Comparing the Bbs10 complete knockout phenotype with a specific renal epithelial knockout one highlights the link between renal defects and systemic inactivation in mice

Abstract

Background: Bardet-Biedl Syndrome (BBS) is a genetically heterogeneous ciliopathy with clinical cardinal features including retinal degeneration, obesity and renal dysfunction. To date, 20 BBS genes have been identified with BBS10 being a major BBS gene found to be mutated in almost 20 percent of all BBS patients worldwide. It codes for the BBS10 protein which forms part of a chaperone complex localized at the basal body of the primary cilium. Renal dysfunction in BBS patients is one of the major causes of morbidity in human patients and is associated initially with urinary concentration defects related to water reabsorption impairment in renal epithelial cells. The aim of this study was to study and compare the impact of a total Bbs10 inactivation (Bbs10 (-/-)) with that of a specific renal epithelial cells inactivation (Bbs10 (fl/fl) ; Cdh16-Cre (+/-)).

Results: We generated the Bbs10 (-/-) and Bbs10 (fl/fl) ; Cadh16-Cre (+/-) mouse model and characterized them. Bbs10 (-/-) mice developed obesity, retinal degeneration, structural defects in the glomeruli, polyuria associated with high circulating arginine vasopressin (AVP) concentrations, and vacuolated, yet ciliated, renal epithelial cells. On the other hand, the Bbs10 (fl/fl) ; Cadh16-Cre (+/-)mice displayed no detectable impairment.

Conclusions: These data highlight the importance of a systemic Bbs10 inactivation to trigger averted renal dysfunction whereas a targeted absence of BBS10 in the renal epithelium is seemingly non-deleterious.

Keywords: Bardet–Biedl Syndrome; Bbs10; Obesity; Renal epithelial-specific KO; Retinal degeneration; Total knockout.

Fig. 1

Generating the Bbs10 ^−/− mouse model. a Schematic representation of the targeting strategy for Bbs10 allele. b Genotyping PCR results for homozygous knock out (Bbs10 ^−/−), heterozygous (Bbs10 ^+/−), wild-type (Bbs10 ^+/+) mice. PCR bands at 475 and 238 bp correspond to the excised allele and to the natural allele, respectively. (bp base pairs). c Relative mRNA expression levels of Bbs10 in kidneys and the eyes of Bbs10 ^+/+ and Bbs10 ^−/−. Reference gene Gapdh (n = 4 per genotype).

Fig. 2

Obesity-associated phenotype in Bbs10 ^−/− mice. a Growth curve of Bbs10 ^+/+ and Bbs10 ^−/− (n = 8). b Photograph of 3-month-old Bbs10 ^+/+ and Bbs10 ^−/− mice. c Food intake measurement of Bbs10 ^+/+ and Bbs10 ^−/− mice (n = 8 per genotype). d Measurement of the circulating concentrations of leptin in Bbs10 ^+/+ and Bbs10 ^−/− (n = 6 per group). e Pictures of AdipoRed-stained visceral adipocytes from Bbs10 ^+/+ and Bbs10 ^−/−. Scale bar 50 µm. Data are expressed as mean ± SEM. f Glucose tolerance test (GTT) and g insulin tolerance test (ITT) for the same mice (n = 7 per group). Values are expressed as mean ± SEM. *p < 0.05.

Fig. 3

Retinal phenotype in Bbs10 ^−/− mice. a Optical coherence tomography (OCT) pictures of retinas from Bbs10 ^+/+ and Bbs10 ^−/− mice. (OS outer segment, IS inner segment, ONL outer nuclear layer) (n = 3 mice per genotype). b Toluidine blue-stained pictures of 7-µm-thick retinal sections from Bbs10 ^+/+ and Bbs10 ^−/− mice (OS outer segment, IS inner segment, ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer) Scale bar 20 µm. c Immunofluorescence of 7-µm-thick retinal sections from Bbs10 ^+/+ and Bbs10 ^−/− mice. Nuclei were counterstained with DAPI (blue), rhodopsin (green), acetylated a-tubulin (red). d Transmission electron microscopy (T.E.M.) pictures of retinal sections in Bbs10 ^+/+ and Bbs10 ^−/− mice (ROS retinal outer segment, RIS retinal inner segment, RPE retinal pigment epithelium). Red arrows indicate the centrioles and the connecting cilium. e Scotopic electroretinograms (ERG) recordings of 2- and 3-month-old Bbs10 ^+/+ and Bbs10 ^−/− mice. Red arrows indicate a-waves on the ERG recordings (n = 6–8 per genotype).

Fig. 4

Renal phenotype of the Bbs10 ^−/− mice and Bbs10 ^fl/fl ; Cadh16-Cre ^+/− mice. a Pictures of hematoxylin and eosin-stained kidney sections of 3-month-old Bbs10 ^+/+ and Bbs10 ^−/− mice. b Transmission electron microscopy picture of kidney sections of the glomerular region from Bbs10 ^+/+and Bbs10 ^−/− mice (P podocyte, BM basement membrane). Scale bars 5 and 2 µm. c Microalbuminuria levels from Bbs10 ^+/+ and Bbs10 ^−/− mice. d Creatinine clearance levels (µL/min) and urinary volume under fluid deprivation of Bbs10 ^+/+ and Bbs10 ^−/− mice (n = 7–9). Values are expressed as mean ± SEM. e Transmission electron microscopy picture of kidney epithelial cells from Bbs10 ^+/+, Bbs10 ^−/− mice. Red arrows indicate the centrioles and the connecting cilium. f Transmission electron microscopy picture of kidney tubular cells from Bbs10 ^+/+, Bbs10 ^−/− mice. Red arrows indicate cytoplasmic vacuoles, A apical side, B Basolateral side. g Pictures of immunostained kidney sections for AQP2 (green) and β-tubulin (red) from Bbs10 ^+/+and Bbs10 ^−/− mice. Nuclei were counterstained with DAPI (blue). Scale bars 20 µm. Values are expressed as mean ± SEM. *p < 0.05

Fig. 5

Renal phenotype of the Bbs10 ^fl/fl ; Cadh16-Cre ^+/− mice. a PCR genotyping of heterozygous WT (Bbs10 ^+/+) (Bbs10 ^fl/+), floxed (Bbs10 ^fl/fl) mice non-Cre (Cre ^−/−) and heterozygous (Cre ^+/−) genotyping (bp base pairs). b 3D image of a kidney section immunostained for Cre (green) and nuclei counterstained with DAPI (blue). c 3D image of a kidney section immunostained for the primary cilia (acetylated α-tubulin in red), cytoskeleton (β-tubulin in green) and nuclei counterstained with DAPI (in blue). d Pictures of immunostained kidney sections for AQP2 (green) and β-tubulin (red) from Bbs10 ^fl/fl ; Cadh16Cre ^+/− mice. Nuclei were counterstained with DAPI (blue). Scale bars 20 µm. e T.E.M. analysis of epithelial cells in the proximal tubule from Bbs10 ^+/+ ; Cadh16Cre ^+/−and Bbs10 ^fl/fl ; Cadh16Cre ^+/− mice. BM basement membrane. f Microalbuminuria levels for of Bbs10 ^fl/fl ; Cadh16Cre ^+/− and control Bbs10 ^+/+ ; Cadh16Cre ^+/− littermates (n = 6–7 per genotype). g T.E.M. analysis of epithelial cells in the distal tubule from Bbs10 ^+/+ ; Cadh16Cre ^+/− and Bbs10 ^fl/fl ; Cadh16Cre ^+/− mice. h Creatinine clearance levels (µL/min) and urinary volume under fluid deprivation of Bbs10 ^fl/fl ; Cadh16Cre ^+/− and control Bbs10 ^+/+ ; Cadh16Cre ^+/− littermates (n = 7–9). Values are expressed as mean ± SEM. *p < 0.05.

Fig. 6

Bbs10^−/− mice but not Bbs10 ^fl/fl ; Cadh16-Cre ^+/− mice display polyuria associated with increased circulating AVP concentrations. a Urinary volume (µL) under 24-h fluid deprivation of Bbs10 ^+/+ and Bbs10 ^−/− mice (n = 6 per genotype). b Relative mRNA expression levels of Aqp2, Aqp3 and Avpr2 in Bbs10 ^+/+ and Bbs10 ^−/− kidneys. Gene of reference Gapdh. c Entire photographs of immunoblots for AQP2, AVPR2 and β-tub in kidneys of Bbs10 ^+/+ and Bbs10 ^−/−. d AVP plasma levels in Bbs10 ^+/+ and Bbs10 ^−/− under 24-h fluid restriction or in normal conditions. (n = 5 per genotype). e Urinary volume (µL) under 24-h fluid deprivation of Bbs10 ^fl/fl ; Cadh16Cre ^+/− and control Bbs10 ^+/+ ; Cadh16Cre ^+/− littermates (n = 6 per genotype). f Relative mRNA expression levels of Aqp2, Aqp3 and Avpr2 in Bbs10 ^+/+ ; Cadh16Cre ^+/− and Bbs10 ^fl/fl ; Cadh16Cre ^+/−. g Entire photographs of the immunoblots for AQP2, AVPR2 and β-tubulin (β-tub) in kidneys of Bbs10 ^+/+ ; Cadh16Cre ^+/− and Bbs10 ^fl/fl ; Cadh16Cre ^+/−; the immunoblots are shown in Additional file 4: Figure S4A, B. h AVP plasma levels in Bbs10 ^fl/fl ; Cadh16Cre ^+/− and control Bbs10 ^+/+ ; Cadh16Cre ^+/− under 24-h fluid restriction or in normal conditions. (n = 5 per genotype). Values are expressed as mean ± SEM. *p < 0.05.