Deregulated Renal Calcium and Phosphate Transport during Experimental Kidney Failure

Impaired mineral homeostasis and inflammation are hallmarks of chronic kidney disease (CKD), yet the underlying mechanisms of electrolyte regulation during CKD are still unclear. Here, we applied two different murine models, partial nephrectomy and adenine-enriched dietary intervention, to induce kidney failure and to investigate the subsequent impact on systemic and local renal factors involved in Ca2+ and Pi regulation. Our results demonstrated that both experimental models induce features of CKD, as reflected by uremia, and elevated renal neutrophil gelatinase-associated lipocalin (NGAL) expression. In our model kidney failure was associated with polyuria, hypercalcemia and elevated urinary Ca2+ excretion. In accordance, CKD augmented systemic PTH and affected the FGF23-αklotho-vitamin-D axis by elevating circulatory FGF23 levels and reducing renal αklotho expression. Interestingly, renal FGF23 expression was also induced by inflammatory stimuli directly. Renal expression of Cyp27b1, but not Cyp24a1, and blood levels of 1,25-dihydroxy vitamin D3 were significantly elevated in both models. Furthermore, kidney failure was characterized by enhanced renal expression of the transient receptor potential cation channel subfamily V member 5 (TRPV5), calbindin-D28k, and sodium-dependent Pi transporter type 2b (NaPi2b), whereas the renal expression of sodium-dependent Pi transporter type 2a (NaPi2a) and type 3 (PIT2) were reduced. Together, our data indicates two different models of experimental kidney failure comparably associate with disturbed FGF23-αklotho-vitamin-D signalling and a deregulated electrolyte homeostasis. Moreover, this study identifies local tubular, possibly inflammation- or PTH- and/or FGF23-associated, adaptive mechanisms, impacting on Ca2+/Pi homeostasis, hence enabling new opportunities to target electrolyte disturbances that emerge as a consequence of CKD development.


Introduction
Chronic kidney disease (CKD) is a major public health issue with a high prevalence currently affecting millions of people worldwide [1]. CKD results in a significant morbidity and mortality and highly associates with the development of cardiovascular events, the primary cause of death in CKD patients [2][3][4]. A prominent hallmark of CKD that may contribute to this increased risk is a disturbed electrolyte homeostasis, including calcium (Ca 2+ ) and phosphorus (P i ) deregulation [5]. Physiological Ca 2+ and P i levels in the blood are maintained by complex endocrine systems comprising the parathyroid hormone (PTH) and phosphaturic hormone Fibroblast Growth Factor 23 (FGF23) and vitamin D system [6][7][8]. PTH and FGF23 inhibit renal P i reabsorption and are the primary regulators of renal 1,25-dihydroxy vitamin D 3 (active vitamin-D) synthesis. 1,25-dihydroxy vitamin D 3 itself also regulates urinary calcium excretion by the kidney [6][7][8] .
In addition to bone-derived FGF23, other cell types may contribute to circulating FGF23 levels, including fibroblasts and smooth muscle cells [9]. Moreover, several neuroendocrinologic factors and cytokines directly influence FGF23 secretion into the circulatory system [10,11]. To enable downstream signalling, FGF23 requires membrane-bound αklotho as its obligate co-receptor [12,13]. αklotho is predominantly expressed in renal epithelium and the parathyroid gland, hence providing tissue-specificity for FGF23 signalling [14]. In addition, soluble αklotho present in the circulation or urine can regulate electrolyte transport, through the epithelial Ca 2+ channel (TRPV5) [15] and the renal outer medullary potassium channel (ROMK1) [16][17][18][19]. There are well-described reductions in renal and parathyroid klotho in CKD. It has been shown [20] that CKD associates with reduced αklotho expression and that it is related to elevated FGF23 levels, in response to persistent phosphate retention [21][22][23]. In turn, this may lower systemic vitamin-D levels, which contributes to the pathogenesis of secondary hyperparathyroidism. The consequences of CKD-induced alterations in FGF23αklotho-vitamin-D signalling on renal tubular electrolyte regulatory mechanisms are, however, still unclear. Hence, in the current study, we characterized the impact of two different experimental models of progressive kidney failure on FGF23-αklotho-vitamin-D signalling and local renal Ca 2+ and P i transport regulation. In addition we examined the consequences of inflammatory stimuli and the effect of FGF23 on several mineral-regulating channels on the tubular epithelium. mice expressing enhanced green fluorescent protein (EGFP) driven by the TRPV5 promoter (TRPV5-EGFP) were generated as described previously [24] and housed in the animal facility of the Radboud University Medical Center [25]. The presence of EGFP expression was genetically confirmed by routine PCR screening. All mice received water and food ad libitum and only age-matched male mice were used in this study. All experiments were approved and conducted following the guidelines of the local Animal Ethical Committees, adhering the guidelines of the European animal welfare.

Partial (5/6) nephrectomy and urine collection
Prior to surgery a small volume of peripheral blood was collected into EDTA-coagulated microtainers (BD Biosciences, Breda, The Netherlands). Partial nephrectomy (5/6Nx; n = 9) was performed under standardized sterile conditions and induced as described before [26,27]. Briefly, a small abdominal midline incision of the skin and muscles was made under general anaesthesia (isoflurane) and preoperative analgesia (Buprenorphin; Temgesic (Schering-Plough, Houten, The Netherlands), 0.05 mg/kg intramuscular). The left kidney was decapsulated after which both, the upper and lower pole were ablated by cauterization (High-temperature fine tip Cautery, Bovie Medical Corporation, Clearwater, FL, USA). Subsequently, the contralateral kidney was decapsulated and renal blood vessels and ureter were ligated, after which the entire kidney was removed. The abdomen was closed with sutures in two layers and all mice received subcutaneous injections of postoperative analgesia two days after surgery (Ketoprofen; Ketofen (Merial S.A.S., Velserbroek, The Netherlands), 5 mg/kg). Sham-operated mice were used as controls and underwent the similar protocol including decapsulation of both kidneys, except for the renal ablation (n = 5). After three weeks, mice were placed into individual metabolic cages (Tecniplast, Buguggiate, Italy) enabling 24-hours urine collection. Mice were sacrificed and blood was divided into EDTA-and heparin-coagulated microtainers and centrifuged for 10 minutes (min) at 1,500 g at 4°C. Plasma samples were stored at -80°C. Remnant renal tissue was isolated, subdivided into two equal parts and either directly snap-frozen into liquid nitrogen or fixated in 1% w/v paraformaldehyde-lysine-periodate (PLP) solution for 2 hours (hrs) at room temperature, subsequently incubated overnight at 4°C in phosphate buffered saline (PBS) containing 15% w/v sucrose before snap-frozen in liquid nitrogen.

Adenine-enriched dietary intervention
Prior to the start of diet, a small volume of peripheral blood was isolated by cheek puncture into microvette tubes (Sarstedt, Etten-Leur, The Netherlands). Mice of about 25 gram body weight were fed with a 0.2% w/w adenine (Sigma-Aldrich, Saint-Louis, MO, USA; cat.no. A8626) containing diet (Bio Services; Uden, The Netherlands) during a period of either 2 or 4 weeks (n = 5/group). A control group (n = 5) was fed a standard diet and sacrificed after 4 weeks. Body weights were recorded every other day. After 2 or 4 weeks, mice were placed into individual metabolic cages (Tecniplast) for 24 hrs before sacrificed under general anaesthesia (isoflurane). Blood was isolated from the orbital sinus and collected in microvette tubes. Samples were allowed to clot for 2 hrs at room temperature and subsequently centrifuged 10 min at 9,000 g at 4°C to enable serum isolation, and subsequently stored at -80°C. Kidney tissue was isolated and processed as described above.

Systemic administration of FGF23
Male TRPV5-EGFP mice of 5-7 weeks old received a single intravenous tail vein administration (IV) of 160 μg/kg Carrier-free recombinant mouse FGF23 (rFGF23; R&D Systems, Minneapolis, MN, USA; cat.no 2629-FG-CF) or vehicle (saline) in a total volume of 100 μl (n = 5/ group). These mice were non-CKD mice and after 24 hrs were sacrificed under total anaesthesia (isoflurane). Blood and kidney were collected and processed, as described above.

Systemic administration of Concanavalin A (ConA) and Tumor Necrosis Factor (TNF)
Male mice received a single intravenous retro-orbital administration of 20 ug/kg of bodyweight ConA or vehicle (PBS). A subset of mice received a single intravenous retro-orbital administration of 15 ug/kg of bodyweight TNF or vehicle. These mice were non-CKD mice and were sacrificed either after 8 hrs ConA administration or after 1 hr TNF administration. Kidney were collected and processed as described above.

Electrolyte measurement
Systemic (using either EDTA-coagulated plasma from 5/6Nx mice or serum samples from ADE-treated mice) and urinary concentrations of urea, and P i were determined by in-hospital services using automatic biochemical analyzers. Blood Ca 2+ concentrations were determined in heparin-coagulated plasma (from 5/6Nx mice) or serum (from ADE treated mice) and in urinary samples, verified using a commercial serum standard (Precinorm U, Roche, Switzerland) and measured as described previously [28].
Immunoassays EDTA-coagulated plasma (from 5/6Nx mice) or serum (from ADE treated mice) samples were used in enzyme-linked immunosorbent assays (ELISA) to determine circulating levels of FGF 23 and PTH 1-84 (both Immutopics International). Heparin-coagulated plasma (from 5/6Nx mice) and serum (from ADE treated mice) samples were used to determine circulating 1,25-dihydroxy vitamin D 3 (active vitamin-D) levels using a radioimmunoassay (Immunodiagnostic Systems; IDS, Liege, Belgium). All assays were performed according to the manufacturer's protocols.

RNA isolation and quantitative real-time RT-PCR
Total kidney mRNA was extracted from snap-frozen kidney tissue using Trizol Reagent (Life Technologies; Bleiswijk, The Netherlands), according to the manufacturer's guidelines. All RNA samples were quantified by using an ND-1,000 spectrophotometer (NanoDrop Technologies, Rockland, DE). Total RNA (1.5 μg) was converted into cDNA using random hexamers (Promega, Madison, WI, USA) and M-MLV reverse transcriptase (Life Technologies). Mouse gene mRNA expression was analyzed by real-time quantitative reverse-transcription (RT)-PCR performed on a Bio-Rad CFX96™ Real-Time qPCR or 7900HT system (Applied Biosystems, Warrington, UK), using Bio-Rad iQ™ SYBR1 Green Supermix or Power SYBR Green Master Mix (Roche, Mannheim, Germany). In addition specific gene expression was normalized to housekeeping gene (hypoxanthine-guanine phosphoribosyl transferase; HPRT) and analyzed according the 2^-ddCt method [29]. The mouse gene-specific primer sets used are listed in S1 Table. Immunohistochemistry Immunohistochemical staining was performed on 5 μm cryosections of PLP-fixed renal tissue samples [30]. Sections were stained with guinea pig anti-TRPV5 antibodies (1:2,000; [30]) or mouse anti-calbindinD 28k antibodies (1:2,000; Sigma Aldrich, Saint-Louis, MO, USA), with concomitant tyramide signal amplification (TSA) enhancement (Perkin Elmer, Waltham, MA, USA) following standard procedures. For visualization, sections were subsequently incubated with appropriate Alexa-488-conjugated secondary antibodies (Sigma Aldrich). Fluorescent images were made using a Zeiss fluorescence microscope (Sliedrecht, The Netherlands) equipped with an AxioCam digital photo camera.

Statistics
Data are presented as mean ± SEM. Differences between groups were analyzed using Mann-Whitney U tests and calculated using Graphpad Prism version 5.0 software. A p-value <0.05 was considered as statistically significant.

Partial nephrectomy and adenine-enriched dietary treatment induced kidney failure
The experimental models of partial nephrectomy (5/6Nx) and adenine-enriched dietary (ADE) treatment both induced renal failure. This is reflected by elevated blood urea levels in 5/6Nx mice compared to sham-operated mice after 3 weeks ( Fig 1A) and the progressive uraemia observed in ADE mice when compared to control mice ( Fig 1B). In addition, renal mRNA expression of tubular injury marker neutrophil gelatinase-associated lipocalin (NGAL) was increased 12-fold in kidney tissue of 5/6Nx mice and >500-fold in ADE-treated mice (Fig 1C  and 1D). Moreover, mice subjected to either 5/6Nx or ADE treatment developed severe polyuria compared to sham-operated or control mice, respectively ( Table 1).

Development of kidney failure disturbed electrolyte homeostasis
To investigate the consequences of induced renal failure on electrolyte homeostasis, blood levels and urinary excretion of Ca 2+ and P i were determined. No variations in blood Ca 2+ and P i levels were found within experimental groups prior to onset of the experiments (data not shown). 5/6Nx mice were hypercalcemic compared to sham-operated mice and additionally displayed elevated 24-hours urinary Ca 2+ excretion ( Table 2). In contrast, no differences were observed in blood P i levels or 24-hours urinary P i excretion. In accordance, ADE-treated mice displayed progressive hypercalcemia and increased urinary Ca 2+ excretion when compared to control mice (Table 2). Blood P i levels progressively increased and were significantly elevated after 4 weeks compared to control mice. No effects were detected on 24-hours urinary P i excretion, whereas after 4 weeks ADE diet the fractional excretion of P i was significantly elevated compared to control mice (26±2 and 40±3% Fractional Excretion of P i ) for control and ADEtreated mice, respectively. In addition, blood Creatinine levels were significantly elevated in both models while urine Creatinine levels were significantly reduced only in 5/6Nx mice ( Table 2).

Induction of experimental CKD altered FGF23-αklotho signalling
In order to determine whether induced kidney failure results in altered FGF23-αklotho signalling, we first measured blood FGF23 levels. FGF23 levels tended to be elevated in 5/6Nx mice when compared to sham-operated mice (Fig 2A), and progressively increased in ADE mice compared to control mice (Fig 2B; P<0.05). 5/6Nx did not affect renal mRNA expression of the primary FGF23 receptor FGFR1, while αklotho expression tended to be decreased, although not statistically significant ( Fig 2C). ADE-treated mice demonstrated a mild raise in FGFR1 (after 4 weeks) and a progressive (>2-fold) reduction in renal αklotho mRNA expression (Fig 2D; P<0.05 for both time points) when compared to control mice. In addition, a 2-fold decline in renal αklotho expression following ADE treatment was confirmed on protein level (Fig 2E and 2F; P<0.05 for both time points).
Since inflammation is another hallmark of CKD we additionally checked whether an acute inflammatory stimulus affects FGF23 levels directly. Interestingly, both ConA and TNF administration in vivo resulted in significantly elevated renal FGF23 mRNA expression in WT mice (S1A Fig; P<0.05).

CKD progression augmented both renal vitamin-D synthesis and blood PTH
CKD induced by either 5/6Nx or ADE-treatment augmented blood PTH levels in both 5/6Nx (Fig 3A; P<0.05) and ADE-treated mice (Fig 3D; P<0.05 for both time points). CKD development resulted in a 5-to 13-fold increased renal mRNA expression of the primary vitamin-D   regulatory enzyme 25-hydroxyvitaminD 3 1α-hydroxylase (Cyp27b1) when compared to shamoperated or control-diet fed mice, respectively (Fig 3B and 3E). In contrast, no effects were observed on renal 1,25-dihydroxyvitamin D 3 24-hydroxylase (Cyp24a1) mRNA expression. In accordance, blood 1,25-dihydroxy vitamin D 3 was higher in 5/6Nx mice compared to shamoperated mice after 3 weeks (Fig 3C; p<0.05), and was as well elevated in elevated in 2 and 4 weeks ADE-treated mice compared to control mice (Fig 3F; P<0.05 for both time points). However 4 weeks ADE-treated mice exhibited a significant reduction in the level of 1,25-dihydroxy vitamin D 3 in compare to 2 weeks ADE-treated mice (Fig 3F; P<0.05; 4 weeks vs. 2 weeks).

CKD altered expression of crucial renal calcium and phosphate transporters
To characterize the impact of CKD on tubular electrolyte transport regulation, the renal expression of Ca 2+ and P i transporters was determined. Renal mRNA expression of TRPV5 and calbindin-D 28k , which are specifically expressed in the distal convoluted tubule, was 2-fold increased in 5/6Nx mice compared to sham-operated mice (Fig 4A). In line, microphotographs of kidney tissue demonstrated concomitant increases on protein expression (Fig 4B). Additionally, ADE treatment induced a 5-fold increase of renal TRPV5, but did not affect renal calbindin-D 28k mRNA expression compared to control mice (Fig 4C). Comparable patterns were also observed on protein level (Fig 4D). Kidney injury induced by either 5/6Nx or ADE treatment reduced renal expression of sodium-dependent P i transporters NaP i 2a and PIT2 more than 2-fold, but elevated renal NaP i 2b expression 2-to 20-fold compared to sham-operated or control mice, respectively (Fig 5A and  5B). On the contrary, renal expression of the thiazide-sensitive sodium-chloride co-transporter (NCC) specifically present in the distal convoluted tubule, was not affected in either 5/6Nx or ADE-treated mice (Data not shown).

FGF23 initiated adaptive mechanisms of renal calcium handling
To gain insight whether inflammatory stimuli directly affect renal Ca 2+ handling, TRPV5 mRNA expression was determined in WT mice following ConA or TNF administration. ConA, but not TNF administration resulted in elevated renal TRPV5 mRNA expression (S1B Fig). Furthermore, to determine whether FGF23 affects renal Ca 2+ handling, total kidney tissue of transgenic mice, expressing EGFP driven by the TRPV5 promoter, was used to determine mRNA expression of TRPV5, calbindin-D 28k and EGFP, as a surrogate marker for TRPV5 transcription. Exogenous FGF23 administration resulted in an increased renal mRNA expression of TRPV5 (Fig 6A; P<0.05) and a tendency towards increased calbindin-D 28k expression (Fig 6A). In addition, EGFP mRNA expression tended to be elevated following FGF23 infusion, although not statistically significant, when compared to control mice (Fig 6A).
TRPV5 protein expression cannot be determined by immunoblotting directly because of the lack of appropriate primary antibodies. We therefore performed Western blotting on kidney lysates from transgenic mice that expresses Enhanced Green Fluorescent Protein (EGFP 31 kD) driven by the TRPV5 promoter. Using this approach, determination of EGFP act as a surrogate marker for TRPV5 expression. Representative pictures are shown in the Fig 6B. Digital quantification analysis showed no significant differences in GFP protein expression level between two groups (Fig 6C).
increased in 2 weeks ADE treated mice in compare to control mice. This was significantly reduced in 4 weeks ADE treated mice in compare to 2 weeks ADE treated mice. Data are mean ± SEM. *: p<0.05 compared to either sham-operated or control mice. Ca 2+ levels in the blood were unaffected after 24 hrs following FGF23 administration ( Fig  6D). Changes in systemic phosphate levels in comparison to the control group were not detected. This might be due to the short-term period of the experiment. Unfortunately, we did not include the use of metabolic cages in the initial experimental setting. Therefore, the main effect of FGF23 infusion (P i excretion) cannot be validated completely. In order to determine the local renal effects of rFGF23 infusion, gene expression of sodium-phosphate cotransporter type 2 (NaP i 2a and NaP i 2b) in renal tissue were measured. No alterations between the groups were however observed (Fig 6F).

Discussion
Ca 2+ and P i disturbances are a hallmark of CKD and an important risk factor for the development of severe cardiovascular complications. In the current study, we characterized the impact of CKD on systemic and local renal factors involved in Ca 2+ and P i regulation. Our data reveal that two different experimental models of kidney failure induce comparable classical features of CKD. A deregulated Ca 2+ and P i homeostasis and a significantly disturbed FGF23-αklothovitamin-D axis were observed in both models. Both models resulted in an increased or decreased expression of specific renal Ca 2+ and P i transporters, respectively.
First, both models of kidney failure, that is 5/6Nx and ADE treatment, induced clinical hallmarks of CKD, as reflected by the development of uraemia and an elevated renal expression of tubular injury marker NGAL. Our results are in accordance with previous studies demonstrating that both partial nephrectomy [31][32][33] and ADE treatment [34][35][36] closely resemble various features of CKD development. The two models we used gave rise to different severity in terms of its consequences on systemic urea and NGAL, electrolyte disturbances and alterations of the FGF23-αklotho-vitamin-D axis, all pointing to more severe damage in the ADE-model.
Second, both experimental models disturbed Ca 2+ homeostasis comparably, as reflected by hypercalcemia and elevated urinary Ca 2+ excretion. Hypercalciuria as a consequence of partial nephrectomy was shown previously [37], and can be explained by increased filtration burden on remnant glomeruli causing increased ion excretion and parallel development of renal osteodystrophy [38]. The hypercalcemia observed in both models is likely due to early elevated PTH levels and aggravated by 1,25-dihydroxy vitamin-D 3 -facilitated intestinal Ca 2+ absorption. In contrast to our data, hypercalcemia was not observed following ADE treatment in mice [39,40], although this might be explained by variation in the amount of adenine supplemented to  the diet, and thus in degree of renal injury induced. While ADE treatment increased blood urea levels to~60 mmol/L after 4 weeks in this study, other studies showed maximal urea levels of around 40 mmol/L, suggesting a distinct degree of renal injury. In agreement with previous studies [36,39,40], ADE treatment dramatically affected P i homeostasis, a common hallmark observed in severe CKD [41]. ADE mice presented a significant rise on P i levels in the blood despite that elevated fractional excretion was initially adequate to manage P i loading. In contract, no effects on systemic P i levels were detected three weeks after 5/6Nx, suggesting that modest elevated levels of PTH and FGF23 are sufficient to maintain physiological blood P i levels.
Third, both experimental models were characterized by a deregulated FGF23-αklotho-vitamin-D axis. We observed an increase in systemic FGF23 levels and a reduction in renal αklotho expression. Previously it has been suggested that lack of αklotho might be a driving force for FGF23 resistance [42][43][44][45][46][47][48]. It is, however, not yet clear whether the downregulation of αklotho can trigger an increase in the expression of FGF23 or vice versa. On the one hand, FGF23 appears to be an early marker of CKD and rises progressively with progression of CKD. On the other hand, loss of renal αklotho occurs early in CKD, and might be the trigger to increase systemic FGF23 in order to maintain P i homeostasis. Additionally, rising FGF23 levels upon CKD might also be due to inflammation [49,50]. Indeed, it has been shown that both experimental models significantly induce renal cytokine levels [51,52] and recent studies point out that elevated blood FGF23 levels are associated with inflammatory components (e.g. cytokines/chemokines) [53][54][55], and in CKD patients FGF23 and inflammatory markers are often both increased [55,56]. Interestingly, a study by Mendoza et al., [57] showed that higher circulatory levels of FGF23 directly correlate with increased blood levels of several proinflammatory markers, suggesting that FGF23 is independently associated with inflammation in CKD. In line, we also observed that two different inflammatory stimuli acutely induce renal FGF23 mRNA expression. Although, in the current study we did not further elucidate the effects of inflammatory pathways on electrolyte regulatory hormones upon kidney failure, as this was beyond the scope of the primary project.
Despite elevated blood FGF23, both kidney failure models augmented renal Cyp27b1 expression, probably due to the observed increase in PTH. Comparable data were shown before in remnant kidneys of nephrectomized rats [58], and following ADE-treatment [59]. In accordance, elevated blood 1,25-dihydroxy vitamin D 3 levels were detected following both 5/6Nx and ADE treatment. Together, these findings indicate that damaged kidneys retain sufficient vitamin-D synthesizing capacity and that an increased Cyp27b1 expression in the remaining epithelium might be effective to compensate for the loss of tissue capable of Cyp27b1 expression due to the reduction in functional kidney mass. Moreover, FGF23-suppressive effects on 1,25-dihydroxy vitamin D 3 either directly via modulation of renal Cyp27b1 and Cyp24a1 expression, or indirectly via modulation of PTH synthesis might be hampered due to lower renal αklotho expression in both kidneys and parathyroid gland, implicating FGF23 resistance [60][61][62]. As a proposed pathway, this points towards early high PTH levels as the likely mechanism responsible for amplifying renal vitamin-D synthesis and the consequent rise in blood 1,25-dihydroxy vitamin D 3 and Ca 2+ levels. Indeed, both CKD models were characterized by the development of secondary hyperparathyroidism. Nevertheless, this was not in line with the observed hypercalcemia, elevated levels of FGF23 and increase in 1,25-dihydroxy vitamin D 3 , which all should have suppress PTH level. However, we did not examine parathyroid tissue to injection. (F) mRNA expression of the renal NaP i 2a and NaP i 2b in mice injected with FGF23 (gray bars), compared to vehicle-treated mice (white bars). Data are mean ± SEM. *: p<0.05 compared to control mice. doi:10.1371/journal.pone.0142510.g006 study local changes that may drive PTH production or secretion. Development of secondary hyperparathyroidism could be due to early disturbances on the local calcium sensing receptor, early decline in parathyroid klotho and possibly lower expression of the vitamin D receptor.
Fourth, to identify local adaptive mechanisms of tubular electrolyte regulation following CKD, renal expression of crucial Ca 2+ and P i transporters was determined. Gene expression of Ca 2+ transporters TRPV5 and calbindin-D 28k was elevated upon CKD. TRPV5 is a crucial rate-limiting Ca 2+ channel expressed in the late distal convoluted and connecting tubule, responsible for apical Ca 2+ influx initiating transcellular reabsorption [63,64]. Calbindin-D 28k is subsequently responsible for the direct intracellular Ca 2+ shuttling towards the basolateral extrusion side [65]. Together, this suggests that elevated distal tubular Ca 2+ reabsorption is likely the consequence of stimulation by calciotropic factors, including PTH, vitamin D and/or FGF23 signalling [28,66,67]. However, Calbindin-D 28k levels were differentially altered in the ADE dietary intervention model; we observed either reduction or no changes in the Calbindin-D 28k gene expression levels, while it increased in the 5/6Nx model. The reason for that is yet unclear but it could be that adenine itself might affect the expression/regulation of intracellular (Ca 2+ ) proteins, that result in a different kinetics compared to TRPV5. In order to determine whether FGF23 directly affects renal Ca 2+ transport, we administered FGF23 to TRPV5-EGFP transgenic mice to verify its effect on renal EGFP expression, as a surrogate marker for TRPV5 transcription. We found, at most, a tendency towards increased renal EGFP expression. In contrast, a previous study indicated a direct effect of FGF23 on renal Ca 2+ transport by increasing TRPV5 gene transcription [68]. A potential explanation for this discrepancy might be that essential upstream enhancer elements for the transcriptional induction of TRPV5 by FGF23 are lacking in the TRPV5-EGFP mice. Interestingly, WT mice exposed to an inflammatory stimulus (ConA, but not TNF treatment) demonstrate elevated renal TRPV5 mRNA expression. Together, these results indicate that both hormonal and inflammatory parameters may influence renal Ca 2+ transport, although the exact stimuli and signalling pathways should be further investigated in future studies.
In concert with the phosphaturic effects of FGF23 and PTH [69], both kidney failure models displayed a reduced renal expression of NaP i 2a and PIT2, the principal renal type II and III sodium-dependent P i transporters, respectively [70][71][72]. NaP i 2a is the main P i co-transporter involved in the phosphaturic response to both PTH and FGF23 and it responded to high FGF23 as expected despite the observed reductions in renal αklotho. On the contrary, renal NaP i 2b was significantly augmented. NaP i 2b as a predominant intestinal P i transporter is also expressed at the basolateral side of renal epithelial cells. Its expression is increased in response to active phosphate loading [73]. Hence NaP i 2b plays major roles in both the kidney and the small intestine. Mice with NaP i 2b deficiency, exhibited predominantly lower P i absorption [74], highlighting a major role of NaP i 2b in body P i homeostasis. As proposed, elevated renal NaP i 2b expression might be an additional mechanism of active tubular P i secretion, although it remains unclear whether this also holds true during progressive renal injury to counteract P i retention. Since 5/6Nx and ADE treatment induced distinct renal pathophysiological mechanisms, its consequences on renal expression of the sodium-chloride co-transporter (NCC) were determined. NCC expression was unaffected within both models, suggesting that the demonstrated effect on the expression kinetics of Ca 2+ and P i transporters within both models was not simply a reflection of distinct kidney tissue composition compared to sham-operated or control mice as a potential confounding factor.
Interestingly, the increased blood Ca 2+ and active 1,25-dihydroxy vitamin D 3 observed in the current study do not correspond with the classical observation of hypocalcemia due to reduced 1,25-dihydroxy vitamin D 3 levels that are frequently observed in clinically diagnosed early stage renal disease patients. The reason for this is unclear but it might be related to the timing of the measurements performed in our experimental models. This is possibly demonstrated by the 1,25-dihydroxy vitamin D 3 levels observed in the ADE mice model, which is increased on week 2, and reduced on week 4. Here we present an early renal failure model, possibly not at steady state yet. Moreover, differences between species may explain why a given response in animal models differs from patients with CKD [75]. Furthermore, several physiological differences between experimental animal models and patients can be identified, including variations in time to develop a certain degree of kidney disease reflecting a particular stage of CKD. Here, we observed hypercalcemia and hypercalciuria in both experimental CKD mice models ( Table 2) and these observations are supported by previously applied experimental CKD animal models [76], but not in patients with CKD [77][78][79]. Human CKD development reflects a gradual and slow decline of vitamin D levels, contributing to hypocalcemia, whereas secondary hyperparathyroidism develops over longer time. On the contrary, experimental animal models are rather short term, due to the methods applied and the shorter lifespan of rodents [80]. One of the limitations of this study is that we did not keep both models for a longer period of time. Furthermore, CKD-induced alterations in intestinal and/or bone Ca 2+ and P i homeostasis might also trigger an increase in blood Ca 2+ and active 1,25-dihydroxy vitamin D 3 observed in these models. Yet, here we primarily focused on renal Ca 2+ and P i regulatory mechanisms; hence the existing experiment was not designed to characterize the impact of CKD-induced alterations on intestinal Ca 2+ and P i transport or bone metabolism. It thus remains difficult to fully elucidate the compensatory mechanisms occurring following CKD, which might contribute to the disturbed electrolyte homeostasis observed.
Taken together, our study demonstrated that murine models of partial nephrectomy and ADE-enriched dietary treatment, both, induce kidney failure that closely resembles CKD development, which is associated with disturbed FGF23-αklotho-vitamin-D signalling and a deregulated Ca 2+ and P i homeostasis. Moreover, this study identified local tubular adaptive mechanisms that are involved in disturbed Ca 2+ and P i regulation, hence enabling new opportunities to target mineral disturbances emerging as a consequence of CKD development.