Evidence for a regulated Ca2+ entry in proximal tubular cells and its implication in calcium stone formation.

Calcium phosphate (CaP) crystals, which begin to form in the early segments of the loop of Henle (LOH), are known to act as precursors for calcium stone formation. The proximal tubule (PT), which is just upstream of the LOH and is a major site for Ca2+ reabsorption, could be a regulator of such CaP crystal formation. However, PT Ca2+ reabsorption is mostly described as being paracellular. Here, we show the existence of a regulated transcellular Ca2+ entry pathway in luminal membrane PT cells induced by Ca2+-sensing receptor (CSR, also known as CASR)-mediated activation of transient receptor potential canonical 3 (TRPC3) channels. In support of this idea, we found that both CSR and TRPC3 are physically and functionally coupled at the luminal membrane of PT cells. More importantly, TRPC3-deficient mice presented with a deficiency in PT Ca2+ entry/transport, elevated urinary [Ca2+], microcalcifications in LOH and urine microcrystals formations. Taken together, these data suggest that a signaling complex comprising CSR and TRPC3 exists in the PT and can mediate transcellular Ca2+ transport, which could be critical in maintaining the PT luminal [Ca2+] to mitigate formation of the CaP crystals in LOH and subsequent formation of calcium stones.

Melamine induces Ca2+-sensing receptor activation and elicits apoptosis in proximal tubular cells.

Melamine causes renal tubular cell injury through inflammation, fibrosis, and apoptosis. Although melamine affects the rise in intracellular Ca2+ concentration ([Ca2+]i), reactive oxygen species (ROS) production, and proapoptotic pathway activation, the mechanism of upstream Ca2+ signaling is unknown. Because melamine has some structural similarities with l-amino acids, which endogenously activate Ca2+-sensing receptors (CSR), we examined the effect of melamine on CSR-induced Ca2+ signaling and apoptotic cell death. We show here that melamine activates CSR, causing a sustained Ca2+ entry in the renal epithelial cell line, LLC-PK1. Moreover, such CSR stimulation resulted in a rise in [Ca2+]i, leading to enhanced ROS production. Furthermore, melamine-induced elevated [Ca2+]i and ROS production caused a dose-dependent increase in apoptotic (by DAPI staining, DNA laddering, and annexin V assay) and necrotic (propidium iodide staining) cell death. Upon examining the downstream mechanism, we found that transforming growth factor ß1 (TGF-ß1), which increases extracellular matrix genes and proapoptotic signaling, was also upregulated at lower doses of melamine, which could be due to an early event inducing apoptosis. Additionally, cells exposed to melamine displayed a rise in pERK activation and lactate dehydrogenase release resulting in cytotoxicity. These results offer a novel insight into the molecular mechanisms by which melamine exerts its effect on CSR, causing a sustained elevation of [Ca2+]i, leading to ROS generation, fibronectin production, proapoptotic pathway activation, and renal cell damage. Together, these results thus suggest that melamine-induced apoptosis and/or necrosis may subsequently result in acute kidney injury and promote kidney stone formation.

Reduction of TRPC1/TRPC3 mediated Ca2+-signaling protects oxidative stress-induced COPD.

Oxidative stress is a predisposing factor in Chronic Obstructive Pulmonary Disease (COPD). Specifically, pulmonary epithelial (PE) cells reduce antioxidant capacity during COPD because of the continuous production of reactive oxygen species (ROS). However, the molecular pathogenesis that governs such ROS activity is unclear. Here we show that the dysregulation of intracellular calcium concentration ([Ca2+]i) in PE cells from COPD patients, compared to the healthy PE cells, is associated with the robust functional expressions of Transient Receptor Potential Canonical (TRPC)1 and TRPC3 channels, and Ca2+ entry (SOCE) components, Stromal Interaction Molecule 1 (STIM1) and ORAI1 channels. Additionally, the elevated expression levels of fibrotic, inflammatory, oxidative, and apoptotic markers in cells from COPD patients suggest detrimental pathway activation, thereby reducing the ability of lung remodeling. To further delineate the mechanism, we used human lung epithelial cell line, A549, since the behavior of SOCE and the expression patterns of TRPC1/C3, STIM1, and ORAI1 were much like PE cells. Notably, the knockdown of TRPC1/C3 in A549 cells substantially reduced the SOCE-induced [Ca2+]i rise, and reversed the ROS-mediated oxidative, fibrotic, inflammatory, and apoptotic responses, thus confirming the role of TRPC1/C3 in SOCE driven COPD-like condition. Higher TRPC1/C3, STIM1, and ORAI1 expressions, along with a greater Ca2+ entry, via SOCE in ROS-induced A549 cells, led to the rise in oxidative, fibrotic, inflammatory, and apoptotic gene expression, specifically through the extracellular signal-regulated kinase (ERK) pathway. Abatement of TRPC1 and/or TRPC3 reduced the mobilization of [Ca2+]i and reversed apoptotic gene expression and ERK activation, signifying the involvement of TRPC1/C3. Together these data suggest that TRPC1/C3 and SOCE facilitate the COPD condition through ROS-mediated cell death, thus implicating their likely roles as potential therapeutic targets for COPD. SUMMARY: Alterations in Ca2+ signaling modalities in normal pulmonary epithelial cells exhibit COPD through oxidative stress and cellular injury, compromising repair, which was alleviated through inhibition of store-operated calcium entry. SUBJECT AREA: Calcium, ROS, Cellular signaling, lung disease.

Underrepresented Populations in Pediatric Epilepsy Surgery.

As awareness of pediatric epilepsy increases, accompanied by advancements in technology and research, it is important to identify certain types of patients that are overlooked for surgical management of epilepsy. Identifying these populations will allow us to study and elucidate the factors contributing to the underutilization and/or delayed application of surgical interventions. Demographically, African-American and Hispanic patients, as well as patients of certain Asian ethnicities, have relatively lower rates of undergoing epilepsy surgery than non-Hispanic and white patients. Among patients with epilepsy, those with higher odds of seizure-freedom following surgery are more likely to be referred for surgical evaluation by their neurologists, with the most common diagnosis being lesional focal epilepsy. However, patients with multifocal or generalized epilepsy, genetic etiologies, or normal (non-lesional) brain magnetic resonance imaging (MRI) are less likely be to referred for evaluation for resective surgery. With an increasing number of high-quality imaging modalities to help localize the epileptogenic zone as well as new techniques for both curative and palliative epilepsy surgery, there are very few populations of patients and/or types of epilepsy that should be precluded from evaluation to determine the suitability of epilepsy surgery. Ultimately, a clearer understanding of the populations who are underrepresented among those considered for epilepsy surgery, coupled with further study of the underlying reasons for this trend, will lead to less disparity in access to this critical treatment among patients with epilepsy.

Vascular Calcification and Stone Disease: A New Look towards the Mechanism.

Calcium phosphate (CaP) crystals are formed in pathological calcification as well as during stone formation. Although there are several theories as to how these crystals can develop through the combined interactions of biochemical and biophysical factors, the exact mechanism of such mineralization is largely unknown. Based on the published scientific literature, we found that common factors can link the initial stages of stone formation and calcification in anatomically distal tissues and organs. For example, changes to the spatiotemporal conditions of the fluid flow in tubular structures may provide initial condition(s) for CaP crystal generation needed for stone formation. Additionally, recent evidence has provided a meaningful association between the active participation of proteins and transcription factors found in the bone forming (ossification) mechanism that are also involved in the early stages of kidney stone formation and arterial calcification. Our review will focus on three topics of discussion (physiological influences-calcium and phosphate concentration-and similarities to ossification, or bone formation) that may elucidate some commonality in the mechanisms of stone formation and calcification, and pave the way towards opening new avenues for further research.