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

The protein klotho is required for the inhibitory effect of FGF23 on 25-hydroxyvitamin D₃ 1-α-hydroxylase (1-α-hydroxylase) and thus 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) production [1]. Klotho may further up-regulate renal epithelial Ca²⁺ channels [2] and down-regulate renal tubular phosphate transport [3]. Klotho-hypomorphic mice (kl/kl) suffer from severe tissue calcification leading to profound growth deficit, premature appearance of several age related disorders and dramatic shortening of life span [1,4]. Conversely, murine life span is extended by klotho overexpression [5]. In humans, klotho gene variants similarly impact on ageing and life span [6].

Chronic kidney disease (CKD) similarly leads to tissue calcification [7] with negative impact on life span [8]. In CKD tissue calcification is primarily a consequence of impaired phosphate excretion but is compounded by klotho deficiency [9] and affected by a klotho gene variant [10]. Vascular calcification is driven by an active pathophysiological process [11] with transition of vascular smooth muscle cells into an osteo- and chondrogenic phenotypes [12]. Osteogenic reprogramming is stimulated by hyperphosphatemia [13] and hyperaldosteronism [14].

CKD patients and kl/kl mice further suffer from acidosis [15,16], which may counteract CaHPO₄ precipitation [17,18]. Along those lines, tissue calcification of kl/kl mice could be prevented by induction of acidosis with acetazolamide [18]. In theory the acidosis could be aggravated by NH₄⁺ intake [19,20]. NH₄⁺ may further dissociate to H⁺ and the cell membrane permeable NH₃, which enters acidic compartments [21], binds H⁺ and is thus trapped as NH₄⁺ in those compartments [22] H⁺ binding of NH₃ is followed by alkalinization of the acidic cellular compartments [23].

Treatment of kl/kl mice with NH₄Cl halts tissue calcification and thus leads to reversal of growth deficit and marked increase of life span [24], effects attributed in large part to disruption of osteogenic signaling due to alkalinization of acidic cellular compartments [24]. The present study explored whether NH₄NO₃ is similarly able to favorably influence tissue calcification and survival of kl/kl mice.

All animal experiments were conducted according to German law for the welfare of animals and were approved by local authorities. Male and female klotho-hypomorphic mice (kl/kl) were compared to male and female wild-type mice (WT). The origin of the mice, breeding and genotyping were described previously [4]. The mice had access to either tap water or a solution of NH₄NO₃ (0.28 M) in tap water ad libitum and were fed a standard chow diet (Sniff, Soest, Germany). The NH₄NO₃ treatment started with the mating of the parental generation and was maintained from pregnancy and weaning until analysis of the mice. The offspring was separated from the mothers at the age of four weeks.

To obtain blood specimens, animals were lightly anesthetized and about 50 - 200 µl of blood was withdrawn into heparinized capillaries by puncturing the retro-orbital plexus. The plasma phosphate concentrations were determined utilizing a photometric method (FUJI FDC 3500i, Sysmex, Norsted, Germany). Blood gas and electrolyte analysis as well as the measurement of ionized calcium was performed with the EDAN i15 Blood Gas and Chemistry Analysis System (EDAN Instruments, Shenzen, China). An ELISA kit was employed to determine plasma 1,25(OH)-vitamin D₃ concentration (IDS, Boldon, UK) [25].

For histological analysis of trachea, lung, kidney, stomach, heart and vessels, tissues from male kl/kl mice (age 8 weeks) with or without treatment with NH₄NO₃ (0.28 M in drinking water) were embedded in paraffin, cut in 2-3 µm sections and stained with hematoxylin and eosin (H&E) and von Kossa [26].

Data are provided as means ± SEM, n represents the number of independent experiments. All data were tested for significance using ANOVA followed by posthoc analysis. For the life span experiments, SAS Jmp version 8.0.1 (SAS Institute Inc., Cary, NC, USA) was used. Only results with p < 0.05 were considered statistically significant.

The present study explored whether NH₄NO₃ treatment (0.28 M in tap water) influenced growth deficit, tissue calcification and survival of klotho-hypomorphic (kl/kl) mice. As shown in Fig. 1A, kl/kl mice were markedly smaller than corresponding wild-type mice (WT). Accordingly, the body weight was significantly less in kl/kl mice than in wild-type mice (Fig. 1B). Following NH₄NO₃ treatment (0.28 M in tap water), the body weight of kl/kl mice was significantly increased. Following NH₄NO₃ treatment the body weight was thus not significantly different between kl/kl mice and wild-type mice.

As illustrated in Fig. 2A, plasma 1,25(OH)₂D₃ concentration was significantly higher in kl/kl mice than in wild-type mice, a difference not significantly affected by NH₄NO₃ treatment. Similarly, plasma phosphate concentration was significantly higher in kl/kl mice than in wild-type mice, a difference again not significantly affected by treatment with NH₄NO₃ (Fig. 2B). Blood ionized Ca²⁺ was again significantly higher in untreated kl/kl mice than in untreated wild-type mice, a difference again not significantly modified by NH₄NO₃ treatment (Fig. 2C). There were no differences in blood Na⁺ levels (Fig. 2D) between kl/kl mice and wild-type mice and between untreated and NH₄NO₃ treated animals. Plasma K⁺ concentration was significantly lower in untreated kl/kl mice than in untreated wild-type mice (Fig. 2E), a difference abolished by NH₄NO₃ treatment. There were no differences in blood Cl^- concentrations between kl/kl mice and wild-type mice (Fig. 2F). NH₄NO₃ treatment significantly increased blood Cl^- concentrations in both, kl/kl mice and wild-type mice (Fig. 2F).

As illustrated in Fig. 3A, blood pH was lower in untreated kl/kl mice than in untreated wild-type mice. NH₄NO₃ treatment significantly decreased blood pH in wild-type mice. NH₄NO₃ treatment tended to decrease blood pH in kl/kl mice, an effect, however, not reaching statistical significance (Fig. 3A). As shown in Fig. 3B, blood pCO₂ was significantly increased in kl/kl mice and significantly decreased to normal values under treatment with NH₄NO₃. Plasma HCO₃^- concentration was significantly higher in untreated kl/kl mice than in untreated wild-type mice, a difference again significantly blunted by NH₄NO₃ treatment (Fig. 3 C,D).

Histology was employed to visualize tissue calcification and histopathology in untreated and NH₄NO₃ treated male kl/kl mice. As illustrated in Fig. 4, excessive calcification was observed in trachea, lung, kidney, and stomach of kl/kl mice. Moreover, histology revealed marked emphysema of lung tissue from kl/kl mice. NH₄NO₃ treatment strongly reduced the tissue calcification and reversed the emphysema of kl/kl mice. As shown in Fig. 5, excessive calcifications were similarly observed in cardiac and vascular tissue of untreated kl/kl mice. Again, NH₄NO₃ treatment blunted the calcification of cardiac and vascular tissue from kl/kl mice.

As illustrated in Fig. 6, NH₄NO₃ treatment was followed by a substantial and significant (Log-Rang p < 0.0004; Wilcoxon p < 0.0016) increase of the life span of male kl/kl mice. Half of untreated kl/kl mice survived 72 days and half of the NH₄NO₃ treated kl/kl mice survived 122 days. Accordingly, NH₄NO₃ treatment extended the median life span of kl/kl mice by a factor of 1.7.

The present study confirms the phenotype of kl/kl mice, i.e. the severe tissue calcification, growth deficit and accelerated aging presumably resulting from unrestricted formation of 1,25(OH)₂D₃ with subsequent marked increase of Ca²⁺ and phosphate concentrations leading to triggering of osteogenic signaling [1]. The present study further reveals that NH₄NO₃ treatment mitigates tissue calcification of kl/kl mice, leading to almost complete reversal of growth deficit, attenuation of tissue injury and substantial extension of life span. All those effects mimic those of NH₄Cl treatment, which has previously been shown to disrupt the excessive osteogenic signaling of kl/kl mice [24].

The tissue calcification of kl/kl mice is most likely due to excessive extracellular phosphate concentrations, which are known to stimulate osteogenic signaling and vascular calcification [13], which is in turn a hallmark of aging [7,27,28,29]. Along those lines plasma phosphate concentration is a determinant of mortality [30]. NH₄NO₃ treatment mitigates tissue calcification in kl/kl mice without significantly modifying plasma 1,25(OH)₂D₃, Ca²⁺ and phosphate concentrations. NH₄NO₃ treatment is further effective despite virtually constant extracellular pH, another determinant of calcification [18,31,32,33].

NH₄NO₃ treatment does not aggravate the acidosis of kl/kl mice, which is due to excessive pCO₂ presumably due to the severe lung emphysema (Fig. 4). The NH₄NO₃ treatment reverses the hypercapnia presumably by counteracting the development of lung emphysema (Fig. 4). At the same time, however, NH₄NO₃ treatment decreases plasma bicarbonate concentration, presumably due to partial incorporation of NH₄⁺ into urea, a metabolic pathway consuming HCO₃^-[34]. Following NH₄NO₃ treatment bicarbonate and pCO₂ decline in parallel leaving extracellular pH virtually unchanged. Accordingly, NH₄NO₃ treatment converts the respiratory acidosis of untreated kl/kl mice into a metabolic acidosis.

Similar to the excessive osteogenic signaling in kl/kl mice [24,35], enhanced osteogenic signaling leads to vascular calcification in CKD patients [35,36]. In CKD vascular calcification increases the risk for cardiovascular events [37], the leading cause of death in this clinical condition [38]. Similar to the osteogenic signaling in kl/kl mice, osteogenic signaling in CKD patients [39] is secondary to hyperphosphatemia and is compounded by decrease of klotho expression [35]. Disruption of osteogenic reprogramming in vascular tissue is thus expected to favourably influence the clinical course of CKD [39].

Treatment with NH₄NO₃ decreases tissue and vascular calcification, reverses the growth deficit and substantially extends the life span of klotho-hypomorphic mice despite continued increase of plasma 1,25(OH)₂D₃, Ca²⁺ and phosphate concentrations. In view of the previous observations following NH₄Cl treatment, NH₄NO₃ treatment is presumably effective by disrupting osteogenic signaling.

The authors state that they do not have any conflicts of interest to disclose.

The authors acknowledge the technical assistance of Dennis Thiele, Elfriede Faber and the meticulous preparation of the manuscript by Tanja Loch. The study was supported by the Deutsche Forschungsgemeinschaft (DFG 315/15-1).