autosomal dominant polycystic kidney disease in Malta.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple renal cysts causing kidney enlargement and end-stage renal disease (ESRD) in half the patients by 60 years of age. The aim of the study was to determine the genetic aetiology in Maltese patients clinically diagnosed with ADPKD and correlate the clinical features. METHODS: A total of 60 patients over 18 years of age clinically diagnosed with ADPKD were studied using a customized panel of genes that had sufficient evidence of disease diagnosis using next generation sequencing (NGS). The genes studied were PKD1, PKD2, GANAB, DNAJB11, PKHD1 and DZIP1L. Selected variants were confirmed by bidirectional Sanger sequencing with specifically designed primers. Cases where no clinically significant variant was identified by the customized gene panel were then studied by Whole Exome Sequencing (WES). Microsatellite analysis was performed to determine the origin of an identified recurrent variant in the PKD2 gene. Clinical features were studied for statistical correlation with genetic results. RESULTS: Genetic diagnosis was reached in 49 (82%) of cases studied. Pathogenic/likely pathogenic variants PKD1 and PKD2 gene were found in 25 and in 23 cases respectively. The relative proportion of genetically diagnosed PKD1:PKD2 cases was 42:38. A pathogenic variant in the GANAB gene was identified in 1 (2%) case. A potentially significant heterozygous likely pathogenic variant was identified in PKHD1 in 1 (2%) case. Potentially significant variants of uncertain significance were seen in 4 (7%) cases of the study cohort. No variants in DNAJB11 and DZIP1L were observed. Whole exome sequencing (WES) added the diagnostic yield by 10% over the gene panel analysis. Overall no clinically significant variant was detected in 6 (10%) cases of the study population by a customized gene panel and WES. One recurrent variant the PKD2 c.709+1G > A was observed in 19 (32%) cases. Microsatellite analysis showed that all variant cases shared the same haplotype indicating that their families may have originated from a common ancestor and confirmed it to be a founder variant in the Maltese population. The rate of decline in eGFR was steeper and progression to ESRD was earlier in cases with PKD1 variants when compared to cases with PKD2 variants. Cases segregating truncating variants in PKD1 showed a significantly earlier onset of ESRD and this was significantly worse in cases with frameshift variants. Overall extrarenal manifestations were commoner in cases segregating truncating variants in PKD1. CONCLUSIONS: This study helps to show that a customized gene panel is the first-line method of choice for studying patients with ADPKD followed by WES which increased the detection of variants present in the PKD1 pseudogene region. A founder variant in the PKD2 gene was identified in our Maltese cohort with ADPKD. Phenotype of patients with ADPKD is significantly related to the genotype confirming the important role of molecular investigations in the diagnosis and prognosis of polycystic kidney disease. Moreover, the findings also highlight the variability in the clinical phenotype and indicate that other factors including epigenetic and environmental maybe be important determinants in Autosomal Dominant Polycystic Kidney Disease.

The Link between Autosomal Dominant Polycystic Kidney Disease and Chromosomal Instability: Exploring the Relationship.

In autosomal dominant polycystic kidney disease (ADPKD) with germline mutations in a PKD1 or PKD2 gene, innumerable cysts develop from tubules, and renal function deteriorates. Second-hit somatic mutations and renal tubular epithelial (RTE) cell death are crucial features of cyst initiation and disease progression. Here, we use established RTE lines and primary ADPKD cells with disease-associated PKD1 mutations to investigate genomic instability and DNA damage responses. We found that ADPKD cells suffer severe chromosome breakage, aneuploidy, heightened susceptibility to DNA damage, and delayed checkpoint activation. Immunohistochemical analyses of human kidneys corroborated observations in cultured cells. DNA damage sensors (ATM/ATR) were activated but did not localize at nuclear sites of damaged DNA and did not properly activate downstream transducers (CHK1/CHK2). ADPKD cells also had the ability to transform, as they achieved high saturation density and formed colonies in soft agar. Our studies indicate that defective DNA damage repair pathways and the somatic mutagenesis they cause contribute fundamentally to the pathogenesis of ADPKD. Acquired mutations may alternatively confer proliferative advantages to the clonally expanded cell populations or lead to apoptosis. Further understanding of the molecular details of aberrant DNA damage responses in ADPKD is ongoing and holds promise for targeted therapies.

Structure-guided discovery of protein and glycan components in native mastigonemes.

Mastigonemes, the hair-like lateral appendages lining cilia or flagella, participate in mechanosensation and cellular motion, but their constituents and structure have remained unclear. Here, we report the cryo-EM structure of native mastigonemes isolated from Chlamydomonas at 3.0 Å resolution. The long stem assembles as a super spiral, with each helical turn comprising four pairs of anti-parallel mastigoneme-like protein 1 (Mst1). A large array of arabinoglycans, which represents a common class of glycosylation in plants and algae, is resolved surrounding the type II poly-hydroxyproline (Hyp) helix in Mst1. The EM map unveils a mastigoneme axial protein (Mstax) that is rich in heavily glycosylated Hyp and contains a PKD2-like transmembrane domain (TMD). Mstax, with nearly 8,000 residues spanning from the intracellular region to the distal end of the mastigoneme, provides the framework for Mst1 assembly. Our study provides insights into the complexity of protein and glycan interactions in native bio-architectures.

The Complexity of Decisions in Genetics: Annotation of Three Novel Variants in the PKD1 and PKD2 Genes.

As nephrology practice is evolving toward precision medicine, and genetic tests are becoming widely available, basic genetic literacy is increasingly required for clinical nephrologists. Yet, decisions based on results of genetic tests are seldom straightforward. We report a 37-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) who was referred for medically assisted reproduction with monogenic preimplantation genetic testing (PGT-M). The PKD1 and PKD2 genes were screened for pathogenic variants. Sequencing analysis revealed the presence of three novel missense single nucleotide variants, two in the PKD1 gene - c.349T>G, p.(Leu117Val) and c.1736C>T, p.(Pro579Leu); and the third in the PKD2 gene - c.1124A>G, p.(Asn375Ser). Bioinformatic predictions of the functional effects of those three missense variants were inconsistent across different software tools. The family segregation analysis, which was mandatory to identify the relevant variant(s) for PGT-M, strongly supported that the disease-causing variant was PKD1 c.349T>G p.(Leu117Val), while the other two were nonpathogenic or, at most, phenotypic modulators. Proving the pathogenicity of novel variants is often complex but is critical to guide genetic counseling and screening, particularly when discussing reproductive alternatives for primary prevention in the progeny of at-risk couples. The family reported herein illustrates those challenges in the setting of ADPKD, and the invaluable importance of a detailed family history and segregation analysis for proper clinical annotation of novel variants. Basic genetic knowledge and proper clinical annotation of novel allelic variants in genes associated with hereditary kidney disorders are increasingly necessary for the contemporary practice of clinical nephrology.

Brain microvascular endothelial cells possess a second cilium that arises from the daughter centriole.

Primary cilia from the brain microvascular endothelial cells (ECs) are specialized cell-surface organelles involved in mediating sensory perception, cell signaling, and vascular stability. Immunofluorescence (IF) analysis of human primary brain microvascular ECs reveals two cilia per cell. To confirm the in vitro observation of the two-cilia phenotype in human primary brain ECs, ECs isolated from mouse brain were cultured and stained for cilium. Indeed, brain ECs from a ciliopathic mouse (polycystic kidney disease or Pkd2 -/-) also possess more than one cilium. Primary cilium emerges from the mother centriole. Centriole analysis by IF suggests that in brain ECs, markers for the mother and daughter centrioles stain both cilia, suggesting that the second cilium in brain ECs arises from the daughter centriole. Further quantification of cilia size in brain ECs revealed that cilia arising from the mother centriole are bigger in size compared with cilia from the daughter centriole. Cell cycle analyses using immunoblotting and flow cytometry suggest that the ciliary proteins ARL13B and IFT88 involved in brain EC ciliogenesis are highly expressed only in the G0/G1 and S phases of the cell cycle. The IF analyses of cells arrested at different cell cycle stages indicate that the two-cilia phenotype is highly specific to the G0/G1 phase. Our findings suggest that in addition to the mother centriole, the daughter centriole also plays a role in ciliogenesis in primary cultured ECs.

Ciliary intrinsic mechanisms regulate dynamic ciliary extracellular vesicle release from sensory neurons.

Cilia-derived extracellular vesicles (EVs) contain signaling proteins and act in intercellular communication. Polycystin-2 (PKD-2), a transient receptor potential channel, is a conserved ciliary EVs cargo. Caenorhabditis elegans serves as a model for studying ciliary EV biogenesis and function. C. elegans males release EVs in a mechanically-induced manner and deposit PKD-2-labeled EVs onto the hermaphrodite vulva during mating, suggesting an active release process. Here, we study the dynamics of ciliary EV release using time-lapse imaging and find that cilia can sustain the release of PKD-2-labeled EVs for a two-hour duration. Intriguingly, this extended release doesn't require neuronal synaptic transmission. Instead, ciliary intrinsic mechanisms regulate PKD-2 ciliary membrane replenishment and dynamic EV release. The ciliary kinesin-3 motor KLP-6 is necessary for both initial and extended ciliary EV release, while the transition zone protein NPHP-4 is required only for sustained EV release. The dihydroceramide desaturase DEGS1/2 ortholog TTM-5 is highly expressed in the EV-releasing sensory neurons, localizes to cilia, and is required for sustained but not initial ciliary EV release, implicating ceramide in ciliary ectocytosis. The study offers a comprehensive portrait of real-time ciliary EV release, and mechanisms supporting cilia as proficient EV release platforms.

The Reissner fiber under tension in vivo shows dynamic interaction with ciliated cells contacting the cerebrospinal fluid.

The Reissner fiber (RF) is an acellular thread positioned in the midline of the central canal that aggregates thanks to the beating of numerous cilia from ependymal radial glial cells (ERGs) generating flow in the central canal of the spinal cord. RF together with cerebrospinal fluid (CSF)-contacting neurons (CSF-cNs) form an axial sensory system detecting curvature. How RF, CSF-cNs and the multitude of motile cilia from ERGs interact in vivo appears critical for maintenance of RF and sensory functions of CSF-cNs to keep a straight body axis, but is not well-understood. Using in vivo imaging in larval zebrafish, we show that RF is under tension and resonates dorsoventrally. Focal RF ablations trigger retraction and relaxation of the fiber's cut ends, with larger retraction speeds for rostral ablations. We built a mechanical model that estimates RF stress diffusion coefficient D at 5 mm2/s and reveals that tension builds up rostrally along the fiber. After RF ablation, spontaneous CSF-cN activity decreased and ciliary motility changed, suggesting physical interactions between RF and cilia projecting into the central canal. We observed that motile cilia were caudally-tilted and frequently interacted with RF. We propose that the numerous ependymal motile monocilia contribute to RF's heterogenous tension via weak interactions. Our work demonstrates that under tension, the Reissner fiber dynamically interacts with motile cilia generating CSF flow and spinal sensory neurons.

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIß activation in gastrointestinal stromal tumor cells.

Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase gene, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes Golgi retention of KIT. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT cannot trigger downstream activation. In the Golgi/trans-Golgi network (TGN), KIT activates the PKD2-phosphatidylinositol 4-kinase IIIß (PKD2-PI4KIIIß) pathway through phospholipase Cγ2 (PLCγ2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in the release of KIT from the Golgi/TGN. Our findings show the molecular mechanisms underlying KIT mislocalization and provide evidence for a strategy for inhibition of oncogenic signaling.

Identified eleven exon variants in PKD1 and PKD2 genes that altered RNA splicing by minigene assay.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic multisystem disease caused primarily by mutations in the PKD1 gene or PKD2 gene. There is increasing evidence that some of these variants, which are described as missense, synonymous or nonsense mutations in the literature or databases, may be deleterious by affecting the pre-mRNA splicing process. RESULTS: This study aimed to determine the effect of these PKD1 and PKD2 variants on exon splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 19 candidate single nucleotide alterations, 11 variants distributed in PKD1 (c.7866C > A, c.7960A > G, c.7979A > T, c.7987C > T, c.11248C > G, c.11251C > T, c.11257C > G, c.11257C > T, c.11346C > T, and c.11393C > G) and PKD2 (c.1480G > T) were identified to result in exon skipping. CONCLUSIONS: We confirmed that 11 variants in the gene of PKD1 and PKD2 affect normal splicing by interfering the recognition of classical splicing sites or by disrupting exon splicing enhancers and generating exon splicing silencers. This is the most comprehensive study to date on pre-mRNA splicing of exonic variants in ADPKD-associated disease-causing genes in consideration of the increasing number of identified variants in PKD1 and PKD2 gene in recent years. These results emphasize the significance of assessing the effect of exon single nucleotide variants in ADPKD at the mRNA level.

Predicting the Risk of Progression in Indian ADPKD Cohort using PROPKD Score - A Single-Center Retrospective Study.

Background: With the variable genotype-phenotype expression of autosomal dominant polycystic kidney disease (ADPKD) and availability of novel targeted therapies, it is important to find predictors for rapid progression. The PROPKD score, consisting of genetic and clinical parameters like sex, hypertension, and urological events, is a useful tool in predicting the risk of progression. This study was aimed to determine the risk of ADPKD progression in Indian patients using the PROPKD score. Materials and Methods: A retrospective study was done from 2006 to 2021. ADPKD patients with ESRD were included in the study. Scoring was done as per the PROPKD score as follows: male sex: 1, onset of hypertension before 35 years: 2, first urological event before 35 years: 2, PKD1 truncating mutation: 4, PKD1 non-truncating mutation: 2, and PKD2 mutation: 0. Two types of risk classifications were done as follows: (a) considering the clinical variables in all 73 patients (male sex, onset of hypertension before 35 years, and first urological event before 35 years), they were classified into three risk groups: low-risk group (0-1), intermediate-risk group (2-3), and high-risk group (4-5) and (b) considering the clinical variables and type of mutation in 39 patients, they were classified into three risk groups: low-risk group (0-3), intermediate-risk group (4-6), and high-risk group (7-9). Results: Total number of patients included was 73, with the median age at ESRD being 54 years. High-risk group of clinical variables with hazard ratio (HR) of 4.570 (2.302-9.075, P < 0.001) and high-risk group of the PROPKD score with HR of 6.594 (1.868-23.284, P = 0.003) were associated with early ESRD. High-risk groups of both classifications were associated with early ESRD. Conclusion: High-risk groups based on the PROPKD scoring and clinical variables were associated with early progression to ESRD.

The cytoplasmic tail of the mechanosensitive channel Pkd2 regulates its internalization and clustering in eisosomes.

Polycystins are a family of conserved ion channels, mutations of which lead to one of the most common human genetic disorders, namely, autosomal dominant polycystic kidney disease. Schizosacchromyces pombe possesses an essential polycystin homologue, Pkd2, which directs Ca2+ influx on the cell surface in response to membrane tension, but its structure remains unsolved. Here, we analyzed the structure-function relationship of Pkd2 based on its AlphaFold-predicted structure. Pkd2 consists of three domains, the extracellular lipid-binding domain (LBD), nine-helix transmembrane domain (TMD) and C-terminal cytoplasmic domain (CCD). Our genetic and microscopy data revealed that LBD and TMD are essential for targeting Pkd2 to the plasma membrane from the endoplasmic reticulum. In comparison, CCD ensures the polarized distribution of Pkd2 by promoting its internalization and preventing its clustering in the eisosome, a caveolae-like membrane compartment. The domains of Pkd2 and their functions are conserved in other fission yeast species. We conclude that both extracellular and cytoplasmic domains of Pkd2 are crucial for its intracellular trafficking and function. We propose that mechanosensitive channels can be desensitized through either internalization or clustering in low-tension membrane compartments.

Modifiers of Autosomal Dominant Polycystic Kidney Disease Severity: The Role of PKD1 Hypomorphic Alleles.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of kidney failure in adult life. Rarely, ADPKD can be diagnosed in utero or in infancy, and the genetic mechanism underlying such severe presentation has been shown to be related to reduced gene dosage. Biallelic PKD1 variants are often identified in early onset ADPKD, with one main pathogenic variant and a modifier hypomorphic variant showing an in trans configuration. We describe two unrelated individuals with early onset cystic kidney disease and unaffected parents, where a combination of next-generation sequencing of cystic genes including PKHD1, HNF1B and PKD1 allowed the identification of biallelic PKD1 variants. Furthermore, we review the medical literature in order to report likely PKD1 hypomorphic variants reported to date and estimate a minimal allele frequency of 1/130 for this category of variants taken as a group. This figure could help to orient genetic counseling, although the interpretation and the real clinical impact of rare PKD1 missense variants, especially if previously unreported, remain challenging.