Intraperitoneal pyrophosphate treatment reduces renal calcifications in Npt2a null mice

Mutations in the proximal tubular sodium-dependent phosphate co-transporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis, however the relative contribution of genotype, dietary calcium and phosphate, and modifiers of mineralization such as pyrophosphate (PPi) to the formation of renal mineral deposits is unclear. In the present study, we used Npt2a-/- mice to model the renal calcifications observed in these disorders. We observed elevated urinary excretion of PPi in Npt2a-/- mice when compared to WT mice. Presence of two hypomorphic Extracellular nucleotide pyrophosphatase phosphodiesterase 1 (Enpp1asj/asj) alleles decreased urine PPi and worsened renal calcifications in Npt2a-/- mice. These studies suggest that PPi is a thus far unrecognized factor protecting Npt2a-/- mice from the development of renal mineral deposits. Consistent with this conclusion, we next showed that renal calcifications in these mice can be reduced by intraperitoneal administration of sodium pyrophosphate. If confirmed in humans, urine PPi could therefore be of interest for developing new strategies to prevent the nephrocalcinosis and nephrolithiasis seen in phosphaturic disorders.


Introduction
Mutations in the sodium phosphate co-transporters NPT2a [1][2][3] and NPT2c [4,5] have been associated with intraluminal stones (nephrolithiasis) and mineral deposits in the renal parenchyma (nephrocalcinosis) in patients with familial forms of hypophosphatemia. In genomewide association studies, NPT2a has also been associated with nephrolithiasis [6] and altered renal function [7,8]. With both genetic abnormalities affected individuals show renal phosphate-wasting, high circulating levels of 1,25(OH) 2 D, and absorptive hypercalciuria as a result of increased intestinal uptake of calcium [4,5,9,10], and oral phosphate supplements are currently thought to reduce the risk for renal mineralization by lowering circulating levels of 1,25 (OH) 2 D and absorptive hypercalciuria [11]. However, the relative contribution of genotype, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 dietary calcium and phosphate, and modifiers of mineralization to the formation of renal mineral deposits is unclear. Our recent work suggests that reduced levels of osteopontin (Opn), an extracellular matrix factor affecting binding of phosphate to hydroxyapatite crystals, contribute to the development of nephrocalcinosis in Npt2a -/mice [12]. This may be due to the fact that Npt2a -/mice respond differently to dietary phosphate when compared to WT mice [13]. Further evaluation in the Npt2a -/cohort on different diets suggests that urinary calcium excretion, plasma phosphate, and FGF23 levels appear to be positively correlated to renal mineral deposit formation, while urine phosphate levels and the urine anion gap, an indirect measure of ammonia excretion, appear to be inversely correlated [13]. In addition, local tissue levels of Pi generated by tissue nonspecific alkaline phosphatase (Tnsalp) and ectonucleoside triphosphate diphosphohydrolase 5 (Entpd5) may be important as suggested by decreased skeletal mineralization in the absence of these enzymes [14,15].
In the present report, we hypothesize that genes involved in the synthesis of pyrophosphate (PPi) in the interstitial matrix may be associated with renal mineralization in these mice [16,17].
PPi is present in plasma at a concentration of 1-6 μM [18] and in urine levels are around 10 μM [19]. Calcium phosphate stone formers appear to have reduced urinary PPi excretion when compared with control subjects [20][21][22][23]. Intravenous 32 PPi is rapidly hydrolyzed in plasma by tissue nonspecific alkaline phosphatase (Tnsalp) that is expressed in the proximal tubules of the kidneys [24] and less than 5% of intravenous 32 PPi appears in urine. These data indicate that urine PPi is generated locally in the kidneys [25,26].
Extracellular nucleotide pyrophosphatase phosphodiesterase 1 (Enpp1) hydrolyzes extracellular ATP into AMP and PPi and may be an important source of extracellular PPi in the body [27,28]. Enpp1 is the founding member of the ENPP or NPP family of enzymes [29]. It has phosphodiesterase activity [27] and is a type II extracellular membrane bound glycoprotein located on the mineral-depositing matrix vesicles of osteoblasts and chondrocytes [30] and the vascular surface of cerebral capillaries [28]. Enpp1 is also expressed in the kidney collecting duct and possibly other segments [25]. The second source of PPi generation in the kidney is the mevalonate pathway inside mitochondria [26]. Intracellular PPi is released into the interstitium and the urine by the transporter progressive ankylosis gene product (Ank) [31]. Ank is located at the apical membrane of collecting ducts suggesting that it may function to inhibit mineralization within the tubule lumen. Additionally, ecto-5-prime nucleotidase (Nt5E/CD73), which inhibits Tnsalp by further hydrolyzing AMP to adenosine, and adenosine triphosphatebinding cassette [32], and subfamily C, member 6 (Abcc 6), recently shown to secrete ATP from hepatocytes [32], may both be involved in PPi generation.
We here report that urine PPi levels are increased in Npt2a -/mice when compared to WT mice, possibly to protect from renal mineralization in the setting of hyperphosphaturia. Presence of two hypomorphic Enpp1 asj/asj alleles decreases urine PPi and worsens renal calcium phosphate deposit formation in Npt2a -/mice. Conversely, development of mineral deposits in these mice can be reduced by intraperitoneal administration of sodium pyrophosphate. These studies suggest that PPi may be a thus far unrecognized factor modulating the development of renal calcifications in Npt2a -/mice which may be, if confirmed in humans, of diagnostic and therapeutic relevance for phosphaturic disorders.

Materials and methods Animals
Male and female C57BL/6 mice were obtained from Charles River Laboratory, MA. Male and female Npt2a -/mice (B6.129S2-Slc34a1 tm1Hten /J, Stock No: 004802), and Enpp1 asj/asj mice (C57BL/6J-Enpp1 asj /GrsrJ, Stock No: 012810) were purchased from The Jackson Laboratory, ME. The Enpp1 asj allele is partially active and shows approximately 15% level of Enpp1 activity compared to wild-type controls [37]. Mice were genotyped by PCR amplification of genomic DNA extracted from tail clippings as described [29,[38][39][40]. Mice were weaned at 3 weeks of age and allowed free access to water and regular chow (1.0% calcium, 0.7% phosphorus, of which 0.3% phosphorus is readily available for absorption, Harlan Teklad TD.2018S). Mice received daily intraperitoneal (i.p.) injection of Hanks Buffered Saline (Gibco, Life Sciences) or sodium pyrophosphate in HBSS for two weeks until age four weeks as previously described (160 micromole/Kg/day) according to [41]. To determine whether renal mineral deposits persist beyond weaning age mice were followed for an additional 10 weeks of age after weaning on regular chow. The background of all mouse lines is C57Bl6, use of littermates for controls further reduced bias based on genetic background. No difference in renal mineral deposits was observed between sexes as previously reported by us [12,36] and thus genders were combined here.
Mice were euthanized following orbital exsanguination in deep anesthesia with isoflurane and vital organs were removed as described [12,36].

Blood and urine parameters
Biochemical analyses were done on blood samples (taken by orbital exsanguination) and spot urines collected following an overnight fast at the same time of day between 10 AM and 2 PM. Following deproteinization of heparinized plasma by filtration (NanoSep 300 K, Pall Corp., Ann Arbor, MI), plasma and urinary total pyrophosphate (PPi) concentrations were determined using a fluorometric probe (AB112155, ABCAM, Cambridge, MA). Urine PPi was corrected for urine creatinine, which was measured by LC-MS/MS or by ELISA using appropriate controls to adjust for inter-assay variability.

Kidney histology
Left kidneys were fixed in 4% formalin/PBS at 4˚C for 12 h and then dehydrated with increasing concentration of ethanol and xylene, followed by paraffin embedding. Mineral deposits were determined on 10 um von Kossa stained sections counterstained with 1% methyl green. Hematoxyline/eosin was used as counterstain for morphological evaluation. Histomorphometric evaluation of sagittal kidney sections that includes cortex, medulla and pelvis was performed blinded by two independent observers using an Osteomeasure System (Osteometrics, Atlanta, GA). Percent calcified area was determined using the formula: % calc. area = 100 Ã calcified area/total area (including cortex, medulla and pelvic lumen), and is dependent on number of observed areas per section. Mineralization size was determined using the formula: calc. size = calcified area/number of observed calcified areas per section.
For transmission electron microscopy, a 1 mm 3 block of the left kidney was fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in phosphate buffered saline for 2 hrs., followed by post-fixation in 1% osmium liquid for 2 hours. Dehydration was carried out using a series of ethanol concentrations (50% to 100%). Renal tissue was embedded in epoxy resin, and polymerization was carried out overnight at 60˚C. After preparing a thin section (50 nm), the tissues were double stained with uranium and lead and observed using a Tecnai Biotwin (LaB6, 80 kV) (FEI, Thermo Fisher, Hillsboro, OR) at the Yale Center for Cellular and Molecular Imaging (YCCMI).

Statistical analysis
Data are expressed as means±SEM and analyzed in Microsoft Excel 2010 or Graphpad Prism 6.0. Differences were considered significant if p-values, calculated using the unpaired, twotailed Student's t-test, linear regression analysis, or one-way ANOVA using Tukey's adjustment for multiple comparisons, were smaller than 0.05.
To further evaluate the role of PPi in renal mineral deposit formation in the setting of renal phosphate wasting we next reduced endogenous PPi production using the hypomorphic murine Enpp1 asj allele [37] or administered sodium pyrophosphate by intraperitoneal injection as previously described [41] to increase PPi.

Intraperitoneal sodium PPi injection decreases renal mineral deposits in
Npt2a -/mice Intraperitoneal injection of sodium pyrophosphate was previously shown to reduce arterial calcification in an uremic mouse model [41]. We used the dose of 160 micromole/Kg/day published by these authors and two weeks old Npt2a -/pups for this experiment, because renal calcification is more pronounced when compared to older mice (Fig 4A and 4C). Size and body weight (BW) of mice in the treatment group were indistinguishable from vehicle and the animals appeared to be thriving well. Following sacrifice at four weeks of age we observed a reduction of renal mineral deposits by 33% in the treatment group (0.4±0.04, n = 9 vs. vehicle 0.7 ±0.06%, n = 12, p = 0.01) (Fig 4C and 4D) while mineralization size again was unaffected ( Fig  4E). Plasma PPi levels at sacrifice were increased, albeit non-significantly (3.9±0.8, n = 9 vs. vehicle 2.0±0.4 micromole/l, n = 5, p = ns) (Fig 4F). Likewise, the U-PPi concentration was increased (244.9±33.2, n = 14 vs. vehicle 149.4 ± 28.8 micromole/l, n = 14, p = 0.039) (Fig 4G  and panel B in S1 Fig).
Histological evaluation showed large interstitial mineral deposits that displaced the surrounding renal tubules. In addition, we observed small intraluminal mineral deposits in cortical and medullary tubular segments of the kidneys of Npt2a -/and double-mutant mice ( Fig  4A). Transmission electron images showed concentric spheres of similar morphology in Npt2a -/and double-knockout mice (Fig 4B) as previously described for Npt2a -/mice by us [13,43] and others [33,34]. No mineralization was observed in renal vasculature or in the renal pelvis of our mice.

Discussion
Oral phosphate supplements are currently thought to be the primary intervention to reduce risk for renal mineralization in human carriers of NPT2a and NPT2c mutations. However, there is concern that oral phosphate therapy might contribute to the formation of renal mineralization despite reduced 1,25(OH) 2 D levels and reduced urinary calcium excretion under certain conditions, for example in patients with X-linked hypophosphatemia (XLH) treated with oral phosphate supplements given multiple times throughout the day [44,45] and in otherwise healthy individuals following treatment with phosphate enema [46]. We recently reported that reduced urine levels of osteopontin (Opn), an extracellular matrix factor affecting binding of phosphate to hydroxyapatite crystals, contribute to the development of nephrocalcinosis in Npt2a -/mice [12]. The present report describes that the urine PPi concentration may be an additional modifier of renal calcifications in this mouse model.
Reduced Enpp1 activity increased the % calcified area in double mutant mice when compared to Npt2a -/mice (Fig 4A), while the size of the calcium phosphate deposits was not affected. Similarly, intraperitoneal sodium PPi treatment reduced % calcified area, while calcification size was unchanged. Although further studies are required to define cause and effect, these data suggest that PPi inhibits nucleation (Figs 2A and 4A), which is different from the effect of Opn reported by us [12], that predominantly decreases mineralization size, consistent with the known role of Opn in calcium phosphate crystal growth. Interventions that increase both PPi and Opn would therefore be predicted to be additive.
Enpp1 expression is positively regulated by phosphate in osteoblast cultures [47], and therefore we expected that expression is likewise increased in Npt2a -/mice to explain the increased urine PPi levels. Instead, we found that Enpp1 expression is unchanged, possibly as a result of reduced Pi sensing in the absence of Npt2a. Furthermore, Ank expression was decreased and Tnsalp was increased, all predicted to reduce local PPi production. These findings suggest that PPi may be generated outside of the kidneys contrary to previous reports [25,26], and elevate urine PPi despite unchanged or decreased local gene expression for Enpp1 and Ank, respectively. Consistent with this hypothesis is our finding that global reduction of Enpp1 activity in Enpp1 asj/asj mutant mice decreased urine PPi levels and that intraperitoneal injection of sodium pyrophosphate increased urine PPi levels ( Fig 4G). Alternatively, PPi production may be regulated locally by increased renal activities of Enpp1 and Ank on a post-transcriptional level.
Interestingly, urine PPi in 10 weeks old Npt2a-/-mice is higher than in 4 weeks old weanlings (1257±272 micromole/l vs. 149.4 ± 28.8 micromole/l). This may be a developmental change of urine PPi over the first 10 weeks of life and could be a contributing factor explaining the initial observation in Npt2a -/mice reported by the Tenenhouse lab [33], that renal calcifications peak with weaning age and subsequently decrease during adult life in these mice.
Tissue specific ablation of Enpp1 (and possibly Ank) could help determine in future studies whether PPi is produced renally or extrarenally. Injection of recombinant Enpp1 may be able to reduce the renal calcifications in Npt2a -/mice [26,29] and provide further evidence of the causal relationship of this extracellular enzyme, urine PPi, and renal mineralization.
Also, separate evaluation of interstitial and luminal mineralization and PPi levels and/or activity of PPi generating enzymes may be of interest in future studies. Finally, determining how urinary pH, anion gap, citrate, oxalate, magnesium, and the expression of uromodulin (Tamm-Horsfall protein, THP) or Opn [48] modify PPi action may help better understanding the pathogenesis of renal mineralization in Npt2a -/mice.
In summary, we show here that urine PPi is increased in Npt2a -/mice. Presence of one or two hypomorphic Enpp1 asj alleles decreases urine PPi and increases renal mineral deposits in Npt2a -/mice. Furthermore, the development of nephrocalcinosis and nephrolithiasis in these mice can be reduced by intraperitoneal administration of sodium pyrophosphate. These studies suggest that PPi may be a thus far unrecognized factor modulating the development of renal calcifications in Npt2a -/mice which may be, if confirmed in humans, of diagnostic and therapeutic relevance for phosphaturic disorders.  Pyrophosphate reduces calcification in kidneys of Npt2a-/-mice mineralization with the ratio of urine pyrophosphate/urine creatinine (U-PPi/U-crea) (% calcified area = 100 Ã calcified area/total area C and calcification size = calcified area/number of mineralization D). The data represent individual animals (closed circles) or means±SEM, pvalues shown above the lines of comparisons were calculated by one-way ANOVA using Tukey's adjustment for multiple comparisons (A) and Student's t-test (B-D