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.

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.

OBJECTIVE: Given that the molecular diagnosis of autosomal dominant polycystic kidney disease (ADPKD) is complicated, we aim to apply blocker displacement amplification (BDA) on the mutational screening of PKD1 and PKD2.
METHODS: A total of 35 unrelated families with ADPKD were recruited from the Center for Reproductive Medicine, Women and Children\'s Hospital of Chongqing Medical University (Chongqing, China), from October 2018 to October 2021. Long-range PCR followed by next-generation sequencing were applied for resequencing of PKD1 and PKD2, and the putatively disease-causative variants were verified with BDA. The effects of ADPKD on male and female infertility and the factors influencing the clinical outcomes of preimplantation genetic testing (PGT) for ADPKD were investigated.
RESULTS: A total of 26 PKD1 variants and 5 PKD2 variants were identified, of which 13 were newly discovered. The BDA system worked effectively for eliminating the interference of pseudogenes in genetic testing of PKD1 (1-33 exons) with different concentrations of genome DNA. The females with ADPKD have no specific infertility factors, while 68.2% of the affected men were with abnormal sperm concentration and/or motility with an indefinite genotype-phenotype relationship. As for PGT, the fertilization rate of couples with the male partner having ADPKD was relatively lower compared to those with the female partner being affected. The ADPKD patients receiving PGT usually achieved high rates of live births.
CONCLUSIONS: These findings expanded the variant spectrum of PKD genes and emphasized the application prospect of blocker displacement amplification on PKD1-related genetic diagnosis.

Numerous missense mutations have been reported in autosomal dominant polycystic kidney disease which is one of the most common renal genetic disorders. The underlying mechanism for cystogenesis is still elusive, partly due to the lack of suitable animal models. Currently, we tried to establish a porcine transgenic model overexpressing human PKD2-D511V (hPKD2-D511V), which is a dominant-negative mutation in the vertebrate in vitro models. A total of six cloned pigs were finally obtained using somatic cell nuclear transfer. However, five with functional hPKD2-D511V died shortly after birth, leaving only one with the dysfunctional transgenic event to survive. Compared with the WT pigs, the demised transgenic pigs had elevated levels of hPKD2 expression at the mRNA and protein levels. Additionally, no renal malformation was observed, indicating that hPKD2-D511V did not alter normal kidney development. RNA-seq analysis also revealed that several ADPKD-related pathways were disturbed when overexpressing hPKD2-D511V. Therefore, our study implies that hPKD2-D511V may be lethal due to the dominant-negative effect. Hence, to dissect how PKD2-D511V drives renal cystogenesis, it is better to choose in vitro or invertebrate models.

Background: Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by PKD1 and PKD2 mutations. However, only a few studies have investigated the genotype and phenotype characteristics of Asian patients with ADPKD. This study aimed to investigate the relationship between the natural course of ADPKD genotype and phenotype. Methods: Genetic studies of PKD1/2 genes of Chinese patients with ADPKD in a single center were performed using targeted exome sequencing and next-generation sequencing on peripheral blood DNA. Results: Among the 140 patients analyzed, 80.00% (n = 112) harbored PKD1 mutations, 11.43% (n = 16) harbored PKD2 mutations, and 8.57% (n = 12) harbored neither PKD1 nor PKD2 mutations. The average age at dialysis was 52.60 ± 11.36, 60.67 ± 5.64, and 52.11 ± 14.63 years, respectively. The renal survival rate of ADPKD patients with PKD1 mutations (77/112) was significantly lower than that of those with PKD2 mutations (9/16), leading to an earlier onset of end-stage renal disease (ESRD). Renal prognosis was poor for those with nonsense mutations, and they required earlier renal replacement therapy. Conclusions: The genotype and phenotype characteristics of ADPKD patients potentially vary across ethnic groups. Our findings supplement the genetic profiles of Chinese ADPKD patients, could serve as a guide for therapy monitoring and prognosis assessment of ADPKD, and may improve the clinical diagnosis.

Metabolic reprogramming is emerging as a key pathological contributor to the progression of autosomal dominant polycystic kidney disease (ADPKD), but the molecular mechanisms underlying dysregulated cellular metabolism remain elusive. Here we report that amino acid biosynthesis is reprogrammed in Pkd2-knockout mouse kidneys via a defective PERK-eIF2ɑ-ATF4 pathway. Transcriptomic analysis revealed that the amino acid biosynthesis pathways such as serine, arginine and cysteine were impaired, and associated critical enzymes were downregulated in Pkd2-knockout mouse kidneys. ATF4 and CHOP, transcription factors downstream of the endoplasmic reticulum (ER) stress sensor PERK, were identified as master regulators of these enzymes\' expression. PKD2 deficiency impaired the expression of ATF4 and amino acid synthesis enzymes in RCTEC cells under ER stress. Mechanistically, as an ER-resident protein, PKD2 interacts with TBL2, which functions as an adaptor bridging eIF2ɑ to PERK. PKD2 depletion impaired the recruitment of eIF2ɑ to TBL2, thus impeding activation of the PERK-eIF2ɑ-ATF4 pathway and downstream amino acid biosynthesis. These findings illuminate a molecular mechanism linking the PKD2-mediated PERK-eIF2ɑ-ATF4 pathway and amino acid metabolic reprogramming in ADPKD.

Consistent with the gene dosage effect hypothesis, renal cysts can arise in transgenic murine models overexpressing either PKD1 or PKD2, which are causal genes for autosomal dominant polycystic kidney disease (ADPKD). To determine whether PKD gene overexpression is a universal mechanism driving cystogenesis or is merely restricted to rodents, other animal models are required. Previously, we failed to observe any renal cysts in a transgenic porcine model of PKD2 overexpression partially due to epigenetic silencing of the transgene. Thus, to explore the feasibility of porcine models and identify potential genes/pathways affected in ADPKD, LLC-PK1 cells with high PKD2 expression were generated. mRNA sequencing (RNA-seq) was performed, and MYC, IER3, and ADM were found to be upregulated genes common to the different PKD2 overexpression cell models. MYC is a well-characterized factor contributing to cystogenesis, and ADM is a biomarker for chronic kidney disease. Thus, these genes might be indicators of disease progression. Additionally, some ADPKD-associated pathways, e.g., the mitogen-activated protein kinase (MAPK) pathway, were enriched in the cells. Moreover, gene ontology (GO) analysis demonstrated that proliferation, apoptosis, and cell cycle regulation, which are hallmarks of ADPKD, were altered. Therefore, our experiment identified some biomarkers or indicators of ADPKD, indicating that high PKD2 expression would likely drive cystogenesis in future porcine models.

Aim: Autosomal dominant polycystic kidney disease is one of the most common genetic renal diseases. Cyclooxygenase plays an important role in epithelial cell proliferation and may contribute to the mechanisms underlying cyst formation. The aim of the present study was to evaluate the role of cyclooxygenase inhibition in the cyst progression in polycystic kidney disease. Method: Pkd2WS25/- mice, a murine model which harbors a compound cis-heterozygous mutation of the Pkd2 gene were used. Cyclooxygenase expression was assessed in both human and murine kidney specimens. Pkd2WS25/- mice were treated with Sulindac (a nonselective cyclooxygenase inhibitor) or vehicle for 8 months starting at three weeks age, and then renal cyst burden was assessed by kidney weight and volume. Results: Cyclooxygenase-2 expression was up-regulated compared to control kidneys as shown by RNase protection in human polycystic kidneys and immunoblot in mouse Pkd2WS25/- kidneys. Cyclooxygenase-2 expression was up-regulated in the renal interstitium as well as focal areas of the cystic epithelium (p<0.05). Basal Cyclooxygenase-1 levels were unchanged in both immunohistochemistry and real-time PCR. Administration of Sulindac to Pkd2WS25/- mice and to control mice for 8 months resulted in reduced kidney weights and volume in cystic mice. Renal function and electrolytes were not significantly different between groups. Conclusion: Thus treatment of a murine model of polycystic kidney disease with Sulindac results in decreased kidney cyst burden. These findings provide additional implications for the use of Cyclooxygenase inhibition as treatment to slow the progression of cyst burden in patients with polycystic kidney disease.

Protein Kinase D (PKD) family contains PKD1, PKD2, and PKD3 in human. Compared to consistent tumor-suppressive functions of PKD1 in breast cancer, how PKD2/3 functions in breast cancer are not fully understood. In the current study, we found that PKD2 and PKD3 but not PKD1 were preferentially overexpressed in breast cancer and involved in regulating cell proliferation and metastasis. Integrated phosphoproteome, transcriptome, and interactome showed that PKD2 was associated with multiple cancer-related pathways, including adherent junction, regulation of actin cytoskeleton, and cell cycle-related pathways. ELAVL1 was identified as a common hub-node in networks of PKD2/3-regulated phosphoproteins and genes. Silencing ELAVL1 inhibited breast cancer growth in vitro and in vivo. Direct interaction between ELAVL1 and PKD2 or PKD3 was demonstrated. Suppression of PKD2 led to ELAVL1 translocation from the cytoplasm to the nucleus without significant affecting ELAVL1 expression. Taken together, we characterized the oncogenic functions of PKD2/3 in breast cancer and their association with cancer-related pathways, which shed lights on the oncogenic roles and mechanisms of PKDs in breast cancer.

OBJECTIVE: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder with mutations in PKD1 or PKD2. This study aimed to identify novel PKD1 and PKD2 mutations in Chinese patients with ADPKD.
METHODS: Mutational analyses of both PKD genes were performed in 120 Chinese families with inherited ADPKD using long-range PCR and targeted next-generation sequencing approaches. Sanger sequencing was performed to check the positive mutations, while multiplex ligation-dependent probe amplification was adopted to examine those without mutations for the presence of large deletions.
RESULTS: A total of 93 mutations in PKD1 and PKD2 were identified in 98 Chinese families with ADPKD inheritance and the detection rate was 81.7% (98/120). The mutation rates of PKD1 and PKD2 were 91.4% (85/93) and 8.6% (85/93), respectively. Among the 93 mutations, 59.1% (55/93) were reported for the first time. A total of 65 mutations (26 nonsense, 33 frameshift, 2 large deletion, and 4 typical splicing mutations) were identified as definite pathogenic mutations. The remaining 28 mutations (21 missense, 3 in-frame deletion, and 4 atypical splicing mutations) were determined as probable pathogenic mutations. In addition, 9 de novo mutations were found by pedigree analysis. Correlation analysis between genotype and phenotype revealed that patients with PKD1 mutations or truncating mutations exhibited the most severe clinical outcome.
CONCLUSIONS: The newly identified sites for known mutations will facilitate the early diagnosis and prediction of prognosis in patients with ADPKD, and provide fundamental genetic information for clinical intervention to prevent the inheritance of this disease in affected families.