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Gen Comp Endocrinol
2022 Sep 15;326:114072. doi: 10.1016/j.ygcen.2022.114072.
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Impaired negative feedback and death following acute stress in glucocorticoid receptor knockout Xenopus tropicalis tadpoles.
Paul B
,
Sterner ZR
,
Bhawal R
,
Anderson ET
,
Zhang S
,
Buchholz DR
.
Abstract Blood glucocorticoid levels are regulated by the hypothalamo-pituitary-adrenal/interrenal axis (HPA axis in mammals, HPI axis in amphibians), and negative feedback by glucocorticoid signaling is a key player in that regulation. Glucocorticoid and mineralocorticoid receptors (GR and MR) mediate negative feedback in mammals, but little is known about nuclear receptor-mediated feedback in amphibians. Because amphibians have only one corticosteroidogenic cell type responsible for glucocorticoid and mineralocorticoid production, we hypothesized that GR knockout (GRKO) tadpoles have elevated levels of glucocorticoids and mineralocorticoids as well as axis components regulating their production. We also examined the response to stress and potential for increased aldosterone signaling in GRKO tadpoles. We found that GRKO tadpoles have severe hyperactivity of the HPI axis, namely high mRNA expression levels of pomc, cyp17a1, cyp21a2, cyp11b2, and star, and high tissue content of corticosterone, aldosterone, 17-hydroxyprogesterone, 21-deoxycortisol, and progesterone. Such aberrant HPI activity was accompanied by reduced survival after acute temperature shock and shaking stress. Like mammalian models of HPA hyperactivity, GRKO tadpoles have high MR mRNA expression levels in brain, kidney, heart, and skin and high levels of the inflammatory cytokine tnf-α and the profibrotic factor tgf-β in kidneys. This study showed GR is critical for negative feedback to the amphibian HPI axis and for survival from acute stressors. This study also showed GRKO tadpoles exhibit altered expression/overproduction of regulators of salt-water homeostasis and associated biomarkers of kidney disease.
Fig. 1. Dysregulated HPI axis in GRKO tadpoles. Total RNA was collected from brains and kidneys of wild-type and GRKO metamorphic tadpoles to analyze mRNA expression of A) corticotropin releasing hormone (crh), B) arginine-vasopressin (avp), C) proopiomelanocortin (pomc), D) steroidogenic acute regulatory protein (star), E) 21-hydroxylase (cyp21a2), F) 17-hydroxylase (cyp17a1), and G) aldosterone synthase (cyp11b2). Bars represent mean mRNA levels relative to a wild-type sample and normalized by the housekeeping gene rpl8. n = 10–12 brain and kidney samples per genotype. Tadpole tails (∼100 mg each) were harvested to measure H) progesterone, I) 17-hydroxyprogesterone (17-OHP), J) 21-deoxycortisol, K) CORT, and L) ALDO via LC-MS/MS in wild-type (WT, white bars), and glucocorticoid receptor knockout (GRKO, black bars) F2 tadpoles at Nieuwkoop and Faber (NF) stage 61 (metamorphic climax). n = 10 tails/genotype. Error bars represent SE. Letters indicate significant groups, p < 0.05.
Fig. 2. GRKO tadpoles have altered MR expression levels. Total RNA was collected from A) brain, B) kidney, C) skin, D) heart, and E) tail of wild-type (WT, white bars) and glucocorticoid receptor knockout (GRKO, black bars) F2 tadpoles at Nieuwkoop and Faber (NF) stage 61 to analyze mRNA expression of mineralocorticoid receptor (MR). Bars represent mean mRNA levels relative to a wild-type sample and normalized by the housekeeping gene rpl8. n = 10–12 brain, kidney, and skin samples per genotype; n = 7 hearts/genotype; n = 10 tails/genotype. Error bars represent SE. Letters indicate significant groups, p < 0.05.
Fig. 3. GRKO tadpole kidneys have altered kidney expression levels of profibrotic and inflammatory factors. Total RNA was collected from kidneys of wild-type (WT, white bars) and glucocorticoid receptor knockout (GRKO, black bars) F2 tadpoles at Nieuwkoop and Faber (NF) stage 61 to analyze mRNA expression of A) transforming growth factor beta (tnf-β), B) tumor necrosis factor alpha (tnf-α), and C) connective tissue growth factor (ctgf). Bars represent mean mRNA levels relative to a wild-type sample and normalized by the housekeeping gene rpl8. n = 10–12 kidney samples per genotype. Error bars represent SE. Letters indicate significant groups, p < 0.05.
