Recent advances in Mineral and Bone Disorders in CKD

Henk van den Broek & Jonathan Elliott, London, UK

Hyperphosphatemia is a well-known risk factor for disease progression and survival in cats and dogs with chronic kidney disease (CKD), and multiple studies have shown the beneficial effects of reducing the serum phosphate concentration by feeding phosphate-restricted clinical renal diets to these animals (Elliott et al., 2000; Jacob et al., 2002; Plantinga et al., 2005; Ross et al., 2006; Boyd et al., 2008; Chakrabarti et al., 2012). Reduced capacity of diseased kidneys to excrete sufficient phosphate on a daily basis as a consequence of CKD is thought to underlie the biochemical abnormalities of calcium-phosphate homeostasis, bone disease, and soft tissue calcification that are grouped together as chronic kidney disease-mineral and bone disorder (CKD-MBD) (Moe et al., 2006; Slatopolsky 2011). For decades the disturbances in calcium-phosphate homeostasis observed in patients with CKD and its consequences have been explained by hyperphosphatemia and (patho)-physiological increases in parathyroid hormone (PTH), i.e. secondary renal hyperparathyroidism, but recent advances in the understanding of phosphate homeostasis have been made by the discoveries of the phosphaturic hormone fibroblast growth factor 23 (FGF23) and its co-receptor, α-Klotho. Recognition of the role of magnesium in CKD-MBD is another recent breakthrough in the field of nephrology.

Fibroblast growth factor 23

The hormone FGF23 is released by osteocytes (Pereira et al., 2009) and decreases the serum concentration of phosphate by downregulating the membrane abundance of the sodium-dependent phosphate transporters (NaPi2a) in the proximal tubules, thus limiting reabsorption of phosphate from the tubular fluid, and by inhibiting the enzyme 1α-hydroxylase which activates vitamin D, hence reducing the serum concentration of calcitriol and thus the intestinal absorption of phosphate (Shimada et al., 2004a; Shimada et al., 2004b). To bind to its receptor and have its effects, FGF23 requires a co-factor, α-Klotho protein (Urakawa et al., 2006; Nakatani et al., 2009). The renal expression of α-Klotho is thought to reduce in CKD, thereby limiting the effects of FGF23 on phosphate excretion (Sakan et al., 2014). No studies have been published yet on the expression of α-Klotho in veterinary patients with CKD, but multiple studies have investigated FGF23 in cats and dogs with CKD.

FGF23 and CKD IRIS Stage

In cats, plasma FGF23 increases in the early stages of CKD before the occurrence of overt azotemia (Finch et al., 2013) and the plasma concentration of FGF23 increases considerably with IRIS CKD stage from a median concentration of 354 pg/mL in IRIS Stage 2 to 1282 pg/mL in Stage 3 and 33478 pg/mL in Stage 4 CKD (Geddes et al., 2013a).

Dogs with CKD also have significantly increased plasma FGF23 concentrations compared to healthy dogs (Dittmer et al., 2017), but this is predominantly true for the IRIS stage 3 and 4 CKD (Harjes et al., 2017). Plasma FGF23 concentration increased in dogs with CKD from a median of 336 pg/mL in IRIS CKD Stage 2, to 2302 pg/mL in Stage 3 and 7733 pg/mL in Stage 4 CKD (Harjes et al., 2017).

FGF23 and hyperphosphatemia

Cats that are hyperphosphatemic in relation to their IRIS stage have significantly higher plasma FGF23 concentrations than normophosphatemic cats with the same severity of CKD (Geddes et al., 2013a). This could not be clearly established in dogs with CKD, probably due to the low numbers of dogs included in each group in this study (Harjes et al., 2017). However, both in cats and dogs with CKD, a significant correlation between plasma FGF23 and phosphate concentrations has been identified (Geddes et al., 2013a; Dittmer et al., 2017). Moreover, feeding a phosphate restricted renal diet was associated with a decrease in plasma FGF23, both in hyperphosphatemic and normophosphatemic cats with stable azotemic CKD (Geddes et al., 2013b). Plasma FGF23 might therefore be used to monitor the effect of treatment in cats with CKD. The effect of dietary phosphate restriction on plasma FGF23 concentrations in dogs with CKD has not yet been examined.

FGF23 and survival

In cats, higher plasma FGF23 concentrations at diagnosis of CKD have been associated with increased risk of death (Geddes et al., 2015, Van den Broek et al., 2018). In the retrospective study by Geddes et al. (2015) on 214 cats with CKD, cats with plasma FGF23 >3000 pg/mL had statistically significant decreased survival compared to cats with plasma FGF23 below the upper limit of its proposed reference interval (i.e. 700 pg/mL) at diagnosis of azotemic CKD, independent of their IRIS stage or age (Geddes et al., 2015). Cats with plasma FGF23 concentrations < 700 pg/mL had a median survival time of 577 days from diagnosis of azotemic CKD, whilst cats with plasma FGF23 concentrations between 3000 and 10,000 pg/mL had a median survival of 277 days Geddes et al., 2015). Median survival time decreased further to 38 days in cats with plasma FGF23 > 10,000 pg/mL at diagnosis of CKD.

A prospective survival study on 27 dogs with variable stages of CKD found a statistically significant increased risk of death for dogs with plasma FGF23 >450 pg/mL compared to dogs with plasma FGF23 <450 pg/mL. (Rudinsky et al., 2018) Dogs with plasma FGF23 <450 pg/mL had a median survival of 26 months, whilst dogs with plasma FGF23 >450 pg/mL had a median survival of 10 months, however multivariable analysis was not performed due to the low number of dogs enrolled onto this study (Rudinsky et al., 2018).

The mechanisms accounting for the association between FGF23 and decreased survival have not been investigated in cats and dogs, but in humans FGF23 excess has been associated with left ventricular hypertrophy, anemia and inflammation (Faul et al., 2011; Scialla et al., 2014; Singh et al., 2016; Mehta et al., 2017).

FGF23 and progression of CKD

In cats, higher plasma FGF23 concentration has been identified as an independent predictor of disease progression within the first 12 months following diagnosis of azotemic CKD (Geddes et al., 2015; Van den Broek et al., 2018). In the study by Geddes et al. (2015) cats that showed a plasma creatinine increase >25% within a year of diagnosis had a median baseline plasma FGF23 concentration of 1243 pg/mL, whilst cats with stable disease had a median concentration of 504 pg/mL.

Plasma FGF23 is elevated prior to the development of overt renal azotemia in cats. Finch et al. (2013) showed that plasma FGF23 concentration was significantly higher in non-azotemic cats that progressed to azotemic CKD within a 12-month follow-up period than in cats that remained non-azotemic (Finch et al., 2013). Such observations have not been reported for dogs.

Measuring FGF23

Intact FGF23 can be measured by a sandwich-ELISA (Kainos, Tokyo, Japan) in a small volume (0.1 mL) of heparinized plasma, although currently this test is not clinically available. In geriatric cats the reference interval generated for the plasma concentration of FGF23 appears much higher than in humans: 56-700 pg/mL compared to 8.2-54.3 pg/mL (Geddes et al., 2013a). In the abovementioned study by Finch et al. (2013), cats that remained non-azotemic over a 12-month period generally had plasma FGF23 concentrations below 200 pg/mL, which suggests that the reference interval upper limit for FGF23 in cats of 700 pg/mL is too high. In dogs, no reference interval has been established, but the upper plasma FGF23 concentration from healthy dogs (i.e. 449 pg/mL) has been used as the cutoff value to define FGF23 excess in this species (Harjes et al., 2017).

Magnesium

Magnesium is an important mineral for various physiologic processes, such as ATP generation and bone formation, and an inhibitor of soft tissue calcification (Jahnen-Dechent & Ketteler, 2012; Louvet et al., 2013). It is primarily stored in bone and muscle tissue, and only 1% of the total body magnesium stores is located in serum, in which it can be found in 3 fractions: ionized, protein-bound, and complexed with anions (Elin, 2010; Jahnen-Dechent & Ketteler, 2012; Humphrey et al., 2014).

Magnesium and CKD IRIS stage

Plasma total magnesium concentrations are relatively stable in cats with IRIS stage 2 and 3 CKD, and becomes elevated when renal excretory function is severely impaired in IRIS stage 4 CKD (Barber & Elliott, 1998; Van den Broek et al., 2018). In our recent study involving 174 cats with azotemic CKD, we identified hypomagnesemia (defined as serum total magnesium concentration <0.71 mmol/l) in 9%, 18% and 10% of cats with IRIS stage 2, 3, and 4 CKD, respectively (Van den Broek et al., 2018). Hypermagnesemia was only identified in 3% of cats with IRIS stage 2 CKD and 6% of cats with IRIS stage 3 CKD, but in 50% of cats with IRIS stage 4 CKD. Information on ionized magnesium was unavailable (Van den Broek et al., 2018).

Magnesium and FGF23

In human hemodialysis patients, an inverse relationship between serum magnesium and FGF23 concentrations has been observed (Iguchi et al., 2014). We had very similar findings in cats with azotemic CKD, in which plasma total magnesium concentrations were inversely associated with plasma FGF23 concentrations in each CKD IRIS stage (Van den Broek et al., 2018). Rodent studies suggest that dietary magnesium intake influences plasma FGF23 concentrations (Matsuzaki et al., 2013). Dietary magnesium supplementation could potentially be an additional management strategy to lower plasma FGF23, just as dietary phosphate restriction is efficient in reducing plasma FGF23 concentrations (Geddes et al., 2013b).

Magnesium and survival

Various observational studies have identified hypomagnesemia (based on serum total magnesium) as a risk factor for death in human patients with dialysis-dependent CKD and non dialysis-dependent CKD (Ishimura et al., 2007; Kanbay et al., 2012; Van Laecke et al., 2013; Sakaguchi et al., 2014a). Moreover, higher serum magnesium appears to attenuate the mortality risk associated with hyperphosphatemia in human CKD patients (Sakaguchi et al., 2014b). In cats, hypomagnesemia at diagnosis of azotemic CKD was also significantly associated with an increased risk of death, independent of age, packed cell volume, plasma FGF23, and severity of CKD and proteinuria (Van den Broek et al., 2018). Our results also suggested that higher plasma magnesium attenuates the risk of death associated with hyperphosphatemia in cats, as it does in humans, but this observation needs further investigation.

Hypermagnesemia was also associated with an increased risk of death, but not after correction for CKD IRIS stage. Therefore, this association was likely due to the increased prevalence of hypermagnesemia in cats with end-stage CKD (Van den Broek et al., 2018).

In unadjusted analysis, hypomagnesemia was not only associated with reduced survival but also with increased risk of progression of azotemia (Van den Broek et al., 2018). This association was lost, however, after correction for other variables and further studies are required to assess this possible association.

Measuring magnesium

Plasma total magnesium can be measured by most laboratories in patient serum or heparinized plasma samples, but EDTA plasma needs to be avoided as it will lead to an erroneously low result (Sharratt et al., 2009). Ionized magnesium is biologically-active and therefore might reflect magnesium status better than total magnesium (Bateman, 2012; Humphrey et al., 2014). However, there is no consensus whether total or ionized magnesium best reflects body magnesium status and the current knowledge derived from the studies on humans and cats presented above is based on measurement of total magnesium. We derived a reference interval for plasma total magnesium from 120 apparently-healthy senior cats of 1.73-2.57 mg/dL or 0.71-1.06 mmol/L (Van den Broek et al., 2018).

Bone disease and soft tissue calcification

The clinical focus in veterinary medicine has mainly been on the biochemical abnormalities associated with CKD in cats and dogs. Less attention has been paid to bone disease (i.e. renal osteodystrophy) and soft tissue calcification, which do occur in cats and dogs with CKD - albeit in the more advanced stages of disease. Osteitis fibrosa cystica and bone resorption most likely occur as a result of high bone turnover caused by secondary renal hyperparathyroidism, and have been reported in early radiographic and necropsy studies in cats with CKD (Lucke, 1968; DiBartola et al., 1987). In dogs, osteodystrophy affecting the maxillofacial and mandible bones, and ultimately "rubber jaw" is observed (Davis, 2015). Two recent studies using micro-CT compared the bone structure of cats and dogs with CKD to that of healthy controls. Bones of cats with IRIS Stage 3 and 4 CKD had decreased mineral density and an increased cortical porosity (Shipov et al., 2014). A similar study on dogs found relatively mild changes in bone quality in animals with CKD, which is probably due to the shorter duration of disease in dogs compared to that of cats and humans (Shipov et al., 2018).

Vascular calcification, by the deposition of calcium phosphate salts in the medial layer of the vessel wall, is an important predictor of patient mortality due to cardiovascular disease in human CKD patients (London et al., 2003; Górriz et al., 2015). Increases in serum phosphate and calcium stimulate calcification of vessel walls, which leads to increased vascular stiffness, elevated pulse pressure and left ventricular hypertrophy (London et al., 2003; Lomashvili et al., 2006; Shanahan et al., 2011). In cats, radiographic evidence exists for soft tissue calcification of the thoracic and abdominal aorta, as well as of other soft tissues such as the gastric wall, kidneys and interdigital space (DiBartola et al., 1987; Jackson & Barber, 1998; Bertazzolo et al., 2003; Chakrabarti et al., 2013; McLeland et al., 2014). Chronic kidney disease leads to an imbalance of serum promotors (e.g. phosphate, calcium) and inhibitors (e.g. magnesium, fetuin-A) of calcification, and the effects of these imbalances on the propensity of individual patient's serum to calcify can be measured by an assay, the T50 test (Pasch et al., 2012). Increased serum calcification propensity has been associated with increased risk of death and mineral imbalances in humans with CKD (Smith et al., 2014; Bielesz et al., 2017). Recently, we presented data showing that more severe CKD-MBD appears associated with an increased tendency of individual cat's serum to calcifya. Whether vascular calcification is of clinical significance and what its relationship is to systemic arterial hypertension in dogs and cats with CKD is unknown.

In conclusion, the importance of restricting dietary phosphate intake in the CKD patient has been recognized for many years and is a key component of the management of CKD in veterinary and human medicine. Recent advances in understanding the role of FGF23 - α-Klotho axis in the pathophysiology of bone and mineral disorders associated with chronic kidney disease and recognition of the way in which serum magnesium impacts on this hormonal system and influences the propensity for vascular calcification are important. Routine measurement of serum FGF23 and magnesium could identify those veterinary patients that will benefit most from effective management of these bone mineral disturbances.

a (Van den Broek, D.H.N., Chang, Y.-M., Elliott, J., and Jepson, R.E. (2018) Serum calcification propensity in cats with chronic kidney disease. Abstract presented at ACVIM Forum 2018, Seattle, USA)

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