Sex-specific Stone-forming Phenotype in Mice During Hypercalciuria/Urine Alkalinization.

Sex differences in kidney stone formation are well known. Females generally have slightly acidic blood and higher urine pH when compared with males, which makes them more vulnerable to calcium stone formation, yet the mechanism is still unclear. We aimed to examine the role of sex in stone formation during hypercalciuria and urine alkalinization through acetazolamide and calcium gluconate supplementation, respectively, for 4 weeks in wild-type (WT) and moderately hypercalciuric [TRPC3 knockout [KO](-/-)] male and female mice. Our goal was to develop calcium phosphate (CaP) and CaP+ calcium oxalate mixed stones in our animal model to understand the underlying sex-based mechanism of calcium nephrolithiasis. Our results from the analyses of mice urine, serum, and kidney tissues show that female mice (WT and KO) produce more urinary CaP crystals, higher [Ca2+], and pH in urine compared to their male counterparts. We identified a sex-based relationship of stone-forming phenotypes (types of stones) in our mice model following urine alkalization/calcium supplementation, and our findings suggest that female mice are more susceptible to CaP stones under those conditions. Calcification and fibrotic and inflammatory markers were elevated in treated female mice compared with their male counterparts, and more so in TRPC3 KO mice compared with their WT counterparts. Together these findings contribute to a mechanistic understanding of sex-influenced CaP and mixed stone formation that can be used as a basis for determining the factors in sex-related clinical studies.

The Rise in Tubular pH during Hypercalciuria Exacerbates Calcium Stone Formation.

In calcium nephrolithiasis (CaNL), most calcium kidney stones are identified as calcium oxalate (CaOx) with variable amounts of calcium phosphate (CaP), where CaP is found as the core component. The nucleation of CaP could be the first step of CaP+CaOx (mixed) stone formation. High urinary supersaturation of CaP due to hypercalciuria and an elevated urine pH have been described as the two main factors in the nucleation of CaP crystals. Our previous in vivo findings (in mice) show that transient receptor potential canonical type 3 (TRPC3)-mediated Ca2+ entry triggers a transepithelial Ca2+ flux to regulate proximal tubular (PT) luminal [Ca2+], and TRPC3-knockout (KO; -/-) mice exhibited moderate hypercalciuria and microcrystal formation at the loop of Henle (LOH). Therefore, we utilized TRPC3 KO mice and exposed them to both hypercalciuric [2% calcium gluconate (CaG) treatment] and alkalineuric conditions [0.08% acetazolamide (ACZ) treatment] to generate a CaNL phenotype. Our results revealed a significant CaP and mixed crystal formation in those treated KO mice (KOT) compared to their WT counterparts (WTT). Importantly, prolonged exposure to CaG and ACZ resulted in a further increase in crystal size for both treated groups (WTT and KOT), but the KOT mice crystal sizes were markedly larger. Moreover, kidney tissue sections of the KOT mice displayed a greater CaP and mixed microcrystal formation than the kidney sections of the WTT group, specifically in the outer and inner medullary and calyceal region; thus, a higher degree of calcifications and mixed calcium lithiasis in the kidneys of the KOT group was displayed. In our effort to find the Ca2+ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca2+ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca2+ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca2+ entry) inhibitors (Pyr10), Ca2+ entry by WTT cells was moderately inhibited, suggesting that the Ca2+ and pH levels exerted sensitivity changes in response to ROCE and SOCE. An assessment of the gene expression profiles in the PT cells of WTT and KOT mice revealed a safeguarding effect of TRPC3 against detrimental processes (calcification, fibrosis, inflammation, and apoptosis) in the presence of higher pH and hypercalciuric conditions in mice. Together, these findings show that compromise in both the ROCE and SOCE mechanisms in the absence of TRPC3 under hypercalciuric plus higher tubular pH conditions results in higher CaP and mixed crystal formation and that TRPC3 is protective against those adverse effects.

Ablation of TRPC3 compromises bicarbonate and phosphate transporter activity in mice proximal tubular cells.

Proximal tubular (PT) cells reabsorb most calcium (Ca2+ ), phosphate (PO4 3- ), bicarbonate (HCO3 - ), and oxalate (C2 O4 2- ) ions. We have shown that mice lacking Transient Receptor Potential Canonical 3 (TRPC3-/- ) channel are moderately hypercalciuric with presentation of luminal calcium phosphate (CaP) crystals at the loop of Henle (LOH). However, other predisposing factors for such crystal deposition are unknown. Thus, we examined the distinctions in functional status of HCO3 - , PO4 3- , and C2 O4 2- transporters in PT cells of wild type (WT) and TRPC3-/- mice by whole-cell patch clamp techniques to assess their contribution in the development of LOH CaP crystals. Here we show the development of concentration dependent HCO3 - -induced currents in all PT cells, which was confirmed by using specific HCO3 - channel inhibitor, S0859. Interestingly, such activities were diminished in PT cells from TRPC3-/- mice, suggesting reduced HCO3 - transport in absence of TRPC3. While PO4 3- -induced currents were also concentration dependent in all PT cells (confirmed by PO4 3- channel inhibitor, PF-06869206), those activities were reduced in absence of TRPC3, suggesting lower PO4 3- reabsorption that can leave excess luminal PO4 3- . Next, we applied thiosulfate (O3 S2 2 - ) as a competitive inhibitor of the SLC26a6 transporter upon C2 O4 2- current activation and observed a reduced C2 O4 2- -induced conductance which was greater in TRPC3-/- PT cells. Together, these results suggest that the reduced activities of HCO3 - , PO4 3- , and C2 O4 2- transporters in moderately hypercalciuric (TRPC3-/- ) PT cells can create a predisposing condition for CaP and CaP tubular crystallization, enabling CaP crystal formation in LOH of TRPC3-/- mice.

Stabilization of uric acid mixed crystals by melamine.

Melamine stabilizes heterogeneous nucleation of calcium crystals by increasing the retention time and decreasing the rate of dissolution. Stabilization of such mixed crystals limit the efficacy of non-invasive treatment options for kidney stones. Crystalline forms of uric acid (UA) are also involved in urolithiasis or UA kidney stones; however, its interactions with contaminating melamine and the resulting effects on the retention of kidney stones remain unknown. Since melamine augments calcium crystal formation, it provides an avenue for us to understand the stability of UA-calcium phosphate (CaP) crystals. We show here that melamine facilitates UA+CaP crystal formation, resulting in greater aggregates. Moreover, melamine induced mixed crystal retention through a time-dependent manner in presence and/or absence of hydroxycitrate (crystal inhibitor), indicating its abridged effectiveness as conventional remedy. CaP was also shown to modify optical properties of UA+CaP mixed crystals. Differential staining of individual crystals revealed enhanced co-aggregation of UA and CaP. The dissolution rate of UA in presence of melamine was faster than its heterogeneous crystallization form with CaP, although the size was comparatively much smaller, suggesting disparity in regulation between UA and CaP crystallization. While melamine stabilized UA, CaP and mixed crystals in relatively physiological conditions (artificial urine), the retentions of those crystals were further augmented by melamine, even in presence of hydroxycitrate, thus reducing treatment efficacy.

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.

Modulation of Tubular pH by Acetazolamide in a Ca2+ Transport Deficient Mice Facilitates Calcium Nephrolithiasis.

Proximal tubular (PT) acidosis, which alkalinizes the urinary filtrate, together with Ca2+ supersaturation in PT can induce luminal calcium phosphate (CaP) crystal formation. While such CaP crystals are known to act as a nidus for CaP/calcium oxalate (CaOx) mixed stone formation, the regulation of PT luminal Ca2+ concentration ([Ca2+]) under elevated pH and/or high [Ca2+] conditions are unknown. Since we found that transient receptor potential canonical 3 (TRPC3) knockout (KO; -/-) mice could produce mild hypercalciuria with CaP urine crystals, we alkalinized the tubular pH in TRPC3-/- mice by oral acetazolamide (0.08%) to develop mixed urinary crystals akin to clinical signs of calcium nephrolithiasis (CaNL). Our ratiometric (λ340/380) intracellular [Ca2+] measurements reveal that such alkalization not only upsurges Ca2+ influx into PT cells, but the mode of Ca2+ entry switches from receptor-operated to store-operated pathway. Electrophysiological experiments show enhanced bicarbonate related current activity in treated PT cells which may determine the stone-forming phenotypes (CaP or CaP/CaOx). Moreover, such alkalization promotes reactive oxygen species generation, and upregulation of calcification, inflammation, fibrosis, and apoptosis in PT cells, which were exacerbated in absence of TRPC3. Altogether, the pH-induced alteration of the Ca2+ signaling signature in PT cells from TRPC3 ablated mice exacerbated the pathophysiology of mixed urinary stone formation, which may aid in uncovering the downstream mechanism of CaNL.

Microstructural densification and alignment by aspiration-ejection influence cancer cell interactions with three-dimensional collagen networks.

Extracellular matrix microstructure and mechanics are crucial to breast cancer progression and invasion into surrounding tissues. The peritumor collagen network is often dense and aligned, features which in vitro models lack. Aspiration of collagen hydrogels led to densification and alignment of microstructure surrounding embedded cancer cells. Two metastasis-derived breast cancer cell lines, MDA-MB-231 and MCF-7, were cultured in initially 4 mg/ml collagen gels for 3 days after aspiration, as well as in unaspirated control hydrogels. Videomicroscopy during aspiration, and at 0, 1, and 3 days after aspiration, epifluorescence microscopy of phalloidin-stained F-actin cytoskeleton, histological sections, and soluble metabolic byproducts from constructs were collected to characterize effects on the embedded cell morphology, the collagen network microstructure, and proliferation. Breast cancer cells remained viable after aspiration-ejection, proliferating slightly less than in unaspirated gels. Furthermore, MDA-MB-231 cells appear to partially relax the collagen network and lose alignment 3 days after aspiration. Aspiration-ejection generated aligned, compact collagen network microstructure with immediate cell co-orientation and higher cell number density apparently through purely physical means, though cell-collagen contact guidance and network remodeling influence cell organization and collagen network microstructure during subsequent culture. This study establishes a platform to determine the effects of collagen density and alignment on cancer cell behavior, with translational potential for anticancer drug screening in a biomimetic three-dimensional matrix microenvironment, or implantation in preclinical models.

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.

Titanium Dioxide Promotes the Growth and Aggregation of Calcium Phosphate and Monosodium Urate Mixed Crystals.

The increased utilization of titanium dioxide (TiO2) nanoparticles (TNPs) in various industrial and consumer products has raised concerns regarding its harmful effect due to its accumulation within the different systems of the human body. Here, we focused on the influence of TNPs on the growth and aggregation of two crucial crystalline substances, calcium phosphate (CaP) and monosodium urate (MSU), particularly its implications in gout disease. In this study, we adopted microscopic techniques and generated kinetic models to examine the interactions between TNPs, CaP and MSU, and crystallization, under controlled laboratory conditions. Our findings reveal that TNPs not only facilitate the growth of these crystals but also promote their co-aggregations. Crystal dissolution kinetics also exhibit that an increase in TNPs concentration corresponds to a reduction in the dissolution rate of CaP and MSU crystals in presence of the dissoluting agent hydroxycitrate (Hcit). These observations suggest that TNPs can stabilize CaP+MSU mixed crystals, which underscores the significance of TNPs' exposure in the pathogenesis of gout disease.

Extracellular Ca(2+) sensing in salivary ductal cells.

Ca(2+) is secreted from the salivary acinar cells as an ionic constituent of primary saliva. Ions such as Na(+) and Cl(-) get reabsorbed whereas primary saliva flows through the salivary ductal system. Although earlier studies have shown that salivary [Ca(2+)] decreases as it flows down the ductal tree into the oral cavity, ductal reabsorption of Ca(2+) remains enigmatic. Here we report a potential role for the G protein-coupled receptor, calcium-sensing receptor (CSR), in the regulation of Ca(2+) reabsorption by salivary gland ducts. Our data show that CSR is present in the apical region of ductal cells where it is co-localized with transient receptor potential canonical 3 (TRPC3). CSR is activated in isolated salivary gland ducts as well as a ductal cell line (SMIE) by altering extracellular [Ca(2+)] or by aromatic amino acid, L-phenylalanine (L-Phe, endogenous component of saliva), as well as neomycin. CSR activation leads to Ca(2+) influx that, in polarized cells grown on a filter support, is initiated in the luminal region. We show that TRPC3 contributes to Ca(2+) entry triggered by CSR activation. Further, stimulation of CSR in SMIE cells enhances the CSR-TRPC3 association as well as surface expression of TRPC3. Together our findings suggest that CSR could serve as a Ca(2+) sensor in the luminal membrane of salivary gland ducts and regulate reabsorption of [Ca(2+)] from the saliva via TRPC3, thus contributing to maintenance of salivary [Ca(2+)]. CSR could therefore be a potentially important protective mechanism against formation of salivary gland stones (sialolithiasis) and infection (sialoadenitis).

Anti-inflammatory role of extracellular l-arginine through calcium sensing receptor in human renal proximal tubular epithelial (HK-2) cells.

Renal tubular epithelial cells are capable of synthesizing interleukins (IL) in response to a variety of proinflammatory cytokines. Moreover, elevated urinary levels of IL have been shown in patients with various forms of nephritic diseases. However, the underlying intracellular signaling mechanism is unclear. Here we show the immunological signaling role of l-Arginine (l-Arg) through Ca2+-sensing receptor (CaSR) in human kidney 2 (HK-2) renal proximal tubular epithelial cells, using Ca2+ imaging and patch clamp techniques and its mechanistic link to the downstream cellular function. Both pharmacological and siRNA inhibitors support the activation CaSR by extracellular l-Arg to induced Ca2+ entry via a Transient receptor potential canonical (TRPC) channel in HK-2 cells mainly through the receptor operated Ca2+ entry (ROCE). Activation of CaSR by l-Arg led to the rise in p-p38/p38 expression suggesting [Ca2+]i as a regulator for p38-signaling pathways. Notably, l-Arg activated CaSR-induced Ca2+ signaling reduced the expressions of key fibrotic, inflammatory, and apoptotic genes, suggesting its nephroprotective role via Ca2+ signaling through CaSR in HK-2 cells. Since we found that the IL-6 expressions were inversely proportional to the increasing concentrations of l-Arg in HK-2 cells, we measured the release of IL-6, which steadily decreased as the concentrations of l-Arg were elevated. Taken together, extracellular l-Arg is a negative regulator for IL-6-induced inflammatory process, through the activation of CaSR and TRPC channel by ROCE pathway and can have a potential to alleviate inflammatory renal diseases.

Ablation of TRPC3 disrupts Ca2+ signaling in salivary ductal cells and promotes sialolithiasis.

Clinical studies and structural analyses of salivary stones strongly suggest a linkage between higher saliva calcium (Ca2+) and salivary stone formation, sialolithiasis; however, the process and the mechanism leading to Ca2+ overload during sialolithiasis is not well understood. Here, we show that TRPC3 null (-/-) mice presented with a reduction in Ca2+ entry and current in ductal cells with higher saliva [Ca2+] suggesting diminished transepithelial Ca2+ flux across the salivary ductal cells, leaving more Ca2+ in ductal fluid. Significantly, we found that TRPC3 was expressed in mice and human salivary ductal cells, while intraductal stones were detected in both mice (TRPC3-/-) and patient (sialolithiasis) salivary glands. To identify the mechanism, we found that TRPC3 was crucial in preventing the expression of calcification genes (BMP2/6, Runx2) in ductal cells which may be due to higher extracellular Ca2+ in SMG tissues. Similarly, inflammatory (IL6, NLRP3), fibrotic (FN1, TGFß1) and apoptotic (Bax1/Bcl2) markers were also elevated, suggesting that the loss of TRPC3 induces genetic changes that leads to salivary gland cell death and induction of inflammatory response. Overall, ablation of TRPC3-/- leads to higher saliva [Ca2+], along with elevated detrimental gene expressions, altogether contributing to salivary gland 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.

Enhanced carbonic anhydrase expression with calcification and fibrosis in bronchial cartilage during COPD.

Pulmonary cartilage plays a crucial structural role determining the physiologic airway compressibility and distensibility, necessary for proper mechanical function. This functionality deteriorates with aging due to increased stiffness of both airway muscle and cartilage, as well as, decreased renewal capacity. Altered airway remodeling has been suggested as a pathogenic driver of chronic obstructive pulmonary disease (COPD) through mechanisms still incompletely understood. Using paraffin-embedded lung tissue sections from archived autopsy material from COPD with non-COPD age matched controls a histopathologic analysis focused on inflammation, fibrosis and calcification was performed with special stains (Masson's trichrome and Von Kossa) and immunohistochemistry for carbonic anhydrase IV (CA IV) and Ki-67. COPD lung tissues showed increased peribronchial inflammation compared to the non-COPD. Coarse amphophilic crystalline deposits in bronchial cartilage were more frequently observed in COPD sections, which were compatible with early dystrophic calcification of the extracellular matrix and chondrocytes. Moreover, Von Kossa staining revealed a significant calcium deposition in the cartilages from COPD in comparison to the controls. Interestingly, Ki-67 immunostains demonstrated a higher overall proliferative rate, including epithelial cells, in COPD. Furthermore, Masson's trichrome staining revealed relatively increased peribronchial collagen deposition associated with a fibrotic stromal response, which may be secondary to the inflammatory milieu in COPD. To further characterize the tissue microenvironment associated with dystrophic calcification, immunohistochemistry for CA IV was used, revealing significantly increased expression in chondrocytes and peribronchial tissue in COPD. Our findings demonstrate that dystrophic calcification of the extracellular matrix and chondrocytes can be linked to CA IV expression in COPD and suggest that pH changes in pulmonary tissue associated with inflammation and calcification may play an active role in COPD.

Hypercalciuria switches Ca2+ signaling in proximal tubular cells, induces oxidative damage to promote calcium nephrolithiasis.

Proximal tubule (PT) transports most of the renal Ca2+, which was usually described as paracellular (passive). We found a regulated Ca2+ entry pathway in PT cells via the apical transient receptor potential canonical 3 (TRPC3) channel, which initiates transcellular Ca2+ transport. Although TRPC3 knockout (-/-) mice were mildly hypercalciuric and displayed luminal calcium phosphate (CaP) crystals at Loop of Henle (LOH), no CaP + calcium oxalate (CaOx) mixed urine crystals were spotted, which are mostly found in calcium nephrolithiasis (CaNL). Thus, we used oral calcium gluconate (CaG; 2%) to raise the PT luminal [Ca2+]o further in TRPC3 -/- mice for developing such mixed stones to understand the mechanistic role of PT-Ca2+ signaling in CaNL. Expectedly, CaG-treated mice urine samples presented with numerous mixed crystals with remains of PT cells, which were pronounced in TRPC3 -/- mice, indicating PT cell damage. Notably, PT cells from CaG-treated groups switched their mode of Ca2+ entry from receptor-operated to store-operated pathway with a sustained rise in intracellular [Ca2+] ([Ca2+]i), indicating the stagnation in PT Ca2+ transport. Moreover, those PT cells from CaG-treated groups demonstrated an upregulation of calcification, inflammation, fibrotic, oxidative stress, and apoptotic genes; effects of which were more robust in TRPC3 ablated condition. Furthermore, kidneys from CaG-treated groups exhibited fibrosis, tubular injury and calcifications with significant reactive oxygen species generation in the urine, thus, indicating in vivo CaNL. Taken together, excess PT luminal Ca2+ due to escalation of hypercalciuria in TRPC3 ablated mice induced surplus CaP crystal formation and caused stagnation of PT [Ca2+]i, invoking PT cell injury, hence mixed stone formation.

Tannic acid attenuates vascular calcification-induced proximal tubular cells damage through paracrine signaling.

Vascular calcification is common in chronic kidney disease; however, the extent to which such condition can affect the renal microvasculature and the neighboring cell types is unclear. Our induced-calcification model in renal proximal tubular (PT) cells exhibited endoplasmic reticulum (ER) stress and oxidative damage, leading to apoptosis. Here, we utilized such calcification in mouse vascular smooth muscle (MOVAS-1) cells as a vascular calcification model, because it exhibited reactive oxygen species (ROS) generation, ER and oxidative stress, inflammatory, and apoptotic gene expressions. To demonstrate whether the vascular calcification condition can dictate the function of the adjacent PT cell layer, we utilized a Transwell multilayer culture system by combining those MOVAS-1 cells in the bottom chamber and polarized PT cells in the upper chamber to show the dimensional cross-signaling effect. Interestingly, calcification of MOVAS-1 cells, in this co-culture, induced H2O2 and lactate dehydrogenase (LDH) release leading to store-operated Ca2+ entry, ROS generation, and activation of oxidative, inflammatory, and apoptotic gene expressions in PT cells through paracrine signaling. Interestingly, application of tannic acid (TA) to either calcified MOVAS-1 or uncalcified PT cells diminished such detrimental pathway activation. Furthermore, the TA-mediated protection was much higher in the PT cells when applied on the calcified MOVAS-1 cells, and the delayed the pathological effects in neighboring PT cells can well be via paracrine signaling. Together, these results provide evidence of vascular calcification-induced PT cell damage, and the protective role of TA in preventing such pathological consequences, which can potentially be used as a nephroprotective remedy.

Immunohistochemical localization of carbonic anhydrase IV in the human parotid gland.

Carbonic anhydrases (CAs) catalyze the hydration and dehydration of carbon dioxide. They are important for regulating ions, fluid and acid-base balance in many tissues. The location of CAs by cell type is important for understanding their roles in these functions. CAs II and VI have been demonstrated using immunohistochemistry (IHC) in the serous acinar cells of human salivary glands and ducts of rat salivary glands. CA IV has been localized by IHC to the ducts of rat salivary glands. CA IV also is present in human parotid glands as shown by real time-polymerase chain reaction (RT-PCR), but this method does not show the distribution of the CA isozymes by cell type. We investigated the cell-specific distribution of CA IV in the human parotid gland. Sections from five formalin fixed, paraffin embedded specimens of human parotid gland were subjected to IHC for CA IV using a commercial antibody. Moderate to strong reactions were found in the cell membranes and cytoplasm of the intercalated, striated and excretory ducts and capillaries, and reactions in the acini were limited to faint areas in some cells. These results indicate that CA IV participates in the regulation of bicarbonate/carbon dioxide fluxes in the ductal system of the human parotid gland.

Differential biomolecular recognition by synthetic vs. biologically-derived components in the stone-forming process using 3D microfluidics.

Calcium phosphate (CaP) biomineralization is the hallmark of extra-skeletal tissue calcification and renal calcium stones. Although such a multistep process starts with CaP crystal formation, the mechanism is still poorly understood due to the complexity of the in vivo system and the lack of a suitable approach to simulate a truly in vivo-like environment. Although endogenous proteins and lipids are engaged with CaP crystals in such a biological process of stone formation, most in vitro studies use synthetic materials that can display differential bioreactivity and molecular recognition by the cellular component. Here, we used our in vitro microfluidic (MF) tubular structure, which is the first completely cylindrical platform, with renal tubular cellular microenvironments closest to the functional human kidney tubule, to understand the precise role of biological components in this process. We systematically evaluated the contribution of synthetic and biological components in the stone-forming process in the presence of dynamic microenvironmental cues that originated due to cellular pathophysiology, which are critical for the nucleation, aggregation, and growth of CaP crystals. Our results show that crystal aggregation and growth were enhanced by immunoglobulin G (IgG), which was further inhibited by etidronic acid due to the chelation of extracellular Ca2+. Interestingly, biogenic CaP crystals from mice urine, when applied with cell debris and non-specific protein (bovine serum albumin), exhibited a more discrete crystal growth pattern, compared to exposure to synthetic CaP crystals under similar conditions. Furthermore, proteins found on those calcium crystals from mice urine produced discriminatory effects on crystal-protein attachment. Specifically, such biogenic crystals exhibited enhanced affinity to the proteins inherent to those crystals. More importantly, a physiological comparison of crystal induction in renal tubular cells revealed that biogenic crystals are less effective at producing a sustained rise in cytosolic Ca2+ compared to synthetic crystals, suggesting a milder detrimental effect to downstream signaling. Finally, synthetic crystal-internalized cells induced more oxidative stress, inflammation, and cellular damage compared to the biogenic crystal-internalized cells. Together, these results suggest that the intrinsic nature of biogenically derived components are appropriate to generate the molecular recognition needed for spatiotemporal effects and are critical towards understanding the process of kidney stone formation.

Novel Mutations in a Lethal Case of Lymphomatous Adult T Cell Lymphoma with Cryptic Myocardial Involvement.

The autopsy of a 65-year-old diabetic African American male revealed significant left myocardial involvement by adult T-cell leukemia/lymphoma (ATLL) despite normal pre-mortem fluorodeoxyglucose (FDG) uptake by positron emission tomography/computed tomography (PET/CT). Due to pre-existing diabetic cardiomyopathy with reduced ejection fraction (EF) and compatible imaging studies, cardiac lymphomatous involvement was not suspected. While peripheral blood was negative for leukemia, next-generation sequencing of a lymph node revealed at least eight novel mutations (AXIN1, R712Q, BARD1 R749K, CTNNB1 I315V, CUX1 P102T, DNMT3A S199R, FGFR2 S431L, LRP1B Y2560C and STAG2 I771M). These findings underscore a diagnostic pitfall in a rare lymphomatous variant of ATLL infiltrating myocardium and contribute to its molecular characterization.

l-ornithine activates Ca2+ signaling to exert its protective function on human proximal tubular cells.

Oxidative stress and reactive oxygen species (ROS) generation can be influenced by G-protein coupled receptor (GPCR)-mediated regulation of intracellular Ca2+ ([Ca2+]i) signaling. ROS production are much higher in proximal tubular (PT) cells; in addition, the lack of antioxidants enhances the vulnerability to oxidative damage. Despite such predispositions, PT cells show resiliency, and therefore must possess some inherent mechanism to protect from oxidative damage. While the mechanism in unknown, we tested the effect of l-ornithine, since it is abundantly present in PT luminal fluid and can activate Ca2+-sensing receptor (CaSR), a GPCR, expressed in the PT luminal membrane. We used human kidney 2 (HK2) cells, a PT cell line, and performed Ca2+ imaging and electrophysiological experiments to show that l-ornithine has a concentration-dependent effect on CaSR activation. We further demonstrate that the operation of CaSR activated Ca2+ signaling in HK-2 cells mediated by the transient receptor potential canonical (TRPC) dependent receptor-operated Ca2+ entry (ROCE) using pharmacological and siRNA inhibitors. Since PT cells are vulnerable to ROS, we simulated such deleterious effects using genetically encoded peroxide-induced ROS production (HyperRed indicator) to show that the l-ornithine-induced ROCE mediated [Ca2+]i signaling protects from ROS production. Furthermore, we performed cell viability, necrosis and apoptosis assays, and mitochondrial oxidative gene expression to establish that presence of l-ornithine rescued the ROS-induced damage in HK-2 cells. Moreover, l-ornithine-activation of CaSR can reverse ROS production and apoptosis via mitogen-activated protein kinase p38 activation. Such nephroprotective role of l-ornithine can be useful as the translational option for reversing kidney diseases involving PT cell damage due to oxidative stress or crystal nephropathies.