Vitamin D3 suppresses Npt2c abundance and differentially modulates phosphate and calcium homeostasis in Npt2a knockout mice

Vitamin D3 is clinically used for the treatment of vitamin D3 deficiency or osteoporosis, partially because of its role in regulating phosphate (Pi) and calcium (Ca2+) homeostasis. The renal sodium-phosphate cotransporter 2a (Npt2a) plays an important role in Pi homeostasis; however, the role of vitamin D3 in hypophosphatemia has never been investigated. We administered vehicle or vitamin D3 to wild-type (WT) mice or hypophosphatemic Npt2a−/− mice. In contrast to WT mice, vitamin D3 treatment increased plasma Pi levels in Npt2a−/− mice, despite similar levels of reduced parathyroid hormone and increased fibroblast growth factor 23. Plasma Ca2+ was increased ~ twofold in both genotypes. Whereas WT mice were able to increase urinary Pi and Ca2+/creatinine ratios, in Npt2a−/− mice, Pi/creatinine was unchanged and Ca2+/creatinine drastically decreased, coinciding with the highest kidney Ca2+ content, highest plasma creatinine, and greatest amount of nephrocalcinosis. In Npt2a−/− mice, vitamin D3 treatment completely diminished Npt2c abundance, so that mice resembled Npt2a/c double knockout mice. Abundance of intestinal Npt2b and claudin-3 (tight junctions protein) were reduced in Npt2a−/− only, the latter might facilitate the increase in plasma Pi in Npt2a−/− mice. Npt2a might function as regulator between renal Ca2+ excretion and reabsorption in response to vitamin D3.


Vitamin D 3
Active vitamin D 3 or 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) is produced via the combined actions of skin, liver and kidneys 1 .Under normal conditions, it plays an essential role in the regulation of calcium (Ca 2+ ) and phosphate (P i ) homeostasis in the body 2 .A lack of vitamin D 3 can lead to rickets and other potential processes beyond bone health, including immune system dysregulation, development of cancer, or progression of cardiovascular disease 3 .The actions of vitamin D 3 are complex and involve hormones such as parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) [4][5][6] .PTH and FGF23 are both phosphaturic hormones (via action on the renal Na + -P i transporters, Npt2a and Npt2c) which work collaboratively to maintain P i homeostasis; however, this process involves a complicated regulatory role of 1,25(OH) 2 D 3 4,7 .1,25(OH) 2 D 3 has opposing effects on these hormones: it enhances the production of FGF23 in bone, while simultaneously suppressing the synthesis of PTH 8,9 .In this complex feedback loop, where PTH and FGF23 normally promote renal P i excretion, 1,25(OH) 2 D 3 may act as a switch between P i excretion/absorption in order to maintain total body P i 4 .The precursor of vitamin D 3 , previtamin D 3 , is formed by the skin and subsequently undergoes spontaneous isomerization to vitamin D 3 , which has a half-life of ~ 26 h 10 .Any excess vitamin D 3 is stored mainly within fat tissue 11 .In the liver, an initial hydroxylation step takes place which converts vitamin D 3 into 25(OH) vitamin D 3 .(d).Plasma Ca 2+ levels in both WT and Npt2a −/− mice showed a significant increase following vitamin D 3 treatment (e & f).In WT mice, the urinary Ca 2+ to creatinine ratio significantly increased after vitamin D 3 treatment (g).In contrast, this ratio significantly decreased in Npt2a −/− mice (h).Male mice were used in these studies.In addition to single data summary data are shown and are expressed as mean ± SEM and were analyzed by repeated-measures two-way ANOVA followed by Tukey's multiple comparisons test.*P < 0.05 vs WT same time point, # P < 0.05 vs baseline same genotype, § P < 0.05 vs vehicle same genotype and time point.
Npt2a −/− mice lack PTH responses but FGF23 levels were significantly increased in response to vitamin D 3 treatment Npt2a −/− mice show significantly lower plasma PTH levels under baseline conditions (Fig. 2a,b).In WT mice, plasma PTH showed a small but significant decrease in response to vehicle treatment (Fig. 2a).Vitamin D 3 treatment significantly decreased (~ 85%) plasma PTH (Fig. 2a) in WT mice.No significant changes in plasma PTH were observed in response to vehicle or vitamin D 3 treatment in Npt2a −/− mice (Fig. 2b).In addition to lower PTH levels in Npt2a −/− mice under baseline conditions, FGF23 levels were also significantly lower (~ 50%) compared to WT mice (Fig. 2c,d).Vehicle treatment did not significantly change FGF23 levels in either genotype (Fig. 2c,d).Vitamin D 3 treatment caused a significant increase of FGF23 levels in both genotypes: in WT mice an ~ 80-fold increase was observed, whereas in Npt2a −/− mice a ~ 200-fold increase was observed.The more than double increase of FGF23 in Npt2a −/− compared to WT mice in response to vitamin D 3 treatment is the consequence of the significantly lower baseline levels because FGF23 levels were not significantly different in response to vitamin D 3 treatment between genotypes.Vitamin D 3 has distinct effects on bone remodeling markers in WT and Npt2a −/− mice Since vitamin D 3 is instrumental for bone remodeling, we determined 2 bone formation markers (osteocalcin and PINP) and 2 bone resorption markers (TRAcP 5b and CTX-1) under baseline conditions and in response to vehicle or vitamin D 3 treatment.Under baseline conditions, no significant differences were observed in osteocalcin levels between genotypes, and vehicle and vitamin D 3 treatment resulted in a small but significant increase in osteocalcin levels independent of genotype (Fig. 3a,b).Plasma PINP levels were not significantly different between genotypes under baseline conditions, and vehicle treatment did not significantly change PINP levels in either genotype (Fig. 3c,d).Vitamin D 3 treatment significantly reduced (~ 60%) PINP levels in WT mice, and a similar reduction (~ 44%) was observed in Npt2a −/− mice.Plasma TRAcP 5b levels were not significantly different between genotypes under baseline conditions, and in both genotypes TRAcP 5b levels slightly but significantly decreased in response to vehicle treatment.Vitamin D 3 treatment significantly increased (~ 1.5-fold) TRAcP 5b levels in WT mice but was without significant effect in Npt2a −/− mice (Fig. 3e,f).Plasma CTX-1 levels were not significantly different between genotypes under baseline conditions, and vehicle treatment did not significantly affect CTX-1 levels in either genotype (Fig. 3g,h).Vitamin D 3 treatment significantly increased (~ twofold) CTX-1 levels in WT mice and even further increased (~ 3.5-fold) CTX-1 in Npt2a −/− mice.Consequently, CTX-1 levels were significantly higher in Npt2a −/− compared to WT mice after vitamin D 3 treatment (Fig. 3g,h).

Acute oral P i loading results in greater plasma P i levels in response to vitamin D 3 treatment compared to vehicle
In vehicle-treated WT and Npt2a −/− mice, acute oral P i loading resulted in a significant increase in plasma P i levels (2.4 ± 0.1 and 3.1 ± 0.1 mmol L −1 , respectively).In vitamin D 3 -treated WT and Npt2a −/− mice, acute oral P i loading resulted in a significantly greater increase in plasma P i levels (3.9 ± 0.2 and 4.2 ± 0.3 mmol L −1 , respectively) compared to their respective vehicle-treated genotype (Fig. 4).Vitamin D 3 treatment causes significant Ca 2+ accumulation in the kidney of Npt2a −/− mice Determination of Ca 2+ and P i amounts were conducted on ashed tissue from vehicle and vitamin D 3 -treated WT and Npt2a −/− mice.Amounts of P i or Ca 2+ in bone were not significantly different between genotypes in response to vehicle or vitamin D 3 treatment (Fig. 5A,B).Similarly, kidney P i levels were not significantly different between genotypes in response to vehicle or vitamin D 3 treatment (Fig. 5c).Of note, kidney Ca 2+ levels in response to vehicle treatment were significantly greater (~ threefold) in Npt2a −/− compared to WT mice (Fig. 5d).Kidney Ca 2+ levels were not significantly different between vehicle-and vitamin D 3 -treated WT mice.In contrast, vitamin D 3 treatment in Npt2a −/− mice resulted in the highest kidney Ca 2+ content observed between groups and genotypes and was ~ 3.5-fold greater compared to vitamin D 3 -treated WT mice.Plasma creatinine shows the highest levels in vitamin D 3 -treated Npt2a −/− mice, no differences were observed between the other groups (Fig. 6a).Urinary albumin/creatinine ratios also showed significantly increased levels in vitamin D 3 -treated Npt2a −/− mice compared to vehicle-treated Npt2a −/− mice (Fig. 6b).To visualize Ca 2+ -P i deposits, we used von Kossa staining and performed semi-quantitative analysis (Fig. 6c-g).Consistent with plasma creatinine levels, vitamin D 3 -treated Npt2a −/− mice show the most severe amount (> 50%) of crystal deposits in the tubular lumen.The tubules show evidence of damage with attenuation of the epithelial lining.

Npt2a maintains Npt2c and claudin-3 expression in response to vitamin D 3
Confirming the specificity of the Npt2a antibody, no Npt2a band was observed in kidney tissue of Npt2a −/− mice (Fig. 7a).In response to vitamin D 3 treatment in WT mice, Npt2a abundance was significantly lower (~ 65%) compared to vehicle-treated mice (Fig. 7a).Consistent with a compensatory response of Npt2c abundance in Npt2a −/− mice, Npt2c abundance was ~ 3.5-fold greater in vehicle-treated Npt2a −/− mice compared to vehicletreated WT mice (Fig. 7b).In response to vitamin D 3 treatment in WT mice, Npt2c abundance was significantly  www.nature.com/scientificreports/lower (~ 40%) compared to vehicle-treated WT mice.Of note, vitamin D 3 treatment completely diminished Npt2c abundance in Npt2a −/− mice.Since vitamin D 3 affects intestinal P i transport, we further analyzed intestinal abundance of Npt2b and claudin-3, the latter being a paracellular tight junction protein involved in P i transport 21 .The majority of intestinal P i uptake in the mouse occurs in the distal small intestine 22,23 .The abundance of Npt2b in the proximal small intestine was not significantly different between genotypes or treatments (Fig. 8a).No differences were observed in the abundance of Npt2b in the distal small intestine between genotypes in response to vehicle treatment.In WT mice (Fig. 8b), Npt2b abundance was not significantly different in response to vitamin D 3 compared to vehicle treatment.In contrast, Npt2b expression in the distal small intestine of Npt2a −/− mice was significantly lower (~ 77%) in response to vitamin D 3 compared to vehicle treatment (Fig. 8b).
In the proximal small intestine of WT mice, claudin-3 protein abundance was not significantly different between vehicle or treatment groups (Fig. 8c).In the distal small intestine, no significant differences were observed in claudin-3 abundance between genotypes in response to vehicle treatment (Fig. 8d) and in WT mice no significant differences were observed between vehicle and vitamin D 3 treatment.In contrast, in Npt2a −/− mice, claudin-3 protein abundance was significantly lower (~ 72%) in response to vitamin D 3 compared to vehicle treatment (Fig. 8d).

Discussion
The role of Npt2a in regulating renal P i transport has been extensively studied.However, there are significant knowledge gaps when it comes to the complex hormonal regulation of this transporter, in particular the role of vitamin D 3 .To gain further mechanistic insight, we studied P i and Ca 2+ homeostasis when the body is challenged by exogenous administration of a high dose of vitamin D 3 in the absence and presence of hyposphosphatemia, the latter caused by lack of Npt2a.Surprisingly, despite lack of Npt2a which should have facilitated renal P i excretion, these mice show signs of impaired P i excretion, possibly a results of greater nephrocalcinosis and a reduction of kidney function, in response to vitamin D 3 administration (for a summary see Fig. 9).
Lack of Npt2a causes hypophosphatemia 16,18,20 , a finding confirmed in the current study.A similar situation can be induced by administration of a Npt2a inhibitor 20,24,25 .In WT mice, the administration of vitamin D 3 did not affect plasma P i levels; in contrast, Npt2a −/− mice had a very uniform increase in plasma P i following administration of vitamin D 3. Regarding the former, other studies which administered vitamin D 3 at a dose of 400,000 (IU kg −1 ) to C57Bl/6 mice did not report changes in plasma P i levels 26 .It is conceivable that in WT mice, the increase in urinary P i /creatinine ratio served to stabilize plasma P i levels.Consistent with this, vitamin D 3 treatment resulted in lower Npt2a and Npt2c expression in WT mice.
Under baseline conditions, we did not find a clear P i wasting phenotype in Npt2a −/− mice.This could be related to a substantial compensatory greater Npt2c expression (seen on the protein but not mRNA level) which might www.nature.com/scientificreports/mitigate the P i wasting phenotype.It is notable that plasma P i significantly increased in response to vitamin D 3 treatment despite the absence of Npt2a.This corroborated with greater urinary albumin/creatinine ratios and greater plasma creatinine levels, possibly implying that reduced kidney function might have contributed to this finding.In addition, Npt2c abundance was also diminished in Npt2a −/− mice under these conditions, both of which should facilitate urinary P i excretion and prevent a rise in plasma P i .In terms of renal Npt2 transporter expression in response to vitamin D 3 , Npt2a −/− mice resemble Npt2a/c double knockout mice.What could explain the lack of increase in urinary P i /creatinine ratios in response to vitamin D 3 in Npt2a −/− mice?One possible explanation could be that urinary P i excretion has reached a maximum.Consistent with this, we have previously shown in short-term metabolic cage experiments that urinary P i /creatinine ratios in Npt2a −/− mice were of the same magnitude, and only acute Npt2a inhibition in control mice was able to double urinary P i /creatinine 20 , suggesting that in Npt2a −/− mice (chronically), despite the presence of compensatory mechanisms, no further increase in urinary P i excretion can be achieved.
In terms of Ca 2+ , our data are consistent with the previously published Npt2a −/− phenotype as well as the role of vitamin D 3 in Ca 2+ homeostasis 16,18 .Npt2a −/− mice have higher plasma Ca 2+ levels and greater urinary Ca 2+ / creatinine ratios.Vitamin D 3 is a well-known regulator of intestinal Ca 2+ absorption 27 and knockout of Npt2a is associated with significantly increased intestinal Ca 2+ absorption, possibly as a consequence of greater intestinal mRNA expression of epithelial Ca 2+ channels (ECaC1 and ECaC2) and the Ca 2+ binding protein calbindin-D9k 28 .Vitamin D 3 treatment increased plasma Ca 2+ in both genotypes, but to a greater amount in Npt2a −/− mice.Of note, one of the most interesting findings in this study relates to the response of the kidney after administration of vitamin D 3 .In WT mice, urinary Ca 2+ /creatinine was appropriately increased possibly as a consequence of significant hypercalcemia.In contrast to WT mice, in Npt2a −/− mice urinary Ca 2+ /creatinine was reduced in response to vitamin D 3 administration, reaching levels seen in WT mice under baseline conditions.Consequently, the reduction of urinary Ca 2+ /creatinine ratio could have contributed to the greater increase in plasma Ca 2+ in Npt2a −/− mice.Despite vitamin D response elements being present in the CaSR gene causing up-regulation of CaSR expression 29 , our study provides evidence that vitamin D 3 treatment can reduce CaSR expression.
Ultrastructural studies in Npt2a −/− mice showed that at early age Ca 2+ /P i deposits develop that were purged during later stages of life 30,31 .Along those lines, our tissue analysis showed that vitamin D 3 -treated Npt2a −/− mice had the highest kidney Ca 2+ content of all studied groups, without significant differences in kidney P i content.Of note, Npt2a mutations in humans seem fairly common in a large cohort of Ca 2+ -stone forming pedigrees, but they do not seem to corroborate with clinically significant P i or Ca 2+ handling abnormalities 32 .Our studies expand this knowledge and show that vitamin D 3 -treated Npt2a −/− mice show the greatest amount of Ca 2+ -P i crystal deposits in the tubule lumen.Of note, vitamin D 3 -treated WT mice show a similar pattern of Ca 2+ -P i crystal deposits compared to vehicle-treated Npt2a −/− mice.Taken together, Npt2a −/− mice have a significant problem in excreting Ca 2+ in their urine and, considering also the lack of Npt2c in response to vitamin D 3 administration, their kidney Ca 2+ content is further consistent with the phenotype of Npt2a/c double knockout mice, which show severe renal calcifications 18 .
Possibly because of a combination of hypophosphatemia and hypercalcemia in Npt2a −/− mice, PTH and FGF23 levels are significantly lower compared to WT mice [16][17][18] which is still present when a high P i or high P i / Ca 2+ diet is provided 17 .Under baseline conditions, these findings were confirmed in our study.PTH synthesis and release under these conditions seems to be under a dual control: (1) hypercalcemia inhibits the synthesis and secretion of PTH from the parathyroid gland via activation of the CaSR and (2) active vitamin D 3 suppresses the synthesis and release of PTH via activation of the VDR 33 .Of note, the changes observed cannot explain the paradoxical response of urinary Ca 2+ /creatinine in Npt2a −/− mice in response to vitamin D 3 .The situation in vitamin D 3 -treated Npt2a −/− mice is similar to hereditary hypophosphatemic rickets with hypercalciuria 34 a physiology also resembled in Npt2a/c double knockout mice 18 .One notable difference is the accumulation of Ca 2+ in the kidney of vitamin D 3 -treated Npt2a −/− mice rather than the development of a hypercalciuric response.Along those lines, PTH was already substantially reduced in Npt2a −/− mice under baseline conditions possibly in the face of lower plasma P i and elevated plasma Ca 2+ levels.The decrease of PTH in WT mice in response to vehicle treatment possibly relates to the presence of ethanol which has been shown to decrease PTH levels 35,36 .
Consistent with previous reports, our study confirms the lower FGF23 levels in Npt2a −/− mice, a possible consequence of lower plasma P i levels 18 .Our study did not determine 1,25(OH) 2 D 3 levels but levels were found to be significantly greater in Npt2a −/− compared with WT mice 16,18 .Our data show that vitamin D 3 treatment does not affect Cyp24a1 and Cyp27b1 mRNA expression in WT mice.In chronic kidney disease, expression of Cyp24a1 is increased possibly accounting for decreased 1,25(OH) 2 D 3 due to degradation 37 .Vehicle treatment in Npt2a −/− mice showed significantly lower expression compared to WT mice, possibly explaining the body's effort to increase 1,25(OH) 2 D 3 levels.Only in vitamin D 3 -treated Npt2a −/− mice was Cyp27b1 mRNA expression significantly increased, which is consistent with greater 1,25(OH) 2 D 3 production.Study participants treated with vitamin D 3 normally do not show increases in 1,25(OH) 2 D 3 levels 38 , which is reflected in unchanged Cyp27b1 levels in vitamin D 3 -treated WT mice compared to vehicle treatment.However, in the absence of Npt2a this can be offset, and our data imply that Cyp27b1 mRNA expression is paradoxically increased.
When VDR are knocked out in chondrocytes of mice, FGF23 expression in osteoblasts and consequently FGF23 plasma levels are significantly reduced, implying that VDR is a prerequisite in this signaling pathway 39 .Of note, exogenous administration of vitamin D 3 is a powerful stimulator of FGF23, leading to ~ 80-200-fold increase; however, the exact signaling pathway(s) causing this increase remain unclear and the relationship between these hormones is complex.Our results dispute the role of PTH being a major determining factor for FGF23 production (which was drastically suppressed in both genotypes), or suggest additional regulatory mechanisms, which has been demonstrated in vivo and in vitro as well as in mice with hyperparathyroidism 40,41 .Vice versa, our data are consistent with the notion that FGF23 reduces PTH synthesis directly 42 16 , these effects are even more exaggerated in Npt2a/c double knockout mice 18 .Our study used highly sensitive bone remodeling markers as estimators, which, to our knowledge, have never been determined in Npt2a −/− mice.Despite significant differences in P i and Ca 2+ homeostasis between genotypes, our study did not identify changes in any of the bone remodeling markers studied under baseline conditions.This might relate to the fact that Npt2a −/− mice were of adult age when our studies were performed.Osteocalcin is predominantly produced and secreted by osteoblasts during bone formation 43 .Although low doses of vitamin D 3 can stimulate bone turnover, high doses can cause bone resorption 44 .Despite these results, our study did not provide any differences between treatment or genotype in terms of osteocalcin levels.A possible explanation could be the short-term experimental setup we employed.PINP is considered the most sensitive marker of bone formation 45 , which has been reported to be under the control of PTH 46 and shows an inverse relationship with active vitamin D 3 47 .Consistent with this, both genotypes decreased PINP levels after vitamin D 3 administration, consistent with a role of reduced bone formation.Of note, this occurred despite a significant decrease of PTH in both genotypes.
TRAcP 5b is an osteoclast-derived marker of bone resorption. 48.Our findings show that vitamin D 3 treatment increased TRAcP 5b in WT mice.So far, no correlations have been described between FGF23 and TRAcP 5b under normal conditions; however, in patients on evocalcet treatment (CaSR agonist) PTH, FGF23 and TRAcP 5b decreased over the 30 week treatment period 49 .Our data point toward a role of Npt2a in this process since Npt2a −/− mice lack a response in TRAcP 5b in response to vitamin D 3 .CTX-1 is a marker for bone remodeling that is released when type 1 collagen is degraded 50 .The role of vitamin D 3 on CTX-1 is ambiguous, with several human studies showing no effect of vitamin D 3 supplementation on CTX-1 levels 51,52 whereas others show a positive correlation 47 .This might relate to the pre-existing conditions that were studied, e.g.presence or absence of vitamin D 3 deficiency.Of note, and consistent with our study, a study in humans showed a dose-dependent effect of vitamin D 3 bolus administration (up to 600,000 IU) on CTX-1 levels 1 day after administration 53 .This might explain an increase in fracture risk when elderly women are treated annually with a single high dose (500,000 IU) of vitamin D 3 54 .In addition, daily doses of 10,000 IU for 3 years also resulted in a significant increase of CTX-1 in healthy adults 44 .Lack of Npt2a possibly unravels that these mice are more susceptible for disturbed bone remodeling.
The intestine plays a vital role in P i and Ca 2+ absorption in order to regulate homeostasis in the body and vitamin D 3 has been implicated in this regulation 14,28 .Our acute P i loading experiments confirm these findings: independent of genotype, the intestinal uptake of P i was significantly greater in vitamin D 3 -treated mice compared to vehicle-treated mice as evidenced by greater increases in plasma P i levels in the face of reduced renal Npt2a/c abundance.Our studies on Npt2b abundance also expand the knowledge on spatial regulation, where abundance in response to Npt2b was unaffected in the proximal small intestine, which contrasts with the distal small intestine.Of note, the contribution of transcellular versus paracellular intestinal P i transport is a highly debated topic 15,23 , and claudin-3 has been implicated in the paracellular process.Supporting this hypothesis are data from claudin-3 knockout mice, which have enhanced intestinal P i uptake 21 .Similar to Npt2b, no regulation of claudin-3 abundance in the proximal small intestine was found in our studies, but in the distal small intestine of Npt2a −/− mice, claudin-3 abundance was significantly reduced, possibly contributing to greater plasma P i levels in Npt2a −/− mice.In the kidney we find evidence that claudin-16, expressed in the thick ascending limb and distal convoluted tubule, was significantly reduced in vitamin D 3 -treated Npt2a −/− mice.Claudin-16 inactivating mutations in humans are associated with hypercalciuria and nephrocalcinosis 55,56 , possibly suggesting that significantly reduced claudin-16 expression in our studies might have contributed to the phenotype of vitamin D 3 -treated Npt2a −/− mice.Along those lines, claudin-16 interacts with Trpv5 since knockdown of claudin-16 delocalized Trpv5 from the luminal membrane 57 .In our study, Trpv5 was also significantly reduced in vitamin D 3 -treated Npt2a −/− mice compared to vehicle-treated Npt2a −/− mice.Our data provide information on the regulation of Atp2b4, which was reduced in response to vitamin D 3 in both genotypes; however, knockout of Atp2b4 in mice did not cause a Ca 2+ phenotype 58 .
In summary, our data provide novel insight into the role of vitamin D 3 in the regulation of P i and Ca 2+ homeostasis in the context of Npt2a.One limitation of using mice to study P i homeostasis relates to distinct differences in intestinal and renal P i handling compared to humans.Despite the vitamin D 3 dose used in our studies is supraphysiological, significant differences were observed between genotypes that pinpoint to an important role of Npt2a (and possibly claudin-16) in renal calcification and consequently kidney function decline.It is noteworthy that vitamin D 3 treatment in Npt2a −/− mice resulted in a complete loss of Npt2c, and mice in terms of renal P i transporter expression resembled Npt2a/c double knockout mice.Despite a complete lack of renal P i transporters, Npt2a −/− mice experience greater plasma P i levels, possibly a consequence of reduced intestinal claudin-3 abundance.Further, Npt2a −/− mice develop significantly greater plasma Ca 2+ levels in response to vitamin D 3 , possibly a consequence of impaired renal Ca 2+ excretion with tissue accumulation of Ca 2+ , implying that Npt2a can function as a switch between renal Ca 2+ excretion and reabsorption.However, the contribution of Npt2c in this process cannot be excluded considering its absence in abundance in response to vitamin D 3 treatment in Npt2a −/− mice.

Methods
The animal experiments were conducted in compliance with the NIH Guide for Care and Use of Laboratory Animals, set by the National Institutes of Health (Bethesda, MD), received approval from the Institutional Animal Care and Use Committee (11201R) at the University of South Florida, and are reported in accordance with ARRIVE guidelines.Npt2a −/− mice were obtained from the Jackson Laboratory (strain# 004802, Bar Harbor, ME) and propagated by heterozygote breeding.Mice have been backcrossed to C57BL/6J for 9 generations.Only www.nature.com/scientificreports/male WT and Npt2a −/− mice, 3-5 months old, were used for the study.The specific pathogen free mice were group housed and kept in a controlled environment with a 12-h light-dark cycle (light off at 18:00) in isolated ventilated cages.They were provided with free access to standard rodent diet (TD.2018, containing 0.7% P i and 1% Ca 2+ , Envigo, Madison, WI) and drinking water.Genotype was determined by PCR amplification of genomic DNA, which was extracted from ear tissue samples.The genotyping was carried out in accordance with protocol # 29530 published on the Jackson Laboratory website.

Vitamin D 3 treatment
Wild-type and Npt2a −/− mice were randomized into two treatment groups: one vehicle (5% Ethanol, 5% Cremophor EL, and 90% water) or vitamin D 3 (3,000 and 300,000 IU/kg body weight, Alfa Aesar, Haverhill, MA) dissolved in vehicle 26 .Treatments were administered on 4 consecutive days via subcutaneous injections (2 µL g −1 body weight) by and investigator blinded to genotype and treatment.Blood samples were collected under brief isoflurane anesthesia from the retrobulbar plexus before and after the 4-day treatment period.Spontaneously voided urine was collected at the same time.

Analysis of plasma and urine samples
Clinical chemistry was performed utilizing commercially available assays, adapted for use with small sample volumes 20,23 .Concentrations of P i and Ca 2+ in both plasma and urine were measured using inorganic phosphorous reagent and calcium arsenazo III reagent respectively, (Pointe Scientific, Canton, MI) 59 .Urinary creatinine was measured by infinity creatinine liquid stable reagent (Thermo Fisher Scientific, Middletown, VA).Urinary albumin and plasma creatinine were determined as described previously 60,61 .PTH and intact FGF23 were measured according to the manufacturer instructions (Quidel, San Diego, CA).Markers for bone resorption (tartrateresistant acid phosphatase isoform 5b [TRAcP 5b, Quidel] and type I collagen cross-linked C-telopeptide [CTX-1, Immunodiagnostic Systems]) and bone formation (procollagen type I N-propeptide [PINP, Immunodiagnostic Systems, Gaithersburg, MD] and osteocalcin [Quidel]) were measured using ELISAs.

Acute hyperphosphatemic model
Four days after the administration of either vehicle or vitamin D 3 , WT and Npt2a −/− mice were subjected to gavage of 0.5 mol L −1 NaH 2 PO 4 , 1% of body weight by an investigator blinded to genotype 23,62 .Before gavage and 60 min after administration, blood samples were collected under brief isoflurane anesthesia.Plasma P i was measured as described above.

Determination of Ca 2+ and P i content in the kidney
In another set of WT and Npt2a −/− mice, femurs and kidneys were harvested under terminal isoflurane anesthesia 4 days after the last administration of vehicle or vitamin D 3 .The collected tissues were dried for 24 h at 50 °C.Following the drying process, the weight of each tissue was determined.Next, the tissues were incinerated at a temperature of 560 °C for 12 h in a muffle furnace (Thermolyne F48015-60, Thermo Fisher Scientific).The ashes from the incineration were dissolved in 0.75 mol L −1 HCl.Concentrations of Ca 2+ and P i in the dissolved samples were determined as described above.

Histological analysis of kidneys
In a separate cohort of mice kidneys were perfused in vivo through the left ventricle with 4% PFA in phosphate buffered saline under isoflurane anesthesia.After kidneys were removed, they were fixed overnight in the same solution and subsequently paraffin embedded and sectioned at 4-6 μm.After deparaffinization and rehydration, sections were stained with hematoxylin and eosin (H&E) and von Kossa (to determine mineral deposits).Sectioning and staining were performed by Reliance Pathology Partners, LLC (Tampa, FL).Quantification of Ca 2+ -P i deposits were performed using the following scheme: none, mild (< 10%), moderate (10-50%), or severe (> 50%).The highest score seen in sections was reported for each mouse.All scoring was performed by a pathologist (M.T.) blinded to sample identity.

Isolation of intestinal epithelial cells
Another cohort of mice was randomized to administration of either vehicle or vitamin D 3 as described above.Following the 4-day treatment, mice were anesthetized by isoflurane and their kidneys and small intestines removed.Isolation of intestinal epithelial cells (IEC) via Ca 2+ chelation was performed as described previously 23,63,64 .The IEC pellets were prepared for immunoblotting as described below.

Western blotting
Collected IEC and kidneys were homogenized in a buffer composed of 250 mmol L −1 sucrose and 10 mmol L −1 triethanolamine (Sigma-Aldrich, St. Louis, MO) containing Halt protease inhibitor cocktail and Halt phosphatase inhibitor cocktail (both Thermo Fisher Scientific).The homogenate was then subjected to a centrifugation process at 1000×g for 15 min followed by generation of plasma membrane-enriched samples (by centrifugation of the supernatant at 17,000×g) for 30 min.The pellets that emerged from this process were then resuspended and prepared for Western blotting.Protein quantity was determined using a bicinchoninic acid assay (Thermo Fisher Scientific).Samples of equal concentration were made by the addition of Laemmli sample buffer (final concentration of 0.1 mol L −1 SDS and 15 mg L −1 DTT).Samples were heated at 65 0 C for 15 min before immunoblotting.The samples were resolved on either NuPAGE 4-12% or 12% Bis-Tris gels in MOPS.Proteins were transferred to polyvinylidene difluoride membranes and immunoblotted with rabbit polyclonal antibodies against www.nature.com/scientificreports/Npt2a, Npt2b, Npt2c (each with a dilution of 1:1500, generous gift from M. Levi) 23,24,62 , rabbit anti claudin-3 (dilution 1:1000, also rabbit-sourced, Thermo Fisher Scientific) 23 , and mouse anti β-actin (dilution 1:30,000, Sigma-Aldrich).These targets were then detected with secondary antibodies designed for rabbit (IRDye® 800CW donkey anti-rabbit IgG, at a dilution of 1:5000) or mouse (IRDye® 680RD donkey anti-mouse IgG, also at a dilution of 1:5000), using an Odyssey® CLx detection system (LI-COR Biosciences, Lincoln, NE).Quantification of the band intensities was carried out using Image Studio Lite for densitometric analysis (LI-COR Biosciences).

Quantitative polymerase chain reaction from kidney and bone
Total RNA from kidney homogenates was extracted using Tri Reagent (Sigma-Aldrich) using a protocol adapted from the manufacturer's recommendations.Total RNA was quantified using a Synergy Neo2 plate reader (Agilent, Santa Clara, CA).One thousand ng RNA of kidney sample were used to produce cDNA using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific).Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific) was used in conjunction with a QuantStudio 6 Pro (Applied Biosystems, Thermo Fisher Scientific) for amplification.Template concentration was 1 ng µl −1 cDNA per 10 µl reaction (performed in triplicate) and used in conjunction with primer pairs specific for Slc8a1, Slc34a1, Slc34a3, Trpv5, Atp2b4, Cyp25a1, Cyp27b1, Cldn2, Cldn14, Cldn16, Cldn19, and CaSR with actin used as a reference gene (all primer sequences are provided in the Supplementary Information).Data analysis used the ΔΔ Ct method, i.e. cycle thresholds (Ct), were normalized to actin expression, and compared with control.

Statistical analyses
Data are expressed as mean ± S.E.M. Two-way ANOVA or repeated-measures two-way ANOVA followed by Tukey's multiple comparison tests, or two-way mixed-effects ANOVA followed by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, as indicated in the figure legends, were used to test for significant differences between genotype and/or treatment.All data were analyzed via GraphPad Prism (Version 10.1, Boston, MA) or SigmaPlot (Version 14, San Jose, CA, USA).Significance was considered at P < 0.05.

Figure 1 .
Figure 1.Lack of Npt2a unravels a link of vitamin D 3 on plasma P i .Measurements of plasma and urinary P i and Ca 2+ were conducted in WT and Npt2a −/− mice after 4 days of treatment with either a vehicle or vitamin D 3 (n = 6-10 per genotype).(a) In WT mice, plasma P i levels remained unchanged following vitamin D 3 treatment.(B) In contrast, lower plasma P i levels under baseline conditions in Npt2a −/− mice significantly increased in response to vitamin D 3 treatment.(c) The urinary P i /creatinine ratio in WT mice increased significantly in response to vitamin D 3 treatment.(d) This ratio in Npt2a −/− mice was unchanged (d).Plasma Ca 2+ levels in both WT and Npt2a −/− mice showed a significant increase following vitamin D 3 treatment (e & f).In WT mice, the urinary Ca 2+ to creatinine ratio significantly increased after vitamin D 3 treatment (g).In contrast, this ratio significantly decreased in Npt2a −/− mice (h).Male mice were used in these studies.In addition to single data summary data are shown and are expressed as mean ± SEM and were analyzed by repeated-measures two-way ANOVA followed by Tukey's multiple comparisons test.*P < 0.05 vs WT same time point, # P < 0.05 vs baseline same genotype, § P < 0.05 vs vehicle same genotype and time point.

Figure 2 .
Figure 2. Vitamin D 3 induces divergent responses in plasma PTH and increases FGF23 in both WT or Npt2a −/− mice.Measurements of plasma PTH and FGF23 were performed in WT and Npt2a −/− mice following 4 days of treatment with either vehicle or vitamin D 3 (n = 6 per genotype).(a) In WT mice, vitamin D 3 treatment led to a decrease in plasma PTH levels.(b) In Npt2a −/− mice, plasma PTH levels were lower and unchanged in response to vitamin D 3 treatment.(c & d) FGF23 levels significantly increased in both genotypes in response to vitamin D 3 treatment.Male mice were used in these studies.In addition to single data summary data are shown and are expressed as mean ± SEM and were analyzed by repeated-measures two-way ANOVA followed by Tukey's multiple comparisons test.*P < 0.05 vs WT same time point, # P < 0.05 vs baseline same genotype, § P < 0.05 vs vehicle same genotype and time point.

Figure 3 .
Figure 3. Npt2a determines the effects of Vitamin D 3 on bone remodeling markers.Circulating bone markers, including osteocalcin, PINP, TRAcP 5b, and CTX-1 were measured in WT and Npt2a −/− mice after 4 days of treatment with either a vehicle or vitamin D 3 (n = 6 per genotype).(a & b) Both genotypes show a small but significant increase in osteocalcin levels independent of treatment.(c & d) Vitamin D 3 decreased plasma PINP independent of genotype.(e & f) In both genotypes, vehicle treatment slightly but significantly decreased plasma TRAcP 5b levels but vitamin D 3 only significantly increased TRAcP 5b in WT mice.(g & h) Vitamin D 3 treatment increased CTX-1 levels in both genotypes but to a significantly greater extent in Npt2a −/− mice.Male mice were used in these studies.In addition to single data summary data are shown and are expressed as mean ± SEM and were analyzed by repeated-measures two-way ANOVA followed by Tukey's multiple comparisons test.*P < 0.05 vs WT same time point, # P < 0.05 vs baseline same genotype, § P < 0.05 vs vehicle same genotype and time point.

Figure 4 .
Figure 4. Similar effects of acute oral P i loading on plasma P i levels between genotypes.Plasma P i levels were measured in WT and Npt2a −/− mice before and one hour after oral P i loading via gavage (0.5 mol*L −1 , 1% of body weight).These measurements were performed following 4 days of treatment with either a vehicle or vitamin D 3 (n = 6 per genotype).(a & b) Oral P i loading significantly increased plasma P i levels independent of genotype or treatment; however, in vitamin D 3 -treated mice the increase was significantly greater compared to vehicle-treated mice.Male mice were used in these studies.In addition to single data summary data are shown and are expressed as mean ± SEM and were analyzed by repeated-measures two-way ANOVA followed by Tukey's multiple comparisons test.*P < 0.05 vs WT same time point, # P < 0.05 vs baseline same genotype, § P < 0.05 vs vehicle same genotype and time point.

Figure 5 .
Figure 5. Vitamin D 3 treatment increases kidney Ca 2+ levels in Npt2a −/− mice.Measurements of P i and Ca 2+ levels were carried out in bone and kidney tissues of WT and Npt2a −/− mice following 4 days of treatment with either a vehicle or vitamin D 3 (n = 6 per genotype).(a & b) There was no difference in the levels of P i and Ca 2+ in bone of either genotype in response to vehicle or vitamin D 3 treatment.(c) Kidney P i levels were not significantly different between genotypes or treatment.(d) The kidney Ca 2+ levels were significantly greater in response to vehicle treatment in Npt2a −/− compared to WT mice.Vitamin D 3 treatment was not associated with altered kidney Ca 2+ levels in WT mice; however, resulted in the highest kidney Ca 2+ levels in Npt2a −/− mice.Male mice were used in these studies.In addition to single data summary data are shown and are expressed as mean ± SEM and were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test.*P < 0.05 vs WT same treatment, § P < 0.05 vs vehicle same genotype.

Figure 6 .
Figure 6.Vitamin D 3 -treated Npt2a −/− mice show signs of impaired kidney function and greater renal Ca 2+ -P i deposits.(a) Plasma creatinine levels were the highest in vitamin D 3 -treated Npt2a −/− mice.(b) Similarly, urinary albumin/creatinine ratios showed the biggest increase compared to baseline in vitamin D 3 -treated Npt2a −/− mice.(c) Histological classification of mineral deposits in genotypes with vehicle or vitamin D 3 treatment.The majority of vehicle-treated WT mice show no mineral deposits, vehicle-treated Npt2a −/− mice showed mild deposits (< 10%), vitamin D 3 -treated WT showed greater severity (moderate 10-50%) and only some vitamin D 3 -treated Npt2a −/− mice showed the greatest number of deposits (> 50%).Representative examples of H&E and von Kossa staining are shown for each condition from mice with no mineral deposits (d), mild deposits (e), moderate deposits (f) and severe deposits (g).Von Kossa staining shows that Ca 2+ -P i crystal deposits (black stains) localize within the tubular lumen.The tubules show evidence of damage with attenuation of the epithelial lining.Magnification × 200.Scale bar of 100 µm is shown in each image.Male and female mice were used in these studies.In addition to single data summary data are shown and are expressed as mean ± SEM and were analyzed by two-way ANOVA followed by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli.*P < 0.05 vs WT same treatment, § P < 0.05 vs vehicle same genotype.

Figure 7 .
Figure 7. Npt2c abundance is diminished in Npt2a −/− mice in response to vitamin D 3 treatment.Abundance of Npt2a and Npt2c in kidney tissues of WT and Npt2a −/− mice after 4 days of treatment with vehicle or vitamin D 3 (n = 4-6 per genotype).(a) In this study we confirmed the specificity of the Npt2a antibody in Npt2a −/− mice, which lack the ~ 75-80 kDa band representing Npt2a.An unspecific band was detected.In WT mice, vitamin D 3 treatment showed lower Npt2a expression compared to vehicle treatment.(b) In response to vehicle treatment, Npt2c abundance was significantly greater in Npt2a −/− compared to WT mice.Npt2c abundance was significantly lower in vitamin D 3 -treated mice; however, the level in Npt2a −/− mice was almost undetectable.Male mice were used in these studies.In addition to single data summary data are shown and are expressed as mean ± SEM and were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test.*P < 0.05 vs WT same treatment, § P < 0.05 vs vehicle same genotype.

Figure 8 .
Figure 8. Npt2b abundance is diminished in Npt2a −/− mice in response to vitamin D 3 treatment.(a) Abundance of Npt2b in the proximal small intestine was not different between genotypes or treatment.(b) In the distal small intestine, no differences were detected in Npt2b abundance between vehicle-treated genotypes.In WT mice, Npt2b abundance was similar in response to vitamin D 3 compared to vehicle treatment.Of note, Npt2b abundance in vitamin D 3 -treated Npt2a −/− mice was lower compared to vehicle treatment.(c) Abundance of claudin-3 was somewhat variable in the proximal small intestine and no differences were observed between genotype or treatment.(d) In the distal small intestine, no differences in claudin-3 abundance were detected between vehicle-treated genotypes.In WT mice, claudin-3 abundance was similar in response to vitamin D 3 compared to vehicle treatment.Of note, claudin-3 abundance in vitamin D 3 -treated Npt2a −/− mice was lower compared to vehicle treatment.Male mice were used in these studies.In addition to single data summary data are shown and are expressed as mean ± SEM and were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test.§ P < 0.05 vs vehicle same genotype.

Figure 9 .
Figure 9. Summary figure.In vitamin D 3 -treated Npt2a −/− mice, a circulus vitiosus is observed leading to kidney failure.We hypothesize that vitamin D 3 treatment leads to elevated plasma Ca 2+ levels (possibly via increased bone resorption as indicated by greater CTX-1 levels and/or increased intestinal Ca 2+ absorption) and decreased intestinal P i absorption (via lower Npt2b and claudin-3 levels).A combination of vitamin D 3 , elevated plasma P i , and reduced kidney function causes FGF23 levels to be drastically elevated, subsequently diminishing Npt2c abundance and leading to supersaturation of tubular fluid with Ca 2+ and P i .Formation of Ca 2+ /P i crystals lead to renal calcification and reduced kidney function (increase in plasma creatinine and urinary albumin).Consequently, plasma Ca 2+ and P i levels are further increased.Green arrows indicate an increase, red arrows indicate a decrease.The table on the right summarizes the most significant findings observed between vitamin D 3 treated mice and vehicle treated mice for both genotypes. https://doi.org/10.1038/s41598-024-67839-4 https://doi.org/10.1038/s41598-024-67839-4 Npt2a −/− mice show a skeletal phenotype characterized by delayed secondary ossifications at 21 days of age which are reversed at 45 days of age and are ultimately overcompensated at > 74 days of age . Vol.:(0123456789) Scientific Reports | (2024) 14:16997 | https://doi.org/10.1038/s41598-024-67839-4www.nature.com/scientificreports/