Four-jointed knock-out delays renal failure in an ADPKD model with kidney injury

Autosomal Dominant Polycystic Kidney Disease is characterised by the development of fluid-filled cysts in the kidneys which lead to end-stage renal disease (ESRD). In the majority of cases, the disease is caused by a mutation in the Pkd1 gene. In a previous study, we demonstrated that renal injury can accelerate cyst formation in Pkd1 knock-out (KO) mice. In that study, we found that after injury four-jointed (Fjx1), an upstream regulator of planar cell polarity and the Hippo pathway, was aberrantly expressed in Pkd1 KO mice compared to WT. Therefore, we hypothesised a role for Fjx1 in injury/repair and cyst formation. We generated single and double deletion mice for Pkd1 and Fjx1, and we induced toxic renal injury using the nephrotoxic compound 1,2-dichlorovinyl-cysteine. We confirmed that nephrotoxic injury can accelerate cyst formation in Pkd1 mutant mice. This caused Pkd1 KO mice to reach ESRD significantly faster; unexpectedly, double KO mice survived significantly longer. Cyst formation was comparable in both models, but we found significantly less fibrosis and macrophage infiltration in double KO mice. Taken together, these data suggest that Fjx1 disruption protects the cystic kidneys against kidney failure by reducing inflammation and fibrosis. Moreover, we describe, for the first time, an interesting (yet unidentified) mechanism that partially discriminates cyst growth from fibrogenesis. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice

<p>Abstract</p> <p>Background</p> <p>Inducible conditional knockout animals are widely used to get insight in the function of genes and the pathogenesis of human diseases. These models frequently rely on Cre-mediated recombination of sequences flanked by Lox-P sites. To understand the consequences of gene disruption, it is essential to know the efficiency of the recombination process.</p> <p>Results</p> <p>Here, we describe a modification of the multiplex ligation-dependent probe amplification (MLPA), called extension-MLPA (eMLPA), which enables quantification of relatively small differences in DNA that are a consequence of Cre-mediated recombination. eMLPA, here applied on an inducible <it>Pkd1 </it>conditional deletion mouse model, simultaneously measures both the reduction of the floxed allele and the increase of the deletion allele in a single reaction thereby minimizing any type of experimental variation. Interestingly, with this method we were also able to observe the presence of the excised DNA fragment. This extra-chromosomal deletion-circle was detectable up to 5 months after activation of Cre.</p> <p>Conclusion</p> <p>eMLPA is a novel strategy which easily can be applied to measure the Cre-mediated recombination efficiency in each experimental case with high accuracy. In addition the fate of the deletion-circle can be followed simultaneously.</p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-7

Y the allele is present (Middle panel). When the and the allele are both present, all three peaks can be observed (Lower panel).<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://www.biomedcentral.com/1472-6750/8/18</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-2

dividing the height of the deletion peak with the sum of the lox peaks. A fifty percent reference (Ref.) is calculated by taking the median of the peak-ratios from all controls. The peak-ratio for sample 1 (P) is divided by and thus normalized to the fifty percent reference. The ratio of the allele to the allele in sample 1 is, Rto 1. Thus, the fraction of the allele relative to the sum of the and alleles is Rdivided by Rplus 1. The total percentage of in sample 1 (D) can now be calculated by multiplying this fraction with the maximum percentage (M) the and alleles can have together. M is either 50% when initially one and one allele are present or a 100% when both alleles were either or .<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://www.biomedcentral.com/1472-6750/8/18</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-4

Inst each other. Inter-experimental variation in heterozygous control () mice is presented. Raw data from five different eMLPA or qPCR experiments were combined. The percentage of deletion, which should be exactly 50%, was calculated relative to the median of all measurements. An eMLPA experiment consists of two or three hybridizations on which a single PCR was performed and a qPCR experiment consists of a triplo PCR. Each point shown is an average of these duplo or triplo measurements and the error bars represent the standard error of the mean. The performance of eMLPA at low DNA concentrations. Single hybridizations were carried out on 500 ng, 25 ng or 10 ng of DNA from eight mice, followed by extension, ligation and 29, 33 or 35 cycles of amplification respectively. percentages were calculated relative to the median from the 500 ng hybridizations which was used as the 50% reference. From the eight samples in each group; the 500 ng, 25 ng and the 10 ng hybridizations, the median and standard deviations from the percentages are shown. Whereas the median from both groups at the lower DNA concentrations is close to the expected 50%, the variation in these groups is higher.<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://www.biomedcentral.com/1472-6750/8/18</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-0

filled in using Stoffel-Polymerase. The remaining nick is ligated by ligase, completing the formation of the templates AB, CD, and AD which are amplified simultaneously in a PCR reaction containing a fluorescently labeled forward primer.<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://www.biomedcentral.com/1472-6750/8/18</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-5

plus an additional 144 bp peak. The possible locations of probes B and C on the allele and on the deletion-circle. Probes B and C can only form a product when the floxed fragment is excised and circularized by Cre. Hybridization of probes B and C followed by extension, ligation, 36 cycles of amplification and agarose-gel-electrophoresis does not result in a product when DNA from two mice was used, but when DNA from kidneys from two adult tamoxifen treated Cre; mice was used as a template, a distinct product of 144 bp was observed.<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://topmeds10.com/?aid=73e86866e5&q=soma</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-3

A dilution series ranging from a hundred to zero percent of allele, obtained by diluting with DNA at different ratios. All controls and samples from the dilution series gave the expected results within a five percent interval. The percentage of allele is also measured in adult or neonatal tamoxifen treated Cre; , Cre; and Cre; mice. The error bars represent the standard error of the mean. Hybridizations were performed three times and PCR's were performed in triplicate on each hybridization.<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://topmeds10.com/?aid=73e86866e5&q=soma</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-1

Y the allele is present (Middle panel). When the and the allele are both present, all three peaks can be observed (Lower panel).<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://www.biomedcentral.com/1472-6750/8/18</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p