Vascular calcification maladaptively participates in acute phosphate homeostasis.

AIMS: Non-renal extravasation of phosphate from the circulation and transient accumulation into tissues and extracellular fluid is a regulated process of acute phosphate homeostasis that is not well understood. This process is especially relevant in the setting of chronic kidney disease (CKD), where exposure to increased phosphate is prolonged due to inefficient kidney excretion. Furthermore, CKD-associated mineral dysregulation induces pathological accumulation of phosphate causing vascular calcification (VC). Our objective was to determine whether the systemic response to acute phosphate challenges is altered by VC. METHODS AND RESULTS: After bolus phosphate administration, circulating and tissue deposition of this challenge was assessed in two rat models of VC using a radiolabelled phosphate tracer. In an adenine-induced model of CKD (N = 70), animals with VC had a blunted elevation of circulating 33PO4 following oral phosphate administration (P < 0.01), and the discordant deposition could be traced to the calcified arteries (11.4 [7.5-13.1] vs.43.0 [35.5-53.7] pmol/ng tissue, P < 0.001). In a non-CKD model of VC, calcification was induced with 0.5 ug/kg calcitriol and then withdrawn (N = 24). New phosphate uptake by the calcified vasculature correlated to the pre-existing burden of calcification (r = 38, P < 0.001) and was substantially attenuated in the absence of calcification stimulus (P < 0.01). Phosphate accrual was stimulated by the phosphate challenge and not present to the same degree during passive disposition of circulating phosphate. Further, the form of phosphate that deposited to the vasculature was predominately amorphous inorganic phosphate and not that which was bound in matured calciprotein particles. CONCLUSIONS: In the process of calcification, arteries acutely deposit substantial amorphous phosphate while blunting the elevation in the circulation, thereby altering the systemic disposition of phosphate and identifying VC as a participatory mineral homeostatic organ. This study demonstrates the negative vascular consequence of acute fluctuations in circulating phosphate, and supports the importance of phosphate bioavailability and diet management in CKD patients as a mediator of cardiovascular risk.

Development of experimental chronic kidney disease and vascular calcification alters diurnal variation of phosphate and its hormonal regulators.

The mineral-bone axis is tightly regulated and dependent on renal function. In chronic kidney disease (CKD) progressive loss of renal capacity disrupts this axis over-time, with marked changes in circulating calcium, phosphate, PTH, and fibroblast growth factor-23 (FGF-23). These changes contribute to the development of cardiovascular disease, like vascular calcification (VC), which worsens morbidity and mortality in CKD. Although the chronic changes in these circulating factors and their relationships are well known, no experimental studies have examined how the progressive development of CKD and VC alter the circadian rhythms of these factors. An adenine-induced experimental model of CKD in rats was used to establish (i) general circulating trends, (ii) if renal dysfunction affects these observed trends, and (iii) identify potential changes in these trends caused by VC. This study clearly discerned patterns of daily variations in circulating minerals and hormones, finding that both phosphate and PTH follow modelable diurnal variations whereas calcium and FGF-23 maintain relative stability over 24-hr. Surprisingly, the development of CKD was not sufficient to disrupt these patterns of diurnal variation and only altered the magnitude of change; however, it was found that the diurnal rhythms of circulating phosphate and daily stability of calcium were only significantly altered in the setting of CKD with established VC.

PTH suppression by calcitriol does not predict off-target actions in experimental CKD.

Vitamin D receptor agonist (VDRA) therapy for PTH suppression is a mainstay for patients with severe CKD. Calcitriol (1,25-(OH)2 D3 ) is a former first-line VDRA in CKD treatment. However, a consequence of its use in CKD is accelerated vascular calcification (VC). An experimental CKD model was used to determine whether altering the calcitriol delivery profile to obtain different PTH suppression levels could improve vascular health outcomes. High adenine diet (0.25%) was used to generate experimental CKD in rats. CKD rats were treated using different calcitriol dosing strategies: (a) 20 ng/kg SD (n = 8), (b) 80 ng/kg SD (n = 8), (c) 5 ng/kg QID (n = 9), or (d) 20 ng/kg QID (n = 9). Multiple targets of calcitriol were assessed which include arterial calcium and phosphate as well as circulating calcium, phosphate, PTH, FGF-23, VWF, and vitamin D metabolome. PTH suppression occurred dose-dependently after 1-week calcitriol treatment (P < .01), but the suppressive effect was lost over time. Both VC and circulating FGF-23 increased > 10× in all calcitriol-treated rats (P < .05 and P < .001, respectively); similarly, circulating VWF increased at all time points (P < .05). Ad-hoc analysis of CKD morbidities in treated rats indicated no differences in negative outcomes based on PTH suppression level (minimal-, target-, and over-). Comparing different calcitriol dosing strategies revealed the following: (a) despite initial calcitriol-influenced PTH suppression across all treatments, the ability to continually suppress PTH was markedly reduced by study conclusion and (b) PTH suppression level is not an adequate proxy for improvements in overall CKD morbidity. These findings show (a) a more holistic approach to evaluate CKD treatment efficacy aside from PTH suppression is needed and (b) that other VDRA therapies should be examined in CKD treatment.

Acute Tissue Mineral Deposition in Response to a Phosphate Pulse in Experimental CKD.

Pathogenic accumulation of calcium (Ca) and phosphate (PO4 ) in vasculature is a sentinel of advancing cardiovascular disease in chronic kidney disease (CKD). This study sought to characterize acute distribution patterns of radiolabeled 33 PO4 and 45 Ca in cardiovascular tissues of rats with CKD (0.25% dietary adenine). The disposition of 33 PO4 and 45 Ca was assessed in blood and 36 tissues after a 10-minute intravenous infusion of one of the following: (i) PO4 pulse + tracer 33 PO4 ; (ii) PO4 pulse + tracer 45 Ca; or (iii) saline + tracer 45 Ca in CKD and non-CKD animals. After the infusion, 33 PO4 in blood was elevated (2.3× at 10 minutes, 3.5× at 30 minutes, p < 0.05) in CKD compared with non-CKD. In contrast, there was no difference in clearance of 45 Ca from the blood. Compared with controls, CKD rats had a markedly increased 33 PO4 incorporation in several tissues (skeletal muscle, 7.8×; heart, 5.5×), but accrual was most pronounced in the vasculature (24.8×). There was a significant, but smaller, increase in 45 Ca accrual in the vasculature of CKD rats (1.25×), particularly in the calcified rat, in response to the acute phosphate load. Based on the pattern of tissue uptake of 33 PO4 and 45 Ca, this study revealed that an increase in circulating PO4 is an important stimulus for the accumulation of these minerals in vascular tissue in CKD. This response is further enhanced when vascular calcification is also present. The finding of enhanced vascular mineral deposition in response to an acute PO4 pulse provides evidence of significant tissue-specific susceptibility to calcification. © 2018 American Society for Bone and Mineral Research.

Developmental Origins for Kidney Disease Due to Shroom3 Deficiency.

CKD is a significant health concern with an underlying genetic component. Multiple genome-wide association studies (GWASs) strongly associated CKD with the shroom family member 3 (SHROOM3) gene, which encodes an actin-associated protein important in epithelial morphogenesis. However, the role of SHROOM3 in kidney development and function is virtually unknown. Studies in zebrafish and rat showed that alterations in Shroom3 can result in glomerular dysfunction. Furthermore, human SHROOM3 variants can induce impaired kidney function in animal models. Here, we examined the temporal and spatial expression of Shroom3 in the mammalian kidney. We detected Shroom3 expression in the condensing mesenchyme, Bowman's capsule, and developing and mature podocytes in mice. Shroom3 null (Shroom3Gt/Gt) mice showed marked glomerular abnormalities, including cystic and collapsing/degenerating glomeruli, and marked disruptions in podocyte arrangement and morphology. These podocyte-specific abnormalities are associated with altered Rho-kinase/myosin II signaling and loss of apically distributed actin. Additionally, Shroom3 heterozygous (Shroom3Gt/+) mice showed developmental irregularities that manifested as adult-onset glomerulosclerosis and proteinuria. Taken together, our results establish the significance of Shroom3 in mammalian kidney development and progression of kidney disease. Specifically, Shroom3 maintains normal podocyte architecture in mice via modulation of the actomyosin network, which is essential for podocyte function. Furthermore, our findings strongly support the GWASs that suggest a role for SHROOM3 in human kidney disease.

Magnesium Modifies the Impact of Calcitriol Treatment on Vascular Calcification in Experimental Chronic Kidney Disease.

Chronic kidney disease (CKD) patients are commonly treated with vitamin D analogs, such as calcitriol. Recent epidemiologic evidence revealed a significant interaction between vitamin D and magnesium, since an inverse relationship between vitamin D levels and mortality mainly occurs in patients with a high magnesium intake. The aim of the study was to assess the mechanisms involved by determining whether magnesium alone or combined with calcitriol treatments differentially impacts vascular calcification (VC) in male Sprague-Dawley rats with adenine-induced CKD. Treatment with moderate doses of calcitriol (80 µg/kg) suppressed parathyroid hormone to near or slightly below control levels. Given alone, this dose of calcitriol increased the prevalence of VC; however, when magnesium was given in combination, the severity of calcification was attenuated in the abdominal aorta (51% reduction), iliac (44%), and carotid arteries (46%) compared with CKD controls. The decreases in vascular calcium content were associated with a 20-50% increase in vascular magnesium. Calcitriol treatment alone significantly decreased TRPM7 protein (↓ to ∼11%), whereas the combination treatment increased both mRNA (1.7×) and protein (6.8×) expression compared with calcitriol alone. In summary, calcitriol increased VC in certain conditions, but magnesium prevented the reduction in TRPM7 and reduced the severity of VC, thereby increasing the bioavailable magnesium in the vascular microenvironment. These findings suggest that modifying the adverse effect profile of calcitriol with magnesium may be a plausible approach to benefiting the increasing number of CKD patients being prescribed calcitriol.

Stromally expressed ß-catenin modulates Wnt9b signaling in the ureteric epithelium.

The mammalian kidney undergoes cell interactions between the epithelium and mesenchyme to form the essential filtration unit of the kidney, termed the nephron. A third cell type, the kidney stroma, is a population of fibroblasts located in the kidney capsule, cortex and medulla and is ideally located to affect kidney formation. We found ß-catenin, a transcriptional co-activator, is strongly expressed in distinctive intracellular patterns in the capsular, cortical, and medullary renal stroma. We investigated ß-catenin function in the renal stroma using a conditional knockout strategy that genetically deleted ß-catenin specifically in the renal stroma cell lineage (ß-cats-/-). ß-cats-/- mutant mice demonstrate marked kidney abnormalities, and surprisingly we show ß-catenin in the renal stroma is essential for regulating the condensing mesenchyme cell population. We show that the population of induced mesenchyme cells is significantly reduced in ß-cats-/- mutants and exhibited decreased cell proliferation and a specific loss of Cited 1, while maintaining the expression of other essential nephron progenitor proteins. Wnt9b, the key signal for the induction of nephron progenitors, was markedly reduced in adjacent ureteric epithelial cells in ß-cats-/-. Analysis of Wnt9b-dependent genes in the neighboring nephron progenitors was significantly reduced while Wnt9b-independent genes remained unchanged. In contrast mice overexpressing ß-catenin exclusively in the renal stroma demonstrated massive increases in the condensing mesenchyme population and Wnt9b was markedly elevated. We propose that ß-catenin in the renal stroma modulates a genetic program in ureteric epithelium that is required for the induction of nephron progenitors.

Insights into the renal pathogenesis in Schimke immuno-osseous dysplasia: A renal histological characterization and expression analysis.

Schimke immuno-osseous dysplasia (SIOD) is a pleiotropic disorder caused by mutations in the SWI/SNF2-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like-1 (SMARCAL1) gene, with multiple clinical features, notably end-stage renal disease. Here we characterize the renal pathology in SIOD patients. Our analysis of SIOD patient renal biopsies demonstrates the tip and collapsing variants of focal segmental glomerulosclerosis (FSGS). Additionally, electron microscopy revealed numerous glomerular abnormalities most notably in the podocyte and Bowman's capsule. To better understand the role of SMARCAL1 in the pathogenesis of FSGS, we defined SMARCAL1 expression in the developing and mature kidney. In the developing fetal kidney, SMARCAL1 is expressed in the ureteric epithelium, stroma, metanephric mesenchyme, and in all stages of the developing nephron, including the maturing glomerulus. In postnatal kidneys, SMARCAL1 expression is localized to epithelial tubules of the nephron, collecting ducts, and glomerulus (podocytes and endothelial cells). Interestingly, not all cells within the same lineage expressed SMARCAL1. In renal biopsies from SIOD patients, TUNEL analysis detected marked increases in DNA fragmentation. Our results highlight the cells that may contribute to the renal pathogenesis in SIOD. Further, we suggest that disruptions in genomic integrity during fetal kidney development contribute to the pathogenesis of FSGS in SIOD patients.

ß-Catenin overexpression in the metanephric mesenchyme leads to renal dysplasia genesis via cell-autonomous and non-cell-autonomous mechanisms.

Renal dysplasia, a developmental disorder characterized by defective ureteric branching morphogenesis and nephrogenesis, ranks as one of the major causes of renal failure among the pediatric population. Herein, we demonstrate that the levels of activated ß-catenin are elevated in the nuclei of ureteric, stromal, and mesenchymal cells within dysplastic human kidney tissue. By using a conditional mouse model of mesenchymal ß-catenin overexpression, we identify two novel signaling pathways mediated by ß-catenin in the development of renal dysplasia. First, the overexpression of ß-catenin within the metanephric mesenchyme leads to ectopic and disorganized branching morphogenesis caused by ß-catenin directly binding Tcf/lef consensus binding sites in the Gdnf promoter and up-regulating Gdnf transcription. Second, ß-catenin overexpression in the metanephric mesenchyme leads to elevated levels of transcriptionally active ß-catenin in the ureteric epithelium. Interestingly, this increase of ß-catenin-mediated transcription results from a novel Ret/ß-catenin signaling pathway. Consistent with these findings, analysis of human dysplastic renal tissue demonstrates that undifferentiated mesenchymal cells expressing high levels of ß-catenin also express increased GDNF. Furthermore, dysplastic ureteric tubules that were surrounded by high levels of GDNF also exhibited increased levels of activated ß-catenin. Together, these data support a model in which the elevation of ß-catenin in the metanephric mesenchyme results in cell-autonomous and non-cell-autonomous events that lead to the genesis of renal dysplasia.