Accelerated medial calcification is a major cause of premature cardiovascular mortality in patients with chronic kidney disease (CKD). Evidence suggests that extracellular concentration of Ca2+ and vascular smooth muscle cells may play a pivotal role in the pathogenesis of vascular calcification. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that is expressed in a range of tissues, but characterization of its expression and function in the cardiovascular system is limited. Here we report the expression of CaSR mRNA (RT-PCR) and protein (Western blotting and immunocytochemistry) in human aortic smooth muscle cells (HAoSMC). Treatment of HAoSMC with Ca2+ (0-5 mM; 0-30 min) or the CaSR agonists gentamycin and neomycin (0-300 microM; 0-30 min) resulted in a dose- and time-dependent phosphorylation of ERK1/2. Gentamycin- and neomycin-mediated ERK1/2 stimulation was inhibited by pretreatment with PD-98059, an ERK-activating kinase 1 (MEK1) inhibitor, confirming specificity of the observed effects. ERK1/2 activation was inhibited in HAoSMC, with CaSR expression knocked down by transfection with specific small-interference RNA, which confirmed that the observed neomycin/gentamycin-induced MEK1/ERK1/2 activation was mediated via the CaSR. CaSR mRNA and protein were also expressed in large and small arteries from normal subjects (kidney donors) and patients with end-stage renal disease (ESRD). The CaSR was detected in smooth muscle and endothelial cells. Expression was significantly lower in arteries from ESRD patients. In conclusion, these data not only demonstrate the presence of a functional CaSR in human artery but show a correlation between CaSR expression and progression of CKD.

Background/Aim: Diabetic nephropathy is associated with progressive renal damage, leading to impaired function and end-stage renal failure. Secondary hypertension stems from a deranged ability of cells within the kidney to resolve and appropriately regulate sodium resorption in response to hyperglycaemia. However, the mechanisms by which glucose alters sodium re-uptake have not been fully characterised. Methods: Here we present RT-PCR, western blot and immunocytochemistry data confirming mRNA and protein expression of the serum and glucocorticoid inducible kinase (SGK1) and the α conducting subunit of the epithelial sodium channel (ENaC) in a model in vitro system of the human cortical collecting duct (HCD). We examined changes in expression of these elements in response to glucose challenge, designed to mimic hyperglycaemia associated with type 2 diabetes mellitus. Changes in Na⁺ concentration were assessed using single-cell microfluorimetry. Results: Incubation with glucose, the Ca²⁺-ionophore ionomycin and the cytokine TGF-β1 were all found to evoke significant and time-dependent increases in both SGK1 and αENaC protein expression. These molecular changes were correlated to an increase in Na⁺-uptake at the single-cell level. Conclusion: Together these data offer a potential explanation for glucose-evoked Na⁺-resorption and a potential contributory role of SGK1 and ENaCs in development of secondary hypertension, commonly linked to diabetic nephropathy.

Aberrant sodium absorption has been linked to the development of hypertension in both renal disease and diabetes. Efficient absorption depends on coordination of cellular activity across the entire epithelium via cell-to-cell coupling. In the current study we have utilized a model human collecting duct cell line (HCD) to assess the role of connexin43 (Cx43)-mediated gap junctions in the transfer of intracellular Ca(2+) transients within coupled cell clusters. HCD cells express Cx43 mRNA and protein, as well as that for the mechanosensitive transient receptor potential receptor (TRPV4). Mechanical stimulation of individual cells within a cluster evoked a transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) that propagated between cells via a heptanol-sensitive mechanism. The rise in [Ca(2+)](i) was dependent on both store release and Ca(2+)-influx pathways. Lucifer yellow dye transfer and Cx43 knockdown experiments confirmed direct cell-to-cell communication. Application of the Ca(2+) ionophore ionomycin, or an increase in glucose (5 to 25 mM), produced a time-dependent (48 h) increase in Cx43 protein expression. The transmission rate of touch-evoked Ca(2+) transients between coupled cells was accelerated after exposure to high glucose, providing a functional correlate to increased Cx43 expression. These data suggest a pivotal role for Cx43-mediated gap junctions in the synchronization of activity between HCD cells in response to stimuli that mimic osmotic and physical changes. Cx43 expression and cell-to-cell communication increased in response to high glucose and may protect the collecting duct from renal damage associated with more established diabetic nephropathy.

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