bims-bicyki Biomed News
on Bicaudal-C1 and interactors in cystic kidney disease
Issue of 2020‒11‒29
nine papers selected by
Céline Gagnieux
École Polytechnique Fédérale de Lausanne (EPFL)

  1. Am J Nephrol. 2020 Nov 23. 1-10
      BACKGROUND: Tolvaptan was approved for the treatment of autosomal dominant polycystic kidney disease (ADPKD). However, the official indication of "rapidly progressive disease" is described differently in the clinical guidelines. We aim to define "rapidly progressive disease" by risk of ESRD, which is evaluated using estimated height-adjusted total kidney volume (HtTKV) growth rate.METHODS: The risk of ESRD was retrospectively analyzed in 617 initially non-ESRD adults with ADPKD and observed with standard of care between 2007 and 2018. The estimated annual growth rate of the HtTKV, termed as eHTKV-α (%/year), is derived from the following equation: [HtTKV at age t] = K(1 + eHTKV-α/100)t, where K = 150 mL/m is used in Mayo Imaging Classification and K = 130 mL/m is proposed for individually stable eHTKV-α value from baseline. The accuracy of eHTKV-α to predict ESRD for censored ages was analyzed using time-dependent receiver-operating characteristic curves (ROC). The cutoff point of initially measured eHTKV-α to predict ESRD was assessed using Kaplan-Meier and Cox's proportional hazards models. Performance characteristics of the cutoff point for censored ages were calculated using time-dependent ROC and validated by the bootstrap method.
    RESULTS: The area under the time-dependent ROC of eHTKV-α to predict ESRD at age 65 was 0.89 ± 0.04 (K = 130). The mean renal survival was less than 70 years at eHTKV-α ≥4.0%/year (K = 130). Mean renal survival was approximately 12 years shorter, and hazard ratio of ESRD was more than 5-time higher at this cutoff point than at lower point. Time-dependent sensitivity for age 65 and cutoff point of 4.0%/year (K = 130) was 93.4 ± 0.3%. Between cutoff points ≥4.0%/year (K = 130) and ≥3.5%/year (K = 150), there was no significant difference in performance characteristics and accuracy to predict ESRD.
    CONCLUSION: eHTKV-α well predicts ESRD. Initially, measured eHTKV-α ≥4.0%/year (K = 130) defines high-risk ESRD. Without additional conditions, a single eHTKV-α cutoff point identifies subjects that are most likely to benefit from tolvaptan.
    Keywords:  Autosomal dominant polycystic kidney disease; End-stage renal disease; Estimated HtTKV growth rate; Guidelines; Height-adjusted TKV; Tolvaptan; Total kidney volume
  2. Ann Transl Med. 2020 Sep;8(18): 1175
      Background: DJ-1 is critical for the mitochondrial function associated with autosomal dominant polycystic kidney disease (ADPKD). We aimed to investigate DJ-1's function in the pathogenesis of ADPKD.Methods: DJ-1 was knocked-down in IMCD3 cells to evaluate the effects of DJ-1 on cell phenotype and mitochondrial function in vitro. Furthermore, we generated three groups of mice with different expression levels of DJ-1 within an established ADPKD model: ADPKD, ADPKDpcDNA, and ADPKDpcDNA-DJ-1.
    Results: DJ-1 knock-down significantly increased oxidative stress as well as the proliferation and apoptosis rate of IMCD3 cells, along with Bcl-2 down-regulation and the up-regulation of Ki67, PCNA, Bax, cleaved caspase-3, and cleaved caspase-9. DJ-1 knock-down suppressed the cellular respiration, Ca2+ absorption, and mitochondrial complex I activity in mitochondria. In vivo, we verified that DJ-1 was down-regulated in ADPKD models, and its overexpression attenuated the renal dysfunction in ADPKD models. The transgenic mice had a significantly smaller renal cyst and less interstitial fibrosis than control, accompanied byα-SMA, fibronectin, and TGF-β1 up-regulation. Moreover, in vivo results confirmed DJ-1 overexpression inhibited the proliferation and apoptosis of tubular epithelial cells along with down-regulation of Ki67, PCNA, p53, intracellular Cyt c, cleaved caspase-3, and cleaved caspase-9 and the up-regulation of Bcl-2.
    Conclusions: DJ-1 was down-regulated in ADPKD models, and its overexpression may attenuate the renal dysfunction and pathological damage by regulating the proliferation, apoptosis, oxidative stress and mitochondrial metabolism, which may be mediated by the p53 signaling pathway.
    Keywords:  Autosomal dominant polycystic kidney disease (ADPKD); DJ-1; mitochondrial dysfunction; mitochondrial metabolism
  3. Molecules. 2020 Nov 23. pii: E5477. [Epub ahead of print]25(22):
      Autosomal dominant polycystic kidney disease (ADPKD) causes progressive cystic degeneration of the renal tubules, the nephrons, eventually severely compromising kidney function. ADPKD is incurable, with half of the patients eventually needing renal replacement. Treatments for ADPKD patients are limited and new effective therapeutics are needed. Melatonin, a central metabolic regulator conserved across all life kingdoms, exhibits oncostatic and oncoprotective activity and no detected toxicity. Here, we used the Bicaudal C (BicC) Drosophila model of polycystic kidney disease to test the cyst-reducing potential of melatonin. Significant cyst reduction was found in the renal (Malpighian) tubules upon melatonin administration and suggest mechanistic sophistication. Similar to vertebrate PKD, the BicC fly PKD model responds to the antiproliferative drugs rapamycin and mimics of the second mitochondria-derived activator of caspases (Smac). Melatonin appears to be a new cyst-reducing molecule with attractive properties as a potential candidate for PKD treatment.
    Keywords:  Drosophila; longevity; melatonin; oxidative stress; polycystic kidney disease; renal cysts
  4. Nat Biotechnol. 2020 Nov 23.
      Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn7-like transposon from Vibrio cholerae, which uses a Type I-F CRISPR-Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA-assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.
  5. Nat Protoc. 2020 Nov 27.
      The liver is composed of two epithelial cell types: hepatocytes and liver ductal cells. Culture conditions for expansion of human liver ductal cells in vitro as organoids were previously described in a protocol; however, primary human hepatocytes remained hard to expand, until recently. In this protocol, we provide full details of how we overcame this limitation, establishing culture conditions that facilitate long-term expansion of human fetal hepatocytes as organoids. In addition, we describe how to generate (multi) gene knockouts using CRISPR-Cas9 in both human fetal hepatocyte and adult liver ductal organoid systems. Using a CRISPR-Cas9 and homology-independent organoid transgenesis (CRISPR-HOT) approach, efficient gene knockin can be achieved in these systems. These gene knockin and knockout approaches, and their multiplexing, should be useful for a variety of applications, such as disease modeling, investigating gene functions and studying processes, such as cellular differentiation and cell division. The protocol to establish human fetal hepatocyte organoid cultures takes ~1-2 months. The protocols to genome engineer human liver ductal organoids and human fetal hepatocyte organoids take 2-3 months.
  6. Nucleic Acids Res. 2020 Nov 24. pii: gkaa1088. [Epub ahead of print]
      Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures.
  7. Arch Biochem Biophys. 2020 Nov 18. pii: S0003-9861(20)30689-5. [Epub ahead of print] 108680
      The study of calmodulin (CaM) functions in living cells has been tackled up to date using cell-permeant CaM inhibitors or interference-RNA methods. CaM inhibitors may lack specificity and the siRNA interference approach is challenging, as all three CaM genes expressing an identical protein in mammals have to be blocked. Therefore, we recently introduced a novel genetic system using CRISPR/Cas9-mediated gene deletion and conditional CaM expression to study the function of CaM in HeLa cells. Here, we describe the effect of CaM downregulation on the basal and epidermal growth factor (EGF)-dependent 2D- and 3D-migration in HeLa cells. CaM downregulation inhibited cell migration on a 2D-surface in the absence but not in the presence of EGF. In contrast, CaM downregulation led to inhibition of 3D-migration across a porous membrane both in the absence and presence of EGF. CaM downregulation decreased the expression of Rac1, Cdc42 and RhoA, all known to play crucial roles in cell migration. These results show that EGF-dependent 2D- and 3D-migration utilize distinct CaM-regulated systems and identify several essential migratory proteins directly or indirectly regulated by CaM.
    Keywords:  Calmodulin; Cdc43; Cell migration; Conditional knock-out cells; Rac1; RhoA
  8. Sci Rep. 2020 Nov 26. 10(1): 20598
      Mechanical cues from the cellular microenvironment are converted into biochemical signals controlling diverse cell behaviours, including growth and differentiation. But it is still unclear how mechanotransduction ultimately affects nuclear readouts, genome function and transcriptional programs. Key signaling pathways and transcription factors can be activated, and can relocalize to the nucleus, upon mechanosensing. Here, we tested the hypothesis that epigenetic regulators, such as methyltransferase enzymes, might also contribute to mechanotransduction. We found that the SMYD3 lysine methyltransferase is spatially redistributed dependent on cell geometry (cell shape and aspect ratio) in murine myoblasts. Specifically, elongated rectangles were less permissive than square shapes to SMYD3 nuclear accumulation, via reduced nuclear import. Notably, SMYD3 has both nuclear and cytoplasmic substrates. The distribution of SMYD3 in response to cell geometry correlated with cytoplasmic and nuclear lysine tri-methylation (Kme3) levels, but not Kme2. Moreover, drugs targeting cytoskeletal acto-myosin induced nuclear accumulation of Smyd3. We also observed that square vs rectangular geometry impacted the nuclear-cytoplasmic relocalisation of several mechano-sensitive proteins, notably YAP/TAZ proteins and the SETDB1 methyltransferase. Thus, mechanical cues from cellular geometric shapes are transduced by a combination of transcription factors and epigenetic regulators shuttling between the cell nucleus and cytoplasm. A mechanosensitive epigenetic machinery could potentially affect differentiation programs and cellular memory.
  9. J Pediatr. 2020 Nov 23. pii: S0022-3476(20)31452-9. [Epub ahead of print]
    Keywords:  basal body; bronchiectasis; cilia; dynein; primary ciliary dyskinesia