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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 [Ca 2+ ], 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., (Published by Elsevier Inc.)

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 ([Ca 2+ ] 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 Ca 2+ 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 [Ca 2+ ] 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 Ca 2+ 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 [Ca 2+ ] 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 Ca 2+ 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

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 Ca 2+ -sensing receptor (CaSR) in human kidney 2 (HK-2) renal proximal tubular epithelial cells, using Ca 2+ 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 Ca 2+ entry via a Transient receptor potential canonical (TRPC) channel in HK-2 cells mainly through the receptor operated Ca 2+ entry (ROCE). Activation of CaSR by l-Arg led to the rise in p-p38/p38 expression suggesting [Ca 2+ ] i as a regulator for p38-signaling pathways. Notably, l-Arg activated CaSR-induced Ca 2+ signaling reduced the expressions of key fibrotic, inflammatory, and apoptotic genes, suggesting its nephroprotective role via Ca 2+ 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Proximal tubular (PT) cells reabsorb most calcium (Ca 2+ ), phosphate (PO 4 3- ), bicarbonate (HCO 3 - ), and oxalate (C 2 O 4 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 HCO 3 - , PO 4 3- , and C 2 O 4 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 HCO 3 - -induced currents in all PT cells, which was confirmed by using specific HCO 3 - channel inhibitor, S0859. Interestingly, such activities were diminished in PT cells from TRPC3 -/- mice, suggesting reduced HCO 3 - transport in absence of TRPC3. While PO 4 3- -induced currents were also concentration dependent in all PT cells (confirmed by PO 4 3- channel inhibitor, PF-06869206), those activities were reduced in absence of TRPC3, suggesting lower PO 4 3- reabsorption that can leave excess luminal PO 4 3- . Next, we applied thiosulfate (O 3 S 2 2 - ) as a competitive inhibitor of the SLC26a6 transporter upon C 2 O 4 2- current activation and observed a reduced C 2 O 4 2- -induced conductance which was greater in TRPC3 -/- PT cells. Together, these results suggest that the reduced activities of HCO 3 - , PO 4 3- , and C 2 O 4 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., (© 2022 John Wiley & Sons Australia, Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

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 Ca 2+ . 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 Ca 2+ 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.

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 H 2 O 2 and lactate dehydrogenase (LDH) release leading to store-operated Ca 2+ 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., (Published by Elsevier Masson SAS.)

Proximal tubule (PT) transports most of the renal Ca 2+ , which was usually described as paracellular (passive). We found a regulated Ca 2+ entry pathway in PT cells via the apical transient receptor potential canonical 3 (TRPC3) channel, which initiates transcellular Ca 2+ 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 [Ca 2+ ] o further in TRPC3 -/- mice for developing such mixed stones to understand the mechanistic role of PT-Ca 2+ 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 Ca 2+ entry from receptor-operated to store-operated pathway with a sustained rise in intracellular [Ca 2+ ] ([Ca 2+ ] i ), indicating the stagnation in PT Ca 2+ 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 Ca 2+ due to escalation of hypercalciuria in TRPC3 ablated mice induced surplus CaP crystal formation and caused stagnation of PT [Ca 2+ ] i , invoking PT cell injury, hence mixed stone formation.

Proximal tubular (PT) acidosis, which alkalinizes the urinary filtrate, together with Ca 2+ 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 Ca 2+ concentration ([Ca 2+ ]) under elevated pH and/or high [Ca 2+ ] 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 [Ca 2+ ] measurements reveal that such alkalization not only upsurges Ca 2+ influx into PT cells, but the mode of Ca 2+ 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 Ca 2+ 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.

Melamine, which induces proximal tubular (PT) cell damage has a greater nephrotoxic effect when combined with cyanuric and uric acids; however, it is unknown whether such effect can stimulate calcium phosphate (CaP)/calcium oxalate (CaOx) stone formation. Here, we show that melamine acts as an inducer of CaP, CaOx and CaP + CaOx (mixed) crystal formations in a time and concentration-dependent manner by stabilizing those crystals and further co-aggregating with melamine. To explore the physiological relevance of such melamine-augmented calcium crystal formation, we used 2-dimensional (2D) and 3D microfluidic (MF) device, embedded with PT cells, which also resembled the effect of melamine-stimulated CaP, CaOx and mixed crystal formation. Significantly, addition of preformed CaP and/or CaOx crystal in the presence of melamine, further potentiated those crystal formations in 3D MFs, which helped the growth and aggregation of mixed crystals. Our data show that the mechanism of such predisposition of stone formation could be largely due to co-crystallization between melamine and CaP/CaOx and pronounced effect on induction of stone-forming pathway activation in 3D MF. Taken together, melamine-induced CaP and/or CaOx crystal formation ex-vivo will help us in understanding the larger role of melamine as an environmental toxicant in producing the pathology in similar cellular microenvironments.