Guerman Molostvov

Vascular Klotho Deficiency Potentiates the Development of Human Artery Calcification and Mediates Resistance to Fibroblast Growth Factor 23

Background— Klotho is known to function as a cofactor for the phosphatonin, fibroblast growth factor (FGF)-23 at the kidney. FGF-23 levels rise in chronic kidney disease (CKD) despite progression of accelerated vascular calcification. There are currently conflicting data on whether FGF-23 may exhibit direct vasculoprotective effects in CKD. Methods and Results— In this study, we describe for the first time endogenous Klotho expression in human arteries and human aortic smooth muscle cells. We show that CKD is a state of vascular Klotho deficiency promoted by chronic circulating stress factors, including proinflammatory, uremic, and disordered metabolic conditions. Mechanistic studies demonstrated that Klotho knockdown potentiated the development of accelerated calcification through a Runx2 and myocardin-serum response factor–dependent pathway. Klotho knockdown studies further revealed that vascular cells are a Klotho-dependent target tissue for FGF-23. FGF-23 mediated cellular activation of p-ERK, p-AKT, and cellular proliferative effects, which were abrogated following Klotho knockdown. We next showed that vascular Klotho deficiency driven by procalcific stressors could be restored by vitamin D receptor activators, in vitro and further confirmed using human arterial organ cultures from CKD patients, in vivo. Furthermore, restoration of suppressed Klotho expression by vitamin D receptor activators conferred human aortic smooth muscle cells responsive to FGF-23 signaling and unmasked potential anticalcific effects. Conclusions— Chronic metabolic stress factors found in CKD promote vascular Klotho deficiency. Mechanistic studies revealed a bifunctional role for local vascular Klotho, first, as an endogenous inhibitor of vascular calcification and, second, as a cofactor required for vascular FGF-23 signaling. Furthermore, vitamin D receptor activators can restore Klotho expression and unmask FGF-23 anticalcific effects.

Vascular Klotho Deficiency Potentiates the Development of Human Artery Calcification and Mediates Resistance to FGF-23

Background— Klotho is known to function as a cofactor for the phosphatonin, fibroblast growth factor (FGF)-23 at the kidney. FGF-23 levels rise in chronic kidney disease (CKD) despite progression of accelerated vascular calcification. There are currently conflicting data on whether FGF-23 may exhibit direct vasculoprotective effects in CKD. Methods and Results— In this study, we describe for the first time endogenous Klotho expression in human arteries and human aortic smooth muscle cells. We show that CKD is a state of vascular Klotho deficiency promoted by chronic circulating stress factors, including proinflammatory, uremic, and disordered metabolic conditions. Mechanistic studies demonstrated that Klotho knockdown potentiated the development of accelerated calcification through a Runx2 and myocardin-serum response factor–dependent pathway. Klotho knockdown studies further revealed that vascular cells are a Klotho-dependent target tissue for FGF-23. FGF-23 mediated cellular activation of p-ERK, p-AKT, and cellular proliferative effects, which were abrogated following Klotho knockdown. We next showed that vascular Klotho deficiency driven by procalcific stressors could be restored by vitamin D receptor activators, in vitro and further confirmed using human arterial organ cultures from CKD patients, in vivo. Furthermore, restoration of suppressed Klotho expression by vitamin D receptor activators conferred human aortic smooth muscle cells responsive to FGF-23 signaling and unmasked potential anticalcific effects. Conclusions— Chronic metabolic stress factors found in CKD promote vascular Klotho deficiency. Mechanistic studies revealed a bifunctional role for local vascular Klotho, first, as an endogenous inhibitor of vascular calcification and, second, as a cofactor required for vascular FGF-23 signaling. Furthermore, vitamin D receptor activators can restore Klotho expression and unmask FGF-23 anticalcific effects.

Extracellular calcium-sensing receptor mediated signalling is involved in human vascular smooth muscle cell proliferation and apoptosis.

Calcium-sensing receptor (CaSR) plays key role in vascular calcification in patients with chronic kidney disease (CKD). We investigated the role of CaSR in regulating smooth muscle cell (SMC) proliferation and apoptosis. Incubation with 300μM neomycin (CaSR agonist) resulted in 7.5-fold (p<0.05) increase in ERK1,2 phosphorylation. It was reduced (p<0.01) by 10μM PD98059 (MEK1 inhibitor), indicating that CaSR agonist-induced effects were mediated via MEK1/ERK1,2 pathway. ERK1,2 phosphorylation was abolished by 5μM U73122 (PLC inhibitor), indicating that PLC signalling was crucial for MEK1/ERK1,2 activation. Confirming PLC activation, inositol triphosphate (IP3) production was increased by neomycin/gentamycin (p<0.05) and reduced by U73122. To confirm that ERK1,2 and PLC signalling were mediated via CaSR, Human Aortic SMC (HAoSMC) were transfected with CaSR siRNA. CaSR knockdown resulted in lower ERK1,2 neomycin response and IP3 production (p<0.01). Neomycin increased HAoSMC proliferation >3-fold, which was reduced in CaSR knockdown cells (p<0.01) and further inhibited by PD98059 and U73122 (p<0.05). Apoptosis was not affected by neomycin treatment. U73122 produced 3.5-fold increase in HAoSMC apoptosis, which was further increased by CaSR knockdown (5-fold, p<0.05). In conclusion, stimulation of CaSR leads to activation of MEK1/ERK1,2 and PLC pathways and up-regulation of cell proliferation. CaSR-mediated PLC activation is important for SMC survival and protection against apoptosis.

α-Klotho Expression in Human Tissues

Context: α-Klotho has emerged as a powerful regulator of the aging process. To date, the expression profile of α-Klotho in human tissues is unknown, and its existence in some human tissue types is subject to much controversy. Objective: This is the first study to characterize systemwide tissue expression of transmembrane α-Klotho in humans. We have employed next-generation targeted proteomic analysis using parallel reaction monitoring in parallel with conventional antibody-based methods to determine the expression and spatial distribution of human α-Klotho expression in health. Results: The distribution of α-Klotho in human tissues from various organ systems, including arterial, epithelial, endocrine, reproductive, and neuronal tissues, was first identified by immunohistochemistry. Kidney tissues showed strong α-Klotho expression, whereas liver did not reveal a detectable signal. These results were next confirmed by Western blotting of both whole tissues and primary cells. To validate our antibody-based results, α-Klotho-expressing tissues were subjected to parallel reaction monitoring mass spectrometry (data deposited at ProteomeXchange, PXD002775) identifying peptides specific for the full-length, transmembrane α-Klotho isoform. Conclusions: The data presented confirm α-Klotho expression in the kidney tubule and in the artery and provide evidence of α-Klotho expression across organ systems and cell types that has not previously been described in humans.

Glucose-Induced Down Regulation of Thiamine Transporters in the Kidney Proximal Tubular Epithelium Produces Thiamine Insufficiency in Diabetes

Increased renal clearance of thiamine (vitamin B1) occurs in experimental and clinical diabetes producing thiamine insufficiency mediated by impaired tubular re-uptake and linked to the development of diabetic nephropathy. We studied the mechanism of impaired renal re-uptake of thiamine in diabetes. Expression of thiamine transporter proteins THTR-1 and THTR-2 in normal human kidney sections examined by immunohistochemistry showed intense polarised staining of the apical, luminal membranes in proximal tubules for THTR-1 and THTR-2 of the cortex and uniform, diffuse staining throughout cells of the collecting duct for THTR-1 and THTR-2 of the medulla. Human primary proximal tubule epithelial cells were incubated with low and high glucose concentration, 5 and 26 mmol/l, respectively. In high glucose concentration there was decreased expression of THTR-1 and THTR-2 (transporter mRNA: −76% and −53% respectively, p<0.001; transporter protein −77% and −83% respectively, p<0.05), concomitant with decreased expression of transcription factor specificity protein-1. High glucose concentration also produced a 37% decrease in apical to basolateral transport of thiamine transport across cell monolayers. Intensification of glycemic control corrected increased fractional excretion of thiamine in experimental diabetes. We conclude that glucose-induced decreased expression of thiamine transporters in the tubular epithelium may mediate renal mishandling of thiamine in diabetes. This is a novel mechanism of thiamine insufficiency linked to diabetic nephropathy.

Human leukocyte antigen-specific antibodies and gamma-interferon stimulate human microvascular and glomerular endothelial cells to produce complement factor C4

Background The role of the complement system in antibody-mediated rejection has been investigated in relation to circulating complement interacting with renal microvascular endothelium, resulting in the formation of peritubular capillary C4d. However, the possible importance of local complement synthesis is less clear. The aim of this study was to determine whether human vascular endothelium could produce C4 in response to stimulation in vitro. Methods Human microvascular endothelial cells and glomerular endothelial cells were stimulated with endotoxins, cytokines, and human leukocyte antigen-specific antibodies. Synthesis of complement was investigated using western blotting and indirect immunofluorescence. De novo C4 synthesis was confirmed by using C4 small interfering RNA. Results Glomerular and microvascular endothelium, both produce C3 and C4 complement protein. Complement synthesis was stimulant-specific—C3 was produced mainly after stimulation with lipopolysaccharide whereas C4 synthesis occurred on treatment with gamma interferon. Culture with human leukocyte antigen-specific antibodies resulted in a significant increase of C4 protein synthesis by both cell lines. Conclusions We have shown for the first time that human microvascular endothelium can be stimulated to synthesize C4 in vitro. The implications of this for clinical transplantation, especially in the context of antibody-mediated rejection, its histological interpretation and as a potential target for therapy would have to be determined by further studies.

Expression and Role of the Calcium-Sensing Receptor in the Blood Vessel Wall

The calcium-sensing receptor (CaSR), which is involved in systemic calcium homeostasis, has also been found to be functionally expressed on cells of the vascular wall. Its activation on perivascular nerves and endothelial cells has been shown to regulate arterial tone, peripheral vascular resistance and possibly local tissue perfusion. The expression of the CaSR on immune cells involved in vascular inflammation, such as macrophages, and its increased expression in inflammation indicates the central role extracellular calcium plays in vascular inflammation and repair. Further detailed analysis will clarify the role the vascular CaSR plays as a therapeutic target for complex disease conditions such as hypertension, tissue hypoperfusion, atherosclerosis and vascular calcification.

Arterial Expression of the Calcium-Sensing Receptor Is Maintained by Physiological Pulsation and Protects against Calcification

Vascular calcification (VC) is common in chronic kidney disease (CKD) and contributes to cardiovascular mortality. The calcium-sensing receptor (CaSR) is present in human artery, senses extracellular calcium and may directly modulate VC. Objective: to investigate the association between arterial cyclic strain, CaSR expression and VC. Methods and Results: human aortic smooth muscle cells (HAoSMC) were cultured under static or strained conditions, with exposure to CaSR agonists, the calcimimetic R568, and after CaSR silencing and over-expression. High extracellular calcium reduced CaSR expression and promoted osteochondrogenic transformation and calcium deposition. This was partially prevented by cyclic strain and exposure to R568. CaSR silencing enhanced calcification and osteochondrogenic transformation, whereas CaSR over-expression attenuated this procalcific response, demonstrating a central role for the CaSR in the response to cyclic strain and regulation of VC. In arterial explants from CKD patients (n = 11) and controls (n = 9), exposure to R568 did not significantly alter calcium deposition, osteochondrogenic markers or total artery calcium content. Conclusions: physiological mechanical strain is important for arterial homeostasis and may protect arteries from VC. The beneficial effects of cyclic strain may be mediated via the CaSR.

Soluble CD30 and Cd27 levels in patients undergoing HLA antibody-incompatible renal transplantation

HLA antibody-incompatible transplantation has a higher risk of rejection when compared to standard renal transplantation. Soluble CD30 (sCD30) has been shown in many, but not all, studies to be a biomarker for risk of rejection in standard renal transplant recipients. We sought to define the value of sCD30 and soluble CD27 (sCD27) in patients receiving HLA antibody-incompatible transplants. Serum taken at different time points from 32 HLA antibody-incompatible transplant recipients was retrospectively assessed for sCD30 and sCD27 levels by enzyme-linked immunosorbent assay (ELISA). This was compared to episodes of acute rejection, post-transplant donor-specific antibody (DSA) levels and 12 month serum creatinine levels. No association was found between sCD27 and sCD30 levels and risk of acute rejection or DSA levels. Higher sCD30 levels at 4-6 weeks post-transplantation were associated with a higher serum creatinine at 12 months. Conclusion patients undergoing HLA antibody-incompatible transplantation are at a high risk of rejection but neither sCD30 (unlike in standard transplantation) nor sCD27 was found to be a risk factor. High sCD30 levels measured at 4-6 weeks post-transplantation was associated with poorer graft function at one year.

α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells

Endothelial cells (ECs) express fibroblast growth factor (FGF) receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs) and human brain microvascular endothelial cells (HBMECs) were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS) expression, nitric oxide (NO) production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.