Pathophysiology of the Chronic Kidney Disease – Mineral Bone Disorder (CKD-MBD)

Abstract

Purpose of review

The causes of excess cardiovascular mortality associated with chronic kidney disease (CKD) have been attributed in part to the CKD-mineral bone disorder syndrome (CKD-MBD), wherein, novel cardiovascular risk factors have been identified. The causes of the CKD-MBD are not well known and they will be discussed in this review

Recent findings

The discovery of WNT (portmanteau of wingless and int) inhibitors, especially Dickkopf 1 (Dkk1), produced during renal repair as participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical, and whose discovery lead to the finding that activin A is a second renal repair factor circulating in increased levels during CKD. Activin A derives from peritubular myofibroblasts of diseased kidneys, wherein it stimulates fibrosis, and decreases tubular klotho expression.The type 2 activin A receptor, ActRIIA, is induced by CKD in atherosclerotic aortas specifically in vascular smooth muscle cells (VSMC). Inhibition of ActRIIA signaling by a ligand trap inhibited CKD induced VSMC dedifferentiation, osteogenic transition and atherosclerotic calcification. Inhibition of ActRIIA signaling in the kidney decreased renal fibrosis and proteinuria.

Summary

These studies demonstrate that circulating renal repair factors are causal of the CKD-MBD and CKD associated cardiovascular disease, and identify ActRIIA signaling as a therapeutic target in CKD that links progression of renal disease and vascular disease.

Introduction

Kidney diseases are associated with an extremely high mortality, which is related to their production of cardiovascular disease [1]. The kidney disease produced increase in cardiovascular risk even extends to type 2 diabetes, where the presence of mild to moderate kidney disease increases atherosclerotic cardiovascular disease risk by 87% [2]. The causes of the increased cardiovascular risk associated with kidney diseases partly reside in the chronic kidney disease – mineral bone disorder (CKD-MBD) syndrome [3]. Three novel cardiovascular risk factors (hyperphosphatemia, vascular calcification, and elevated fibroblast growth factor 23 (FGF23) levels) have been discovered within the CKD-MBD [4-6], and their risk factor status confirmed in the general population [7-9]. The CKD-MBD begins early in CKD (stage 2) [10-13] consisting of vascular dedifferentiation/calcification, an osteodystrophy, loss of klotho and increased FGF23 secretion [10], and progress into its causes have been made [13-16], but they are mostly unknown.

Discoveries in the Pathogenesis of the CKD-MBD

Multiple investigators and we have shown that kidney diseases reactivate developmental programs involved in nephrogenesis during disease stimulated renal repair [17-21]. Among the nephrogenic factors reactivated in renal repair, the Wnt (portmanteau of Wingless and Integrated) family is critical for tubular epithelial reconstitution [20-22]. In the control of Wnt function, canonical signaling transcriptionally induces the expression of a family of Wnt inhibitory proteins which are secreted proteins that serve to restrict the distances of Wnt stimulation to autocrine or paracrine factors [23-27]. The Wnt inhibitors are circulating factors, and the family includes the Dickkopfs (Dkk). We have shown that various forms of kidney disease increase renal expression of Wnt inhibitors and increase their levels in the circulation [14, 18].

Neutralization of a key Wnt inhibitor elevated in the circulation in CKD, Dkk1, inhibited CKD induced vascular dedifferentiation, vascular calcification, and renal osteodystrophy [14]. This effect was surprising since Wnt signaling in the vascular smooth muscle is implicated in stimulating osteoblastic transition and vascular calcification [28, 29]. However, recent studies demonstrate that Dkk1 mediated inhibition of aortic Wnt7b stimulates smad mediated aortic endothelial-mesenchymal transition (EndMT) and vascular calcification [30]. EndMT is a developmental physiologic process involved in the development of the cardiac valves, the cardiac septum and the aortic root [31, 32], and it may [33] or may not [34] contribute to cardiac fibrosis in various adult disease states. Since EndMT is a process driven by smad transcription factors activated by factors in the transforming growth factor beta (TGFβ) superfamily [35], we investigated whether other factors involved in attempted renal repair during kidney disease derive from the TGFβ superfamily and are increased in the circulation during CKD. Of the TGFβ superfamily members, activin, a known renal developmental factor and circulating hormone, was the primary candidate [19, 36].

Activin is increased in the circulation by CKD associated with increased expression of activin in the kidney [37]. Surprisingly, the activin type 2A receptor (ActRIIA) was induced by CKD in the aortic vascular smooth muscle and not the endothelium. We demonstrated that inhibiting ActRIIA signaling using an ActRIIA ligand trap blocked CKD stimulated vascular smooth muscle osteoblastic transition, vascular calcification, and inhibited renal fibrosis [37]. We found that inhibiting activin signaling decreased renal Wnt activation and circulating Dkk1. As a result a composite vascular effect of indirectly increasing endothelial Wnt signaling through loss of Dkk1 in the circulation, and decreased vascular smooth muscle Wnt activation by blocking direct activin VSMC effects was produced by the ActRIIA ligand trap.

Key Pathogenic Components of the CKD-MBD: DKK1 and Activin

Much work needs to be done to characterize the effects of renal injury and the various kidney diseases on the production and maintenance of circulating factors causing cardiovascular and skeletal diseases, bringing the preclinical studies to human pathobiology. Although elevations in plasma DKK1, sclerostin (another Wnt inhibitor elevated in CKD and partly of renal origin) and activin have been found in human kidney diseases [37, 38], these studies are preliminary and need confirmation and characterization.

FGF23

FGF23 is the original phosphatonin (hormone regulating phosphate excretion) discovered in studies of autosomal dominant hypophosphatemic rickets and oncogenic osteomalacia [39 ]. FGF23 is produced by osteocytes and osteoblasts, and it represents direct bone-kidney and bone–parathyroid connections in the multiorgan systems biology involved in the CKD-MBD [40]. Circulating FGF23 levels rise after mild renal injury and progressively increase several fold during the course of CKD due to increased osteocyte secretion as well as decreased catabolism by the injured kidney. FGF23 levels rise prior to changes in calcium, phosphorus, or PTH levels and are now recognized as one of the earliest detectable biomarkers of the CKD-MBD [11, 41].

FGF23 levels have been associated with cardiovascular risk in CKD, and kidney transplant loss and mortality [42, 43]. In humans and animal models, Faul et al demonstrated that FGF23 is not only a biomarker associated with cardiovascular risk in CKD, but is also a direct pathogenic factor causing left ventricular hypertrophy (LVH) through activation of the calcineurin-NFAT pathway in cardiac myocytes [44].

Recently, the pathogenic nature of circulating FGF23 has become more intriguing. Andrukhova et al showed that FGF23 directly regulates the abundance of the thiazide-sensitive sodium-chloride transporter (NCC) in the distal convoluted tubule, leading to increased distal sodium reabsorption, effective circulating volume expansion, hypertension, and cardiac hypertrophy, effects that were abrogated by a thiazide diuretic [45]. Interestingly, these FGF23-mediated effects on cardiovascular pathophysiology were augmented in animal models ingesting a low sodium diet (which stimulates aldosterone secretion), raising the possibility of an interaction between FGF23 and the renin-angiotensin-aldosterone axis in CKD-stimulated cardiovascular disease. Furthermore, Humalda et al demonstrated that humans with higher baseline FGF23 levels had a reduced antiproteinuric response to dietary sodium restriction and ACE inhibitor therapy, which has been associated with heighted cardiovascular and end-stage renal disease (ESRD) risk in CKD [46]. Andrukhova et al also demonstrated that FGF23 promotes calcium reabsorption through stimulation of the apical calcium entry channel, TRPV5, in the distal tubule [47]. Because the calcium entry channel is also regulated by klotho [48], the Andrukhova et al findings [45, 47] raise the issue of the mechanism of klotho’s actions. Are they direct through glucuronidase activity and FGF23 independent, or as the FGF23 co-receptor and FGF23 dependent?

Klotho

FGF23 signaling through FGF receptors typically requires the co-receptor function of membrane-bound αklotho. Alpha klotho is highly expressed in very few tissues and defines the targets of FGF23 as the proximal and distal renal tubules, the parathyroid glands and the brain [49]. Klotho also circulates as a physiologically active hormone after either being cleaved at the cell surface by ADAM-10 and -17 in the distal renal tubule. Alternative splicing of the klotho gene transcript produces a soluble protein with only one klotho domain of unknown function [50]. Cleaved klotho directly regulates calcium and phosphorus excretion in the kidney and participates in systemic mineral homeostasis by regulating 1-alpha hydroxylase activity, PTH and FGF23 secretion[51, 52]. Klotho expression is significantly reduced by kidney injuries such as acute kidney injury, glomerulonephritis, calcineurin inhibitor use and chronic allograft injury [53]. We have shown that the reduction of klotho is in part related to activing and ActRIIA signaling [37]. The resulting klotho deficiency limits its regulation of FGF23 production and leaves hyperphosphatemia as the principal regulator of FGF23 secretion in CKD. Furthermore, the loss of membrane-bound klotho expression limits FGF23-stimulated signal transduction through FGF receptor/klotho complexes. One result is the loss of negative feedback to FGF23 secretion and the continual production of FGF23 and secretion by the osteocyte. In late CKD, the very high levels of FGF23 permit anomalous FGF receptor activation independent of Klotho and result in unique FGF23-stimulated pathologies such as cardiac myocyte hypertrophy[44]. In addition, recent mechanistic studies have directly linked klotho deficiency with cardiovascular disease including vascular calcification, vascular stiffness, and uremic vasculopathy [13].

Hyperphosphatemia

As renal injury decreases the number of functioning nephrons, phosphate excretion is maintained by reducing the tubular reabsorption of filtered phosphate in the remaining nephrons under the influence of FGF23 and PTH[54]. The effects of FGF23 on phosphate excretion are limited by proximal tubular klotho deficiency in CKD, and PTH becomes the major adaptive mechanism maintaining phosphate homeostasis. In stage 4-5 CKD (GFR < 30% of normal), this adaptation is no longer adequate and hyperphosphatemia develops despite high PTH and FGF23 levels[54].

CKD contributes to hyperphosphatemia and vascular calcification through inhibition of skeletal function. Bone resorption increases phosphate release to the plasma and decreases phosphate deposition resulting in increased serum phosphorus levels [55]. Hyperphosphatemia stimulates osteoblastic transition in the vasculature and directly contributes to extraskeletal mineralization through an elevated calcium-phosphorus product [56].

Hyperphosphatemia exerts other important effects in the CKD-MBD axis. In the kidney, hyperphosphatemia suppresses 1-alpha-hydroxylase activity that further contributes to calcitriol deficiency [57]. In the parathyroid gland, hyperphosphatemia directly stimulates parathyroid cells independent of calcium and calcitriol levels, producing nodular hyperplasia and increasing PTH secretion [58]. In the skeleton, phosphorus stimulates FGF23 secretion from osteocytes [59, 60].

Vitamin D deficiency

In early CKD, the physiologic actions of FGF23 secretion from the osteocyte include inhibition of 1-alpha-hydroxylase and stimulation of 24-hydroxylase in proximal renal tubules, thereby decreasing calcitriol production and producing 25-hydroxyvitamin D deficiency [61]. As CKD advances, the decrease in functioning nephron mass combined with hyperphosphatemia and increased FGF23 levels results in calcitriol (1,25-hydroxyvitamin D) deficiency as well [62]. Calcitriol deficiency decreases intestinal calcium absorption leading to hypocalcemia and diminishes tissue levels of vitamin D receptors, which in the parathyroid gland results in resistance to calcitriol-mediated regulation and stimulation of PTH secretion leading to secondary hyperparathyroidism [63].

Hyperparathyroidism

PTH regulates secretion of FGF23 and is required for the early stimulation of FGF23 secretion [64], which is the earliest detected abnormality of the CKD-MBD [41]. Thus, there is a regulation of PTH secretion early in CKD that remains to be clarified. As CKD progresses, components of the CKD-MBD result in increased production of PTH and nodular hyperplasia of the parathyroid glands. Sustained elevation in PTH levels, while adaptive to maintain osteoblast surfaces, are associated with an abnormal phenotype of osteoblast function and osteocyte stimulation with relatively less type 1 collagen and more RANKL ligand production than anabolic osteoblasts [65]. New studies discussed below indicate that other factors such as FGF23 and activin may impact osteoblast function besides PTH, and produce the mineralization disorder of CKD changing the material properties of bone. The outcome is a high turnover renal osteodystrophy, excess bone resorption, skeletal frailty and elevated fracture risk [66].

Renal osteodystrophy

With progressive loss of renal function, cancellous bone volume may be increased along with a loss of cortical bone, but this is in part due to deposition of woven immature collagen fibrils instead of lamellar mature fibrils. Thus, bone strength suffers despite an apparent increase in mass detected by dual energy x-ray absorptiometry (DXA) [67]. Patients with advanced CKD could have a loss or gain in bone volume depending on overall bone balance. When the bone balance is positive, osteosclerosis may be observed when osteoblasts are active in depositing new bone composed primarily of immature woven collagen. However, this scenario is rare in the 21st century due to improved therapy of secondary hyperparathyroidism. When the bone balance is negative both cortical and cancellous bone loss occurs, resulting in osteopenia or osteoporosis detected by DXA. The prevalence of osteoporosis in CKD patients exceeds that of the general population and is a major public health concern in CKD [68]. With high-turnover renal osteodystrophy, as in secondary hyperparathyroidism with osteitis fibrosa, bone resorption rates are in excess of bone formation and osteopenia progressing to osteoporosis may result [69]. With low-turnover renal osteodystrophy, as occurs with over-treatment of secondary hyperparathyroidism that inappropriately suppresses PTH-stimulated skeletal remodeling, both bone formation and resorption rates may be reduced although resorption is still in relative excess and loss of bone mass occurs [70]. Therefore, osteoporosis may be observed with either high-turnover or low-turnover renal osteodystrophy. The impact of this phenomenon extends far beyond bone health in CKD, as excessive bone resorption rates contribute to hyperphosphatemia with stimulation of heterotopic mineralization including vascular calcification [56]. This disrupted systems biology links kidney, skeletal, and parathyroid dysfunction to cardiovascular risk and mortality through the CKD-MBD.

Cardiovascular disease

The CKD-MBD is a contributing factor to vascular stiffness and calcification that increases the systolic blood pressure, pulse wave velocity, and left ventricular mass, all of which are surrogates for cardiovascular risk in the general population and in those with CKD [71]. Structural and functional abnormalities of the vasculature are seen in early CKD, including vascular stiffness and endothelial dysfunction that progress to vascular calcification, a common phenomenon in the aging general population that is accelerated in CKD to the highest level seen in clinical medicine. Vascular calcification further intensifies vascular stiffness and promotes the development of LVH, all processes that contribute to cardiovascular risk and excess cardiac mortality in native and transplant CKD.

In animal models with mild renal insufficiency (equivalent to human stage 2 CKD), we have demonstrated that expression of proteins involved in the contractile apparatus of aortic smooth muscle cells are decreased, reflecting a dedifferentiated state of the vasculature in early CKD [14]. Within the developmental program of mesenchymal stem cells and early vascular progenitors, dedifferentiated vascular smooth muscle cells are susceptible to osteoblastic transition, which contributes to vascular calcification in CKD. Osteoblastic transition of vascular smooth muscle cells produces CKD-stimulated calcification of atherosclerotic plaques as well as the tunica media, resulting in either neointimal or medial vascular calcification [72].

Emerging Concepts in the Systems Biology of the CKD-MBD: The Wnt pathway and reactivation of renal repair mechanisms in the CKD-MBD

Kidney injuries produce circulating signals that directly affect the vasculature, the myocardium and the skeleton. These signals are derived from reactivation of developmental programs of nephrogenesis in an attempt at kidney repair, which are typically silent in the normal adult kidney [14]. The classic example is the reactivation of the Wnt pathway that controls tubular epithelial proliferation and polarity during nephrogenesis and is a driving force in renal fibrosis [21]. Activation of the canonical Wnt pathway increases expression of downstream transcriptional targets, including Wnt inhibitors that function in a negative feedback loop to autoregulate Wnt activation. These Wnt inhibitors include Dickkopf-1 (Dkk1), soluble frizzled related proteins, Wnt-modulator in surface ectoderm (Wise), and sclerostin among others. While Wnts are strictly autocrine/paracrine factors, the Wnt inhibitors also function as circulating systemic factors [73]. The role of Wnt in renal development largely precedes the invasion of the microcirculation forming the glomerulus and the peritubular capillaries. Therefore, while the Wnt inhibitors did not evolve as circulating factors produced by the normal kidney, during kidney injury and repair they are released into the systemic circulation and may inhibit the physiologic roles of Wnt in extrarenal tissues [14].

We and others have recently shown this “unintended” systemic inhibition of Wnt activity stimulated by kidney disease to have major consequences in the skeleton and vasculature. In animal models of early CKD, incomplete recovery from acute kidney injury led to increased expression of Wnt inhibitors (e.g., Dkk1, sclerostin) in the injured kidney and increased levels in the systemic circulation [14]. The skeleton was affected through both changes in remodeling (decreased bone formation rates) and in secretory properties of the osteocytes (increased FGF23 secretion). The cardiovascular system was affected through loss of vascular contractile machinery and dedifferentiation of vascular smooth muscle cells, stimulation of osteoblastic transition and vascular calcification, and promotion of cardiac hypertrophy [14, 37]. Neutralization of circulating Dkk1 using a monoclonal antibody resulted in increased bone formation rates and bone volume, improved vascular function, and decreased osteoblastic transition and vascular calcification [14].

Conclusion and Future Directions

The CKD-MBD defines a disruption in the systems biology between the injured kidney, skeleton, and cardiovascular system that has a profoundly negative impact on survival in CKD. Recent translational discoveries have introduced a new paradigm where kidney injury directly leads to skeletal and cardiovascular injury through the production of pathogenic circulating factors during attempted renal repair, including molecules that inhibit the canonical Wnt pathway and stimulate endothelial-to-mesenchymal transition, both processes that have been implicated in chronic allograft injury as well as cardiovascular disease. Future studies must clarify whether incomplete recovery from acute kidney injury is sufficient to stimulate these disturbances in the kidney-skeletal-cardiovascular axis that contribute to decreased patient and allograft survival. This would identify the early CKD-MBD as an important therapeutic target for improving long-term outcomes in CKD.