Phosphate restriction using a processed clay mineral reduces vascular pathologies and microalbuminuria in rats with chronic renal failure

Chronic kidney disease (CKD) is defined by the progressive loss of renal function with a severe reduction of the glomerular filtration rate to <60 ml/min/1.73 m² compared to >90 ml/min/1.73 m² in healthy people [1]. Health implications due to CKD are complex, but elevated serum phosphate levels (>5.5 mg/dL) represent a major risk factor for the associated increased cardiovascular morbidity and mortality [2, 3]. Hyperphosphatemia raises calcium release from bones and induces an accelerated progressive vascular calcification and hypertrophy of arterial walls, which results in a mortality rate of approximately 50% in terminal CKD patients due to artery and cardiovascular diseases such as left ventricular hypertrophy (LVH), vascular remodeling, and myocardial fibrosis [4, 5]. Elevated uremic toxins such as phosphate and uric acid cause the transition of vascular smooth muscle cells (SMCs) from a contractile phenotype to an extracellular matrix (ECM) secreting phenotype that contributes to fibrotic cardiac remodeling and increased collagen deposition, both strongly associated with cardiorenal syndrome [6‐8]. Pronounced microalbuminuria is often correlated with hypertension and is a well described prognostic marker for future cardiovascular events [9‐11].

According to the clinical relevance of phosphate in CKD, dietary control of phosphate uptake is crucial throughout all stages of CKD, beginning from earliest stages up to dialysis-dependent end-stage renal disease [12]. The average oral ingestion is about 1.4 to 2.0 g phosphate per day and 40–60% of ingested phosphate is intestinally absorbed [13]. Because of the inability of intermittent dialysis to provide a continuously sufficient phosphate clearance, a low phosphate diet and the elimination of dietary phosphate by phosphate adsorbers within the gastrointestinal tract is considered to be mandatory [14]. Furthermore, some phosphate binders exert pleiotropic effects as they attenuate oxidative stress and inflammation, and reduce the circulating levels of uremic toxins [15]. Contrarily, although phosphate binders have certain intrinsic advantages, they also cause gastrointestinal side effects. Together with a high pill burden, those disadvantages are the main reason for the need of new low compliance-demanding phosphate binders with fewer side effects [16, 17].

This study proves the efficacy of a processed Montmorillonite-Illite clay mineral as a phosphate binding agent in uremia-exhibiting rats, which received a 5/6 nephrectomy, in comparison to lanthanum carbonate. These processed clay minerals provide a high phosphate binding capacity due to their specific four-layer structure, high ion exchange capacity, and high content of iron oxide [18]. Main target parameters of this study were (i) survival, (ii) reduction of kidney retention parameters, (iii) effects on vascular morphology, (iv) changes in cardiac muscle tissue, (v) effects on glomerular filtration rate and (vi) on microalbuminuria. This study indicates an efficient elimination of enhanced dietary phosphate, resulting in reduced vascular pathologies and reduced mortality in CRF rats. The exertion of several beneficial effects in the etiopathology of chronic renal failure reveals a processed Montmorillonite-Illite clay mineral as an interesting and valuable candidate for a new phosphate binding agent.

A balanced phosphate metabolism is essential to maintain health and well-being. Hyperphosphatemia due to reduced phosphate excretion during renal failure in chronic kidney disease (CKD) results in several related complications such as increased vascular calcification and, therefore, enhanced cardiovascular risk [21, 22]. The inefficient ability of dialysis to remediate elevated blood phosphate concentrations to physiological levels, raises the need to reduce the phosphate uptake to a minimum [23, 24]. This is realized by a low phosphate diet supplemented with phosphate adsorbers.

In this study, we present evidence that a specifically processed Montmorillonite-Illite clay mineral (pClM) efficiently binds phosphate and has increased beneficial effects on cardiovascular risk factors in 5/6 nephrectomized Wistar rats in comparison to lanthanum carbonate (LaC), a clinically used phosphate adsorber. The special four-layer structure and the small particle size as well as their mineralogical and chemical composition facilitate the strong ion-exchange capacity of clay minerals (ClM) [18]. If specifically processed to pCIM, ClM possesses a higher phosphate binding capacity compared to LaC (see supplements Fig. S1A). The inability of aqua regia and microwave digestion to sequester the bound phosphate from pClM suggests a strong chemical stability once the binding process is completed (see supplements Fig. S1B). Based on these results, pClM was the only ClM used for further animal experiments in this study.

5/6 nephrectomy in rats resulted in significantly increased plasma phosphate levels whereas both, pClM and LaC, successfully prevented this phenotype (Fig. 1A). Additionally, we found that the 24 h phosphate excretion rate was reduced after 5/6 nephrectomy in rats and even further by treatment with pClM and LaC. This indicates a decreased phosphate uptake by the intestines due to the phosphate binding properties of the adsorbers during the gastrointestinal passage. Additionally, LaC rats showed increased fecal phosphate excretion compared to CRF rats and interestingly also to pClM rats. This is probably due to the fact that the binding between phosphate and pClM is extremely stable as demonstrated by the inability of aqua regia to removed more than 5% of the bound phosphate. In contrast, this method almost completely removed the phosphate bound to lanthanum carbonate.

Consequences of hyperphosphatemia are vascular pathologies e.g. the production of ECM which enhances the resistance of blood vessels and subsequently induces hypertension [25‐27]. In this study, highly elevated formation of ECM was observed in 5/6 nephrectomized rats compared to sham-operated rats (Fig. 2 B). While in pClM-treated animals the amount of ECM was almost reduced to sham levels, LaC-treated animals still revealed a significantly higher quantity than the sham group. The tunica media thickness of the abdominal aorta exhibited a similar pattern (Fig. 2A). Interestingly, only treatment with pClM but not LaC was capable to reduce this pathological alteration. Consistent with these results, the relative amount of α-SMA within vascular smooth muscle cells (VSMC) was significantly lower in CRF rats as shown by Western Blot analyses (Fig. 2C). The loss of α-SMA suggests a phenotypic transition from contractile to secretory cells, as shown by Wang et al. [8, 28]. This is conclusively consistent with increased ECM production as shown for CRF rats. Only pCIM prevented this transition completely whereas LaC showed an incomplete prevention. However, hypertrophy of the whole heart and the left ventricle in particular as well as the increased myocyte size developed after 5/6 nephrectomy were avoided by treatment with pClM or LaC (Figs. 3 and 4). Thus, pClM effectively attenuates the progression of vascular injury which is the most severe consequence of chronic renal failure.

Various studies revealed a pronounced proteinuria as a reliable marker for future cardiovascular events such as stroke in CKD patients [29, 30]. In line with these studies, untreated 5/6 nephrectomy rats displayed a microalbuminuria, which was attenuated by pClM treatment (Fig. 5C). Interestingly, excessive urinary microalbumin concentrations could be observed in those animals (all from the group of untreated CRF rats) that died before the intended sacrifice (Fig. 5D). Likewise, arterial hypertension could have contributed to both: left ventricular hypertrophy and microalbuminuria. In this study, blood pressure measurements could not be conducted. Nevertheless, the phosphate-rich diet and subsequent vascular pathologies might have contributed to potentially increased blood pressure levels. It should also be pointed out that the demonstration of potent phosphate binding in an animal model of CKD may not directly translate to similarly strong effects in humans. Nevertheless, based on our findings, further examinations are indicated due to the promising results in this study. The demonstrated higher binding efficacyi combined with the improved ability of pClM to reduce several cardiovascular risks compared to the clinically used phosphate adsorber LaC strongly suggest to proceed the investigation of pClM to elucidate its full potential.

In summary, pClM and LaC revealed beneficial effects in regard to the reduction of phosphate levels in blood and urine, retention parameters, and attenuating the decline of the GFR in CRF rats. However, pClM exhibited a stronger potential to counteract the development of cardiovascular pathologies. Remarkably, pClM was able to attenuate the decrease in GFR due to 5/6 nephrectomy, to prevent the transition of contractile VSMCs to cells with a secretory phenotype, and left ventricular hypertrophy. These results, especially the reduced excretion of microalbumin, suggest that the processed Montmorillonite-Illite clay mineral bears the potential as a highly potent phosphate binder to treat CKD-associated cardiovascular diseases, which makes it an intriguing candidate for further investigations.