1,25(OH)2D3 dependent overt hyperactivity phenotype in klotho-hypomorphic mice

Klotho, a protein mainly expressed in kidney and cerebral choroid plexus, is a powerful regulator of 1,25(OH)2D3 formation. Klotho-deficient mice (kl/kl) suffer from excessive plasma 1,25(OH)2D3-, Ca(2+)- and phosphate-concentrations, leading to severe soft tissue calcification and accelerated aging. NH4Cl treatment prevents tissue calcification and premature ageing without affecting 1,25(OH)2D3-formation. The present study explored the impact of excessive 1,25(OH)2D3 formation in NH4Cl-treated kl/kl-mice on behavior. To this end kl/kl-mice and wild-type mice were treated with NH4Cl and either control diet or vitamin D deficient diet (LVD). As a result, plasma 1,25(OH)2D3-, Ca(2+)- and phosphate-concentrations were significantly higher in untreated and in NH4Cl-treated kl/kl-mice than in wild-type mice, a difference abrogated by LVD. In each, open field, dark-light box, and O-maze NH4Cl-treated kl/kl-mice showed significantly higher exploratory behavior than untreated wild-type mice, a difference abrogated by LVD. The time of floating in the forced swimming test was significantly shorter in NH4Cl treated kl/kl-mice compared to untreated wild-type mice and to kl/kl-mice on LVD. In wild-type animals, NH4Cl treatment did not significantly alter 1,25(OH)2D3, calcium and phosphate concentrations or exploratory behavior. In conclusion, the excessive 1,25(OH)2D3 formation in klotho-hypomorphic mice has a profound effect on murine behavior

Partial Reversal of Tissue Calcification and Extension of Life Span following Ammonium Nitrate Treatment of Klotho-Deficient Mice

Background/Aims: Klotho is required for the inhibitory effect of FGF23 on 1,25(OH)2D3 formation and Klotho-hypomorphic mice (kl/kl) suffer from severe tissue calcification due to excessive 1,25(OH)2D3 formation with subsequent increase of Ca2+ and phosphate concentrations and stimulation of osteogenic signaling. The excessive tissue calcification dramatically accelerates aging and leads to premature death of the animals. Osteogenic signaling in those mice is disrupted by treatment with NH4Cl, which prevents tissue calcification and early death of kl/kl mice. The present study explored whether the beneficial effects of NH4Cl treatment could be mimicked by NH4NO3 treatment. Methods: The kl/kl mice had free access to tap water either without or with addition of NH4NO3 (0.28 M) starting with the mating of the parental generation. Calcification of trachea, lung, kidney, stomach, heart and vessels was visualized by histology with von Kossa staining. Plasma phosphate concentration was determined utilizing photometry, blood gas and electrolytes utilizing a blood Gas and Chemistry Analysis System and plasma 1,25(OH)2D3 concentration with ELISA. Results: In untreated kl/kl mice plasma 1,25(OH)2D3 and phosphate concentrations were elevated, and the mice suffered from marked calcification of all tissues analyzed. Untreated kl/kl mice further suffered from respiratory acidosis due to marked lung emphysema. NH4NO3-treatment decreased both, blood pCO2 and HCO3-, decreased calcification of trachea, lung, kidney, stomach, heart and vessels and increased the life span of kl/kl mice more than 1.7-fold (♂) or 1.6-fold (♀) without significantly affecting extracellular pH or plasma concentrations of 1,25(OH)2D3, Ca2+, phosphate, Na+, and K+. Conclusions: NH4NO3-treatment turns respiratory acidosis into metabolic acidosis and mitigates calcification thus leading to a substantial extension of kl/kl mice survival

NH4Cl Treatment Prevents Tissue Calcification in Klotho Deficiency

Klotho, a cofactor in suppressing 1,25(OH)(2)D(3) formation, is a powerful regulator of mineral metabolism. Klotho-hypomorphic mice (kl/kl) exhibit excessive plasma 1,25(OH)(2)D(3), Ca(2+), and phosphate concentrations, severe tissue calcification, volume depletion with hyperaldosteronism, and early death. Calcification is paralleled by overexpression of osteoinductive transcription factor Runx2/Cbfa1, Alpl, and senescence-associated molecules Tgfb1, Pai-1, p21, and Glb1. Here, we show that NH(4)Cl treatment in drinking water (0.28 M) prevented soft tissue and vascular calcification and increased the life span of kl/kl mice >12-fold in males and >4-fold in females without significantly affecting extracellular pH or plasma concentrations of 1,25(OH)(2)D(3), Ca(2+), and phosphate. NH(4)Cl treatment significantly decreased plasma aldosterone and antidiuretic hormone concentrations and reversed the increase of Runx2/Cbfa1, Alpl, Tgfb1, Pai-1, p21, and Glb1 expression in aorta of kl/kl mice. Similarly, in primary human aortic smooth muscle cells (HAoSMCs), NH(4)Cl treatment reduced phosphate-induced mRNA expression of RUNX2/CBFA1, ALPL, and senescence-associated molecules. In both kl/kl mice and phosphate-treated HAoSMCs, levels of osmosensitive transcription factor NFAT5 and NFAT5-downstream mediator SOX9 were higher than in controls and decreased after NH(4)Cl treatment. Overexpression of NFAT5 in HAoSMCs mimicked the effect of phosphate and abrogated the effect of NH(4)Cl on SOX9, RUNX2/CBFA1, and ALPL mRNA expression. TGFB1 treatment of HAoSMCs upregulated NFAT5 expression and prevented the decrease of phosphate-induced NFAT5 expression after NH(4)Cl treatment. In conclusion, NH(4)Cl treatment prevents tissue calcification, reduces vascular senescence, and extends survival of klotho-hypomorphic mice. The effects of NH(4)Cl on vascular osteoinduction involve decrease of TGFB1 and inhibition of NFAT5-dependent osteochondrogenic signaling

Impact of bicarbonate, ammonium chloride, and acetazolamide on hepatic and renal SLC26A4 expression

SLC26A4 encodes pendrin, a transporter exchanging anions such as chloride, bicarbonate, and iodide. Loss of function mutations of SLC26A4 cause Pendred syndrome characterized by hearing loss and enlarged vestibular aqueducts as well as variable hypothyroidism and goiter. In the kidney, pendrin is expressed in the distal nephron and accomplishes HCO(3)(-) secretion and Cl(-) reabsorption. Renal pendrin expression is regulated by acid-base balance. The liver contributes to acid-base regulation by producing or consuming glutamine, which is utilized by the kidney for generation and excretion of NH(4)(+), paralleled by HCO(3)(-) formation. Little is known about the regulation of pendrin in liver. The present study thus examined the expression of Slc26a4 in liver and kidney of mice drinking tap water without or with NaHCO(3) (150 mM), NH(4)Cl (280 mM) or acetazolamide (3.6 mM) for seven days. As compared to Gapdh transcript levels, Slc26a4 transcript levels were moderately lower in liver than in renal tissue. Slc26a4 transcript levels were not significantly affected by NaHCO(3) in liver, but significantly increased by NaHCO(3) in kidney. Pendrin protein expression was significantly enhanced in kidney and reduced in liver by NaHCO(3). Slc26a4 transcript levels were significantly increased by NH(4)Cl and acetazolamide in liver, and significantly decreased by NH(4)Cl and by acetazolamide in kidney. NH(4)Cl and acetazolamide reduced pendrin protein expression significantly in kidney, but did not significantly modify pendrin protein expression in liver. The observations point to expression of pendrin in the liver and to opposite effects of acidosis on pendrin transcription in liver and kidney