Milica Bozic

Beyond proteinuria: VDR activation reduces renal inflammation in experimental diabetic nephropathy

Local inflammation is thought to contribute to the progression of diabetic nephropathy. The vitamin D receptor (VDR) activator paricalcitol has an antiproteinuric effect in human diabetic nephropathy at high doses. We have explored potential anti-inflammatory effects of VDR activator doses that do not modulate proteinuria in an experimental model of diabetic nephropathy to gain insights into potential benefits of VDR activators in those patients whose proteinuria is not decreased by this therapy. The effect of calcitriol and paricalcitol on renal function, albuminuria, and renal inflammation was explored in a rat experimental model of diabetes induced by streptozotocin. Modulation of the expression of mediators of inflammation by these drugs was explored in cultured podocytes. At the doses used, neither calcitriol nor paricalcitol significantly modified renal function or reduced albuminuria in experimental diabetes. However, both drugs reduced the total kidney mRNA expression of IL-6, monocyte chemoattractant protein (MCP)-1, and IL-18. Immunohistochemistry showed that calcitriol and paricalcitol reduced MCP-1 and IL-6 in podocytes and tubular cells as well as glomerular infiltration by macrophages, glomerular cell NF-κB activation, apoptosis, and extracellular matrix deposition. In cultured podocytes, paricalcitol and calcitriol at concentrations in the physiological and clinically significant range prevented the increase in MCP-1, IL-6, renin, and fibronectin mRNA expression and the secretion of MCP-1 to the culture media induced by high glucose. In conclusion, in experimental diabetic nephropathy VDR activation has local renal anti-inflammatory effects that can be observed even when proteinuria is not decreased. This may be ascribed to decreased inflammatory responses of intrinsic renal cells, including podocytes, to high glucose.

Glutamatergic Signaling Maintains the Epithelial Phenotype of Proximal Tubular Cells

Epithelial-mesenchymal transition (EMT) contributes to the progression of renal tubulointerstitial fibrosis. The N-methyl-d-aspartate receptor (NMDAR), which is present in proximal tubular epithelium, is a glutamate receptor that acts as a calcium channel. Activation of NMDAR induces actin rearrangement in cells of the central nervous system, but whether it helps maintain the epithelial phenotype of the proximal tubule is unknown. Here, knockdown of NMDAR1 in a proximal tubule cell line (HK-2) induced changes in cell morphology, reduced E-cadherin expression, and increased α-SMA expression. Induction of EMT with TGF-β1 led to downregulation of both E-cadherin and membrane-associated β-catenin, reorganization of F-actin, expression of mesenchymal markers de novo, upregulation of Snail1, and increased cell migration; co-treatment with NMDA attenuated all of these changes. Furthermore, NMDA reduced TGF-β1-induced phosphorylation of Erk1/2 and Akt and the activation of Ras, suggesting that NMDA antagonizes TGF-β1-induced EMT by inhibiting the Ras-MEK pathway. In the unilateral ureteral obstruction model, treatment with NMDA blunted obstruction-induced upregulation of α-SMA, FSP1, and collagen I and downregulation of E-cadherin. Taken together, these results suggest that NMDAR plays a critical role in preserving the normal epithelial phenotype and modulating tubular EMT.

The potential of targeting NMDA receptors outside the CNS

Introduction: NMDA receptor (NMDAR) is an ionotropic glutamate receptor with a high permeability to calcium and a unique feature of controlling numerous calcium-dependent processes. Apart from being widely distributed in the CNS, the presence of NMDAR and its potential significance in a variety of non-neuronal cells and tissues has become an interesting research topic. Areas covered: The current review summarizes prevailing knowledge on the role of NMDARs in the kidney, bone and parathyroid gland, three main organs responsible for calcium homeostasis, as well as in the heart, an organ whose function is highly dependable on balanced intracellular calcium concentrations. The review also examines studies that have advanced our understanding of the therapeutic potential of NMDAR agonists and antagonists in renal, cardiovascular and bone pathologies. Expert opinion: NMDARs have a preeminent role in many physiological and pathological processes outside the CNS. In certain organs and/or disease conditions, activating the NMDAR leads to beneficial effects for the target organ, whereas in other diseases cell signaling downstream of NMDAR activation can exacerbate their pathology. Therefore, targeting NMDARs therapeutically is rather intricate work, and surely requires more extensive investigation in order to properly tune up the diverse NMDAR’s actions translating them into beneficial cellular responses.

Impaired Vitamin D Signaling in Endothelial Cell Leads to an Enhanced Leukocyte-Endothelium Interplay: Implications for Atherosclerosis Development

Endothelial cell activation leading to leukocyte recruitment and adhesion plays an essential role in the initiation and progression of atherosclerosis. Vitamin D has cardioprotective actions, while its deficiency is a risk factor for the progression of cardiovascular damage. Our aim was to assess the role of basal levels of vitamin D receptor (VDR) on the early leukocyte recruitment and related endothelial cell-adhesion-molecule expression, as essential prerequisites for the onset of atherosclerosis. Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, characterized by upregulation of VCAM-1, ICAM-1 and IL-6, decreased peripheral blood mononuclear cell (PBMC) rolling velocity and increased PBMC rolling flux and adhesion to the endothelium. shVDR cells showed decreased IκBα levels and accumulation of p65 in the nucleus compared to shRNA controls. Inhibition of NF-κB activation with super-repressor IκBα blunted all signs of endothelial cell activation caused by downregulation of VDR in endothelial cells. In vivo, deletion of VDR led to significantly larger aortic arch and aortic root lesions in apoE-/- mice, with higher macrophage content. apoE-/-VDR-/-mice showed higher aortic expression of VCAM-1, ICAM-1 and IL-6 when compared to apoE-/-VDR+/+ mice. Our data demonstrate that lack of VDR signaling in endothelial cells leads to a state of endothelial activation with increased leukocyte-endothelial cell interactions that may contribute to the more severe plaque accumulation observed in apoE-/-VDR-/- mice. The results reveal an important role for basal levels of endothelial VDR in limiting endothelial cell inflammation and atherosclerosis.

High phosphate diet increases arterial blood pressure via a parathyroid hormone mediated increase of renin.

Background: There is growing evidence suggesting that phosphate intake is associated with blood pressure levels. However, data from epidemiological studies show inconsistent results. Method and results: The present study was designed to evaluate the effect of high circulating phosphorus on arterial blood pressure of healthy rats and to elucidate the potential mechanism that stands behind this effect. Animals fed a high phosphate diet for 4 weeks showed an increase in blood pressure, which returned to normal values after the addition of a phosphate binder (lanthanum carbonate) to the diet. The expression of renin in the kidney was higher, alongside an increase in plasma renin activity, angiotensin II (Ang II) levels and left ventricular hypertrophy. The addition of the phosphate binder blunted the increase in renin and Ang II levels. The levels of parathyroid hormone (PTH) were also higher in animals fed a high phosphate diet, and decreased when the phosphate binder was present in the diet. However, blood P levels remained elevated. A second group of rats underwent parathyroidectomy and received a continuous infusion of physiological levels of PTH through an implanted mini-osmotic pump. Animals fed a high phosphate diet with continuous infusion of PTH did not show an increase in blood pressure, although blood P levels were elevated. Finally, unlike with verapamil, the addition of losartan to the drinking water reverted the increase in blood pressure in rats fed a high phosphate diet. Conclusion: The results of this study suggest that a high phosphate diet increases arterial blood pressure through an increase in renin mediated by PTH.

Hepatocyte vitamin D receptor regulates lipid metabolism and mediates experimental diet-induced steatosis.

BACKGROUND & AIMS The pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD) is still incompletely understood. Several nuclear receptors play a role in liver lipid metabolism and can promote hepatosteatosis, but the possible role of vitamin D receptor (VDR) in NAFLD has not been investigated. METHODS The expression of liver VDR was investigated in apolipoprotein E knockout (apoE(-/-)) mice on a high fat diet, in wild-type mice on methionine and choline deficient diet and in NAFLD patients with hepatosteatosis and non-alcoholic steatohepatitis. The relevance of VDR was assessed in apoE(-/-) mice by deletion of VDR or paricalcitol treatment and in human HepG2 cells by VDR transfection or silencing. The role of VDR in fibrosis was also determined in VDR knockout mice (VDR(-/-)) treated with thioacetamide. RESULTS Expression of liver VDR was markedly induced in two mouse models of NAFLD, as well as in patients with hepatosteatosis, but decreased in non-alcoholic steatohepatitis. VDR deletion in high fat diet-fed apoE(-/-) mice protected against fatty liver, dyslipidemia and insulin resistance, and caused a decrease in taurine-conjugated bile acids, but did not influence fibrosis by thioacetamide. apoE(-/-)VDR(-/-) mouse livers showed decreased gene expression of CD36, DGAT2, C/EBPα and FGF21, and increased expression of PNPLA2, LIPIN1 and PGC1α. Treatment of apoE(-/-) mice on high fat diet with paricalcitol had modest opposite effects on steatosis and gene expression. Finally, this set of genes showed concordant responses when VDR was overexpressed or silenced in HepG2 cells. CONCLUSIONS Induced hepatocyte VDR in NAFLD regulates key hepatic lipid metabolism genes and promotes high fat diet-associated liver steatosis. Therapeutic inhibition of liver VDR may reverse steatosis in early NAFLD. LAY SUMMARY The amount of vitamin D receptor is induced early in the livers of mice and humans when they develop non-alcoholic fatty liver disease. If the gene for the vitamin D receptor is deleted, hepatic lipid metabolism changes and mice do not accumulate fat in the liver. We conclude that the vitamin D receptor can contribute to the fatty liver disease promoted by a high fat diet.

Sustained activation of renal N-methyl-d-aspartate receptors decreases vitamin D synthesis: a possible role for glutamate on the onset of secondary HPT

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are tetrameric amino acid receptors that act as membrane calcium channels. The presence of the receptor has been detected in the principal organs responsible for calcium homeostasis (kidney, bone, and parathyroid gland), pointing to a possible role in mineral metabolism. The aim of this study was to test the effect of NMDAR activation in the kidney and on 1,25(OH)₂D₃ synthesis. We determined the presence of NMDAR subunits in HK-2 (human kidney cells) cells and proved its functionality. NMDA treatment for 4 days induced a decrease in 1α-hydroxylase levels and 1,25(OH)₂D₃ synthesis through the activation of the MAPK/ERK pathway in HK-2 cells. In vivo administration of NMDA for 4 days also caused a decrease in blood 1,25(OH)₂D₃ levels in healthy animals and an increase in blood PTH levels. This increase in PTH induced a decrease in the urinary excretion of calcium and an increase in urinary excretion of phosphorous and sodium as well as in diuresis. Bone turnover markers also increased. Animals with 5/6 nephrectomy showed low levels of renal 1α-hydroxylase as well as high levels of renal glutamate compared with healthy animals. In conclusion, NMDAR activation in the kidney causes a decrease in 1,25(OH)₂D₃ synthesis, which induces an increase on PTH synthesis and release. In animals with chronic kidney disease, high renal levels of glutamate could be involved in the downregulation of 1α-hydroxylase expression.

Calcium Signaling in Renal Tubular Cells

The kidney handles calcium by filtration and reabsorption. About 60% of the plasma calcium is filterable, and 99% is reabsorbed in the tubule. In the proximal tubule, the reabsorption is passive and paracellular, but in the distal tubule is active and transcellular. Thus, renal tubular cells are exposed to very high concentrations of calcium in both, the extracellular and the intracellular compartments. Extracellular calcium signaling is transmitted by the calcium sensing receptor, located both in the luminal and basolateral sides of tubular cells. This receptor is able to control levels of extracellular calcium and acts in consequence to maintain calcium homeostasis. Furthermore, renal tubular cells possess several calcium channels that regulate some of the cell functions. Among those, voltage gated calcium channels, transient receptor potential channels and N-methyl-D-aspartate receptor channels have been reported to control several functions. Those functions include survival, apoptosis, differentiation, epithelial-mesenchymal transition, and active vitamin D and renin synthesis.

Vascular Calcification Induced by Chronic Kidney Disease Is Mediated by an Increase of 1α-Hydroxylase Expression in Vascular Smooth Muscle Cells

Vascular calcification (VC) is a complication of chronic kidney disease that predicts morbidity and mortality. Uremic serum promotes VC, but the mechanism involved is unknown. A role for 1,25(OH)2D3 in VC has been proposed, but the mechanism is unclear because both low and high levels have been shown to increase it. In this work we investigate the role of 1,25(OH)2D3 produced in vascular smooth muscle cells (VSMCs) in VC. Rats with subtotal nephrectomy and kidney recipient patients showed increased arterial expression of 1α‐hydroxylase in vivo. VSMCs exposed in vitro to serum obtained from uremic rats also showed increased 1α‐hydroxylase expression. Those increases were parallel to an increase in VC. After 6 days with high phosphate media, VSMCs overexpressing 1α‐hydroxylase show significantly higher calcium content and RUNX2 expression than control cells. 1α‐hydroxylase null mice (KO) with subtotal nephrectomy and treated with calcitriol (400 ng/kg) for 2 weeks showed significantly lower levels of vascular calcium content, Alizarin red staining, and RUNX2 expression than wild‐type (WT) littermates. Serum calcium, phosphorus, blood urea nitrogen (BUN), PTH, and 1,25(OH)2D3 levels were similar in both calcitriol‐treated groups. In vitro, WT VSMCs treated with uremic serum also showed a significant increase in 1α‐hydroxylase expression and higher calcification that was not observed in KO cells. We conclude that local activation of 1α‐hydroxylase in the artery mediates VC observed in uremia.

Association of FGF-2 Concentrations with Atheroma Progression in Chronic Kidney Disease Patients

BACKGROUND AND OBJECTIVES Atherosclerosis is highly prevalent in CKD. The rate of progression of atherosclerosis is associated with cardiovascular events. Fibroblast growth factor 2 (FGF-2) is a member of the FGF family with potentially both protective and deleterious effects in the development of atherosclerosis. The role of circulating FGF-2 levels in the progression of atherosclerosis in CKD is unknown. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We used a multicenter, prospective, observational cohorts study of 481 patients with CKD. We determined the presence of atheroma plaque in ten arterial territories by carotid and femoral ultrasounds. Progression of atheromatosis was defined as an increase in the number of territories with plaque after 24 months. Plasma levels of FGF-2 were measured by multiplex analysis. A multivariable logistic regression analysis was performed to determine whether plasma FGF-2 levels were associated with atheromatosis progression. RESULTS Average age of the population was 61 years. The percentage of patients in each CKD stage was 51% in stage 3, 41% in stages 4-5, and 8% in dialysis. A total of 335 patients (70%) showed plaque at baseline. Atheromatosis progressed in 289 patients (67%). FGF-2 levels were similar between patients with or without plaque at baseline (79 versus 88 pg/ml), but lower in patients with atheromatosis progression after 2 years (78 versus 98 pg/ml; P<0.01). In adjusted analyses, higher plasma FGF-2 was associated with lower risk of atheromatosis progression (odds ratio [OR], 0.86; 95% confidence interval [95% CI], 0.76 to 0.96; per 50 pg/ml increment). Analysis of FGF-2 in tertiles showed that atheroma progression was observed for 102 participants in the lowest tertile of FGF-2 (reference group), 86 participants in the middle tertile of FGF-2 (adjusted OR, 0.70; 95% CI, 0.40 to 1.20), and 74 participants in the lowest tertile of FGF-2 (adjusted OR, 0.48; 95% CI, 0.28 to 0.82). CONCLUSIONS Low FGF-2 levels are independently associated with atheromatosis progression in CKD.