Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Peterson, Sarah; Wang, Xuexiang; Johnson, Ashley; Coate, Ian D.; Garrett, Michael R.; Didion, Sean P.

doi:10.3791/58599

“…HCl maceration of whole kidneys was performed according to refs. 22 and 23. Kidneys were isolated from ≥P28 animals, their capsule removed, and the tissue minced using a razor blade and incubated in 6N HCL for 90 minutes.…”

Section: Methodsmentioning

confidence: 99%

Reduced Nephron Endowment in Six2-TGC tg Mice Is Due to Six3 Misexpression by Aberrant Enhancer–Promoter Interactions in the Transgene

Perl,

Liu,

Hass

et al. 2024

JASN

0

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Background Lifelong kidney function relies on the complement of nephrons generated during mammalian development from a mesenchymal nephron progenitor cell population. Low nephron endowment confers increased susceptibility to chronic kidney disease. Reduced nephron numbers in the popular Six2TGC transgenic mouse line may be due to disruption of a regulatory gene at the integration site and/or ectopic expression of a gene(s) contained within the transgene. Methods Targeted locus amplification (TLA) was performed to identify the integration site of the Six2TGC transgene. Genome-wide chromatin conformation capture (Hi-C) datasets were generated from nephron progenitor cells isolated from the Six2TGC +/tg mice, the Cited1 CreERT2/+ control mice, and the Six2TGC +/tg ;Tsc1 +/Flox mice that exhibited restored nephron number compared with Six2TGC +/tg mice. Modified transgenic mice lacking the C-terminal domain of Six3 were used to evaluate the mechanism of nephron number reduction in the Six2TGC +/tg mouse line. Results TLA revealed integration of the Six2TGC transgene within an intron of Cntnap5a on chr1, and Hi-C analysis mapped the precise integration of Six2TGC and Cited1 CreERT2 transgenes to chr1 and chr14, respectively. No changes in topology, accessibility, or expression were observed within the 50-megabase region centered on Cntnap5a in Six2TGC +/tg mice compared with control mice. By contrast, we identified an aberrant regulatory interaction between a Six2 distal enhancer and the Six3 promoter contained within the transgene. Increasing the Six2TGC tg to Six2 locus ratio or removing one Six2 allele in Six2TGC +/tg mice caused severe renal hypoplasia. Furthermore, CRISPR disruption of Six3 within the transgene (Six2TGC ∆Six3CT ) restored nephron endowment to wildtype levels and abolished the stoichiometric effect. Conclusions These findings broadly demonstrate the utility of Hi-C data in mapping transgene integration sites and architecture. Data from genetic and biochemical studies together suggest that in Six2TGC kidneys, SIX3 interferes with SIX2 function in nephron progenitor cell renewal through its C-terminal domain.

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“…Nephron Counts: HCl maceration of whole kidneys was performed according to (MacKay et al, 1987;Peterson et al, 2019). Kidneys isolated from > P28 animals were minced using a razor blade after removal of capsule, and incubated in the presence of 6N HCL for 90 min.…”

Section: Methodsmentioning

confidence: 99%

Revisiting the Role of Notch in Nephron Segmentation: Notch is Required for Proximal, Not Distal Fate Selection During Mouse and Human Nephrogenesis

Duvall

¹

,

Bice

²

,

Perl

³

et al. 2021

Preprint

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Notch signaling promotes maturation of nephron epithelia, but its proposed contribution to nephron segmentation into proximal and distal domains has been called into doubt. We leveraged single cell and bulk RNA-seq, quantitative immunofluorescent lineage/fate tracing, and genetically modified human iPSC to revisit this question in developing mouse kidneys and human kidney organoids. We confirmed that Notch signaling is needed for maturation of all nephron lineages, and thus mature lineage markers fail to detect a fate bias. By contrast, early markers identified a distal fate bias in cells lacking Notch2, and a concomitant increase in early proximal and podocyte fates in cells expressing hyperactive Notch1 was observed. Orthogonal support for a conserved role for Notch signaling in the distal/proximal axis segmentation is provided by the ability of Nicastrin-deficient hiPSCs-derived organoids to differentiate into TFA2B+ distal tubule and CDH1 connecting segment progenitors, but not into HNF4A+ or LTL+ proximal progenitors.SummaryNotch signaling acts in nephron segmentation to select early proximal, but not distal tubule fate downstream of a global role promoting epithelial growth and maturation in mouse and human.

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“…Nephron counts. HCl maceration of whole kidneys was performed according to 49,50 . Kidneys were isolated from > P28 animals, the capsule removed, minced using a razor blade and incubated in 6N HCL for 90 min.…”

Section: ' Bac Junction R2 Cccagaagtggtatagctgagtmentioning

confidence: 99%

Reduced nephron endowment in the commonSix2-TGC^tgmouse line is due toSix3misexpression by aberrant enhancer-promoter interactions in the transgene

Perl,

Liu,

Hass

et al. 2023

Preprint

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Lifelong kidney function relies on the complement of nephrons generated during mammalian development from a mesenchymal nephron progenitor cell (NPC) population. Low nephron endowment confers increased susceptibility to chronic kidney disease. We asked whether reduced nephron numbers in the popularSix2TGCtransgenic mouse line1was due to disruption of a regulatory gene at the integration site or to ectopic expression of a gene(s) contained within the transgene. Targeted locus amplification identified integration of theSix2TGCtransgene within an intron ofCntnap5aon chr1. We generated Hi-C datasets from NPCs isolated from theSix2TGCtg/+mice, theCited1CreERT2/+control mice, and theSix2TGCtg/+;Tsc1+/Flox,2mice that exhibited restored nephron number compared withSix2TGCtg/+mice, and mapped the precise integration ofSix2TGCandCited1CreERT2transgenes to chr1 and chr14, respectively. No changes in topology, accessibility, or expression were observed within the 50-megabase region centered onCntnap5ainSix2TGCtg/+mice compared with control mice. By contrast, we identified an aberrant regulatory interaction between aSix2distal enhancer and theSix3promoter contained within the transgene. Increasing theSix2TGCtgtoSix2locus ratio or removing oneSix2allele inSix2TGCtg/+mice, caused severe renal hypoplasia. Furthermore, CRISPR disruption ofSix3within the transgene (Six2TGCΔSix3CT) restored nephron endowment to wildtype levels and abolished the stoichiometric effect. Data from genetic and biochemical studies together suggest that inSix2TGC,SIX3 interferes with SIX2 function in NPC renewal through its C-terminal domain.SignificanceUsing high-resolution chromatin conformation and accessibility datasets we mapped the integration site of two popular transgenes used in studies of nephron progenitor cells and kidney development. Aberrant enhancer-promoter interactions drive ectopic expression ofSix3in theSix2TGCtgline which was correlated with disruption of nephrogenesis. Disruption ofSix3within the transgene restored nephron numbers to control levels; further genetic and biochemical studies suggest thatSix3interferes withSix2-mediated regulation of NPC renewal.

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Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Cited by 8 publications

References 0 publications

Reduced Nephron Endowment in Six2-TGC tg Mice Is Due to Six3 Misexpression by Aberrant Enhancer–Promoter Interactions in the Transgene

Reduced Nephron Endowment in Six2-TGC tg Mice Is Due to Six3 Misexpression by Aberrant Enhancer–Promoter Interactions in the Transgene

Revisiting the Role of Notch in Nephron Segmentation: Notch is Required for Proximal, Not Distal Fate Selection During Mouse and Human Nephrogenesis

Reduced nephron endowment in the commonSix2-TGC^tgmouse line is due toSix3misexpression by aberrant enhancer-promoter interactions in the transgene

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Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Cited by 8 publications

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Reduced Nephron Endowment in Six2-TGC tg Mice Is Due to Six3 Misexpression by Aberrant Enhancer–Promoter Interactions in the Transgene

Reduced Nephron Endowment in Six2-TGC tg Mice Is Due to Six3 Misexpression by Aberrant Enhancer–Promoter Interactions in the Transgene

Revisiting the Role of Notch in Nephron Segmentation: Notch is Required for Proximal, Not Distal Fate Selection During Mouse and Human Nephrogenesis

Reduced nephron endowment in the commonSix2-TGCtgmouse line is due toSix3misexpression by aberrant enhancer-promoter interactions in the transgene

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Reduced nephron endowment in the commonSix2-TGC^tgmouse line is due toSix3misexpression by aberrant enhancer-promoter interactions in the transgene