Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA357

Poster Communications

Efficient ablation of renin cells in adult kidney using inducible transgenic mouse models, evaluated by a novel automatic histological quantification (FACS on a slide)

A. Steglich1, F. Kessel1, M. Gerlach1, C. Hugo1, V. Todorov1

1. Department of Internal Medicine III, Division of Nephrology, University Hospital Carl Gustav Carus, Dresden, Germany.

In previous experiments, we demonstrated that an inducible knockout of Gsα in renin-producing cells (RPCs) leads to reduced renin production and kidney damage (1). It remained unknown to what extent the RPCs and renin per se are necessary for the maintenance of adult kidney function and morphology. To differentiate between renin deficiency and absence of RPCs, we generated two transgenic mouse models. We crossed our inducible RPC tdTomato-reporter strain (mRen-rtTAm2-LC1-tdT; wildtype controls, WT) and with Gsα-floxed mice (mRen-rtTAm2-LC1-tdT-Gsα; reduced renin production) and DTA mice (mRen-rtTAm2-LC1-tdT-DTA; RPC ablation upon inducible expression of diphtheria toxin A). Adult mice received continuously doxycycline to induce recombination (water: 2mg/ml doxycycline, 10 mg/kg enalapril, 5% saccharose for 18 days; afterwards chow: 625mg/kg doxycycline). Four and twelve weeks after start of induction kidneys were harvested (uninephrectomy/ end biopsy, inhalation narcosis: 2-3% isoflurane) for immunohistochemistry or FACS analysis to evaluate the induction efficiency. Automatic histological analysis of whole kidney sections (up to 100 000 cells) was performed with new algorithms based on Fiji and R software. FACS revealed reduced number of renin positive cells in both transgenic strains compared to WT mice (four weeks: WT 0.15±0.01%, Gsα 0.08±0.03%, DTA 0.05±0.01%; twelve weeks: WT 0.13±0.02%, Gsα 0.03±0.01%, DTA 0.04±0.004%; p<0.05). Evaluation of the knockout efficiency by tdT-positive cells displayed comparable decrease in DTA mice, but no change at the later time point in Gsα mice (four weeks: WT 0.17±0.03%, Gsα 0.09±0.03%, DTA 0.02±0.004%; twelve weeks: WT 0.13±0.02%, Gsα 0.13±0.03%, DTA 0.01±0.01%; p<0.05). This discrepancy was expected because tdT-positive renin cells are continuously recruited in both strains, while cells in which recombination already occurred, persist in Gsα mice (as tdT-positive renin-negative cells) but are ablated in DTA mice. Automatic histological analysis showed similar results for renin (four weeks WT 0.09±0.01%, Gsα 0.02±0.01%, DTA 0.03±0.01%; twelve weeks WT 0.10±0.01%, Gsα 0.02±0.01%, DTA 0.02±0.004%; p<0.001) and tdT (four weeks WT 0.31±0.03%, Gsα 0.08±0.03%, DTA 0.02±0.01%; twelve weeks WT 0.22±0.03%, Gsα 0.27±0.09%, DTA 0.05±0.02%; p<0.05). Data are means±SEM, n=5-9. Statistical test: 2-way ANOVA. Using two independent methods and two different readouts we confirmed the high efficiency of our inducible mouse model for targeting renin cells (71±8% in Gsα and 88±4% in DTA mice). The novel automatic histological analysis is equally reliable to FACS and has the advantage to require less tissue for quantification.

Where applicable, experiments conform with Society ethical requirements