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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. [ABSTRACT FROM AUTHOR]

Objective: To study the association of CaSR gene polymorphism with calcium nephrolithiasis and hypercalciuria in Chinese Dai population. Methods: The Dai patients with calcium containing renal calculi in Dehong Prefecture, Yunnan Province, China were selected as the experimental group, and the healthy Dai people in Dehong Prefecture, Yunnan Province were taken as the control group. The venous blood was drawn for the determination of the three SNP loci rs7652589, rs1801725, and rs1042636 of the CaSR gene. The subjects in the stone group were tested for 24-hour urine calcium value, and the gene correlation was analyzed. Results: There was a statistical difference between the homozygous AA gene and AA+GG gene at rs7652589 locus in the occurrence of calcium nephrolithiasis (P < 0.05). The 24 h urine calcium value of the GG genotype with rs1042636 locus was significantly different from that of the AA based and AG genotype (P < 0.05). There was a significant difference in 24 h urine calcium between those with GG genotype at rs7652589 locus and those with AA and AG genotype (P < 0.05) Conclusion　The rs7652589 polymorphism on the CaSR gene in the Dai population in China is related to the occurrence of calcium nephrolithiasis and hypercalciuria. The GG gene at rs1042636 is related to the occurrence of hypercalciuria in the Dai population. The GG gene at rs7652589 may be a candidate gene for the occurrence of hypercalciuria leading to calcium nephrolithiasis in this ethnic group. [ABSTRACT FROM AUTHOR]

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.

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.)

Calcium-sensing receptor (CaSR) is a plasma-membrane G protein-coupled receptor activated by extracellular calcium and expressed in kidney tubular cells. It inhibits calcium reabsorption in the ascending limb and distal convoluted tubule when stimulated by the increase of serum calcium levels; therefore, these tubular segments are enabled by CaSR to play a substantial role in the regulation of serum calcium levels. In addition, CaSR increases water and proton excretion in the collecting duct and promotes phosphate reabsorption and citrate excretion in the proximal tubule. These CaSR activities form a network in which they are integrated to protect the kidney against the negative effects of high calcium concentrations and calcium precipitates in urine. Therefore, the CaSR gene has been considered as a candidate to explain calcium nephrolithiasis. Epidemiological studies observed that calcium nephrolithiasis was associated with polymorphisms of the CaSR gene regulatory region, rs6776158, located within the promoter-1, rs1501899 located in the intron 1, and rs7652589 in the 5′-untranslated region. These polymorphisms were found to reduce the transcriptional activity of promoter-1. Activating rs1042636 polymorphism located in exon 7 was associated with calcium nephrolithiasis and hypercalciuria. Genetic polymorphisms decreasing CaSR expression could predispose individuals to stones because they may impair CaSR protective effects against precipitation of calcium phosphate and oxalate. Activating polymorphisms rs1042636 could predispose to calcium stones by increasing calcium excretion. These findings suggest that CaSR may play a complex role in lithogenesis through different pathways having different relevance under different clinical conditions. [ABSTRACT FROM AUTHOR]

Abstract: The calcium‐sensing receptor (CaSR) gene plays an important role in regulating the Ca2+ balance and reducing the risk for calcium stones. In this study, we evaluated the association of CaSR polymorphisms with calcium nephrolithiasis in the population of Yi nationality in Southwestern China. Biochemical variables were evaluated in 624 calcium nephrolithiasis patients and 470 age‐matched healthy controls without a history of nephrolithiasis. CaSR polymorphisms rs7652589, rs1501899, rs1801725 (Ala986Ser), rs1042636 (Arg990Gly) and rs1801726 (Gln1011Glu) were investigated between the calcium nephrolithiasis patients and healthy controls, using direct sequencing. Compared with the healthy controls, serum creatinine and 24‐hour urine calcium levels were significantly higher in calcium nephrolithiasis patients. Among these five polymorphisms, the genotypic and allelic frequency distributions of rs7652589 SNP was significantly associated with the risk of calcium nephrolithiasis. However, there were no genotypic or allelic distribution differences for rs1501899, rs1801725, rs1042636, and rs1801726 polymorphisms between calcium nephrolithiasis patients and healthy controls. Moreover, the association between rs7652589 SNP genotypes and the biochemical variables was not found. Our study showed that CaSR rs7652589 polymorphism had a significant effect on the risk of developing calcium nephrolithiasis in the population of Yi nationality in Southwestern China. [ABSTRACT FROM AUTHOR]

Background. Experimental evidence indicate that melatonin regulates some renal tubular functions via specific melatonin receptors (MTNRs) located in the kidney of several avian and mammalian species, including humans. We hypothesized that single nucleotide polymorphisms (SNPs) in the melatonin receptor 1A gene (MTNR1A) might influence the risk of calcium nephrolithiasis. Methods. We performed a systematic analysis of the MTNR1A gene in 246 recurrent calcium stone formers (136 men, 110 women; mean age 40.2 ± 12.0 years; body mass index 25.8 ± 4.5 kg/m2) and 269 healthy controls comparable for age and gender without a history of nephrolithiasis. Results. Two SNPs in Intron 1 of MTNR1A were significantly associated with calcium nephrolithiasis: rs13140012 (P = 0.0004) and rs6553010 (P = 0.009). The haplotypes resulting from the two SNPs were also differently distributed between stone formers and controls, the haplotype A-T being more represented among stone formers (P = 0.00001) and the haplotype T-C being more common in healthy controls (P = 0.00001). Preliminary functional studies showed that the SNP rs13140012 could modify the binding sites for transcription factors. Conclusion. The results of this case–control study indicate a strong association between allelic variants of MTNR1A and recurrent calcium nephrolithiasis. [ABSTRACT FROM PUBLISHER]

Supplementation of certain calcium crystallization inhibitors, such as citrate and magnesium, and the dilution of urine with water are now considered consolidated practice for the prevention of calcium kidney stones. The aim of this study is to verify, using tried and true in vitro methods, whether the effect of these inhibitors can manifest itself in different ways depending on whether the urine is concentrated or diluted. Calcium oxalate crystallization was studied on 4-h urine of 20 male idiopathic calcium oxalate stone formers, first under low hydration conditions (non-diluted urine) and then under high hydration conditions (diluted urine). Both the diluted and the non-diluted urine samples were subjected to three types of load: (a) an oxalate concentration increment of 1.3 mmol/l only; (b) an oxalate concentration increment of 1.3 mmol/l with a citrate concentration increment of 1.56 mmol/l; (c) an oxalate concentration increment of 1.3 mmol/l with a magnesium concentration increment of 2.08 mmol/l. In non-diluted urine, the addition of the citrate and magnesium did not modify the crystallization parameters under study. In contrast, in the diluted urine the addition of the citrate and magnesium led to a reduction in the total quantity of crystals (equivalent to 35–45%) and their aggregates (equivalent to 30–40%); at the same time, there was an increase in the diameter of the monohydrate calcium oxalate crystals, which also underwent a morphological change. In conclusion, the inhibitory effects of citrate and magnesium on the crystallization of calcium oxalate do not manifest themselves in highly concentrated urine. [ABSTRACT FROM AUTHOR]

Increasing urinary volume is an important tool in the prevention of calcium renal stones. However, the mechanism of how it actually works is only partially understood. This study aimed at assessing how urine dilution affects urinary calcium oxalate crystallization. A total of 16 male idiopathic calcium oxalate (CaOx) stone-formers and 12 normal male subjects were studied and 4 h urine samples were taken twice, under low (undiluted urine) and high hydration conditions (diluted urine). An equal oxalate load (1.3 mmol/L) was added to both types of urine and the crystallization parameters were assessed. In both stone-formers and normal subjects, the crystallization processes were significantly (p<0.05 or less) more marked in the undiluted urine than in the diluted urine in terms of: a) total quantity of calcium oxalate dihydrate (COD) and calcium oxalate monohydrate (COM) crystals; b) total quantity of crystalline aggregates; and c) aggregation index (i.e., ratio between the area occupied by crystalline aggregates and the area occupied by all the crystals present). The comparison between stone-formers and normal subjects showed that the greatest difference was for the size of COD crystals, which were larger in the urine of the stone-formers. A further important finding was an inverse relationship between changes in urinary volume and in the aggregation index (r=–0.53, p=0.004). In conclusion, urine dilution considerably reduces crystallization phenomena induced in vitro by an oxalate load in both calcium stone-formers and normal subjects. [ABSTRACT FROM AUTHOR]

Objective: A normal dietary calcium intake to reduce intestinal oxalate absorption is essential to avoid recurrence of calcium oxalate stone formation. It is also important in the prevention of osteopenia in idiopathic hypercalciuria. The calcium content of waters used for hydration may vary from very low to relatively high and is an important factor in prevention or additional risk of stone formation. Therefore, the effect of drinking mineral waters of different calcium concentrations on lithogenic risk factors was studied in normal volunteers. Materials and Methods: Normal subjects were divided into two groups of 11 and 10 individuals each. All followed a prescribed diet with an average calcium content of 800 mg/day. The water intake for hydration consisted of 2 liters of an oligomineral water with a low calcium content, <20 mg/l (group A) or of a bicarbonate alkaline water with a high calcium content, 370 mg/l (group B). Results: Diuresis increased similarly in both groups; urine calcium increased by about 80 mg/day in group B. A rise in urine oxalate was observed in both groups, along with the increased urine volume. Osmolar excretion increased in group B; urine osmolality decreased significantly only in group A. In spite of the increase in calciuria in group B, Ca/citrate ratio was constant, due to an increase in citrate excretion. Inter-group differences in terms of activity products of calcium phosphate, calculated according with Tiselius’s methods, were found. The differences in AP(CaP) index 1 and AP(CaP) index 2 were significant, with higher values in group B, who drank the bicarbonate alkaline mineral water. Conclusions: Increased water intake between meals to prevent renal stone recurrence should preferably be achieved with a relatively low calcium water and calcium-rich mineral waters should be avoided.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background. The aim of this study was to measure the effects of normal (nTHP) and abnormal stone former Tamm–Horsfall protein (SF-THP) on calcium oxalate (CaOx) nucleation and aggregation as well as on crystal morphology, in presence or absence of citrate. [ABSTRACT FROM PUBLISHER]

Calcium nephrolithiasis (CaNL) accounts for more than 70% of all renal stones, and its prevalence has increased in the last decades. Under this definition are included patients passing stones, composed of calcium oxalates and/or calcium phosphates. Current views of the pathogenesis of CaNL are based on the role of metabolic abnormalities which concur to render urines more conducive to crystallization. Therefore, the diagnostic approach is aimed at detecting these abnormalities, and the medical treatment assumes that a decrease in the risk of lithogenesis will result in remission or improvement of recurrences. The workup of the patients with CaNL begins with the analysis of passed stones and X-ray, sonography or other imaging techniques. Eligible patients, that is, both recurrent active stone formers and single-stone formers with individual risk factors, are considered for a metabolic evaluation, by which a number of blood and urine parameters are measured and others calculated. These include estimates of urine state of saturation with calcium and uric acid salts, net gastrointestinal alkali absorption, renal threshold of phosphate and other renal clearances and net acid and total nitrogen excretions. Basically, this screening is informative on renal function, metabolic abnormalities and their pathophysiology, risk of stone formation and dietary habits. During treatment it gives information about patient compliance and adverse effects of therapy. The cost of a comprehensive screening in Piedmont is 192,000 ITL (100 Euro) and rises to 300,000 ITL (154 Euro) if hormones and hydroxyproline are measured. In individual patients second- and third-level studies are performed, in order to detect systemic diseases which account for about 20% of CaNL in our series. Cost-to-benefit analysis has shown that the medical procedures for CaNL yield considerable saving in terms of difference between expenditure for drugs and testing and reduction of stone events. However, the current workup cannot be considered exhaustive, because misleading events may hamper the relation between laboratory findings and clinical outcome, and factors other than urine composition have appeared on the scenario of nephrolithiasis. These represent our challenge for the third millennium. [ABSTRACT FROM AUTHOR]

The calciuric response after an oral calcium load (l000 mg elemental calcium together with a standard breakfast) was studied in 13 healthy male controls and 21 recurrent idiopathic renal calcium stone formers, 12 with hypercalciuria (U×V>7.50 mmol/24 h) and nine with normocalciuria. In controls, serum 1,25(OH) vitamin D3 (calcitriol) remained unchanged 6 h after oral calcium load (50.6±5.1 versus 50.9±5.0 pg/ml), whereas it tended to increase in hypercalciuric (from 53.6±3.2 to 60.6±5.4 pg/ml, =0.182) and fell in normocalciuric stone formers (from 45.9±2.6 to 38.1±3.3 pg/ml, =0.011). The total amount of urinary calcium excreted after OCL was 2.50±0.20 mmol in controls, 2.27±0.27 mmol in normocalciuric and 3.62±0.32 mmol in hypercalciuric stone formers (=0.005 versus controls and normocalciuric stone formers respectively); it positively correlated with serum calcitriol 6 h after calcium load (=0.392, =0.024). Maximum increase in urinary calcium excretion rate, δCa-E, was inversely related to intact PTH levels in the first 4 h after calcium load, i.e. more pronounced PTH suppression predicted a steeper increase in urinary calcium excretion rate. Twenty-four-hour urine calcium excretion rate was inversely related to the ratio of δ calcitriol/δPTH after calcium load (=−0.653, =0.0001), indicating that an abnormally up-regulated synthesis of calcitriol and consecutive relative PTH suppression induce hypercalciuria. Finally, late absorption of calcium as suggested by maximum urinary calcium excretion beyond 4 h after oral calcium load was as rare in hypercalciuric stone formers (2 of 12) as in controls (1 of 13) and did not occur in normocalciuric stone formers. [ABSTRACT FROM PUBLISHER]

Risk factors for low urinary citrate excretion were assessed in 34 consecutive male recurrent idiopathic calcium stone formers (RCSF) who collected two 24-h urines while on free-choice diet. Overt hypocitraturia (hypo-cit) was defined as U×V<1.70 mmol/day, and ‘low’ citraturia (low-cit) as U×V between 1.70 and 2.11 mmol/day. Twenty-three RCSF had normocitraturia (normo-cit), six low-cit and five hypo-cit. U×V positively correlated with urine volume (VOLUME, =0.44, =0.009), vegetable fibre intake (fibers, =0.46, =0.009) and GI-alkali absorption (alkali, =0.47, =0.006), and volume, fibres and alkali tended to be lower among RCSF with low-/hypo-cit. A 3-day NHCl loading test (0.95 mEq/kg BW daily in 3 doses) was performed in RCSF as well as in 14 age-matched healthy male controls (C). On a plot of urine pH versus serum bicarbonate, 10 of 11 RCSF with low-/hypo-cit, but only six of 23 with normo-cit (=0.0004) fell off the normal range, indicating incomplete RTA. Two or more risk factors simultaneously occurred in only four of 23 RCSF with normo-cit, but in eight of 11 with low-/hypo-cit (= 0.002). In conclusion, incomplete RTA is the most prevalent risk factor for low-/hypo-cit in RCSF, and decreases in vegetable fibres and urine volume emerge as two new risk factors for low urinary CIT. [ABSTRACT FROM PUBLISHER]

While the pathogenic mechanisms responsible for calcium nephrolithiasis remain unknown, the influence of heparan sulphate proteoglycan (HSPG) on disease progression of other diseases, such as polycystic kidneys and diabetic glomerulosclerosis, makes it an important candidate for the study of stone formation. Using the indirect immunofluorescence assay and image analysis, we were able to quantify and visualize the loss of HSPG localized in the basement membrane of the glomerulus and the mucosa of ureter or renal pelvis in patients with recurrent calcium nephrolithiasis as compared to normal subjects. However, no significant change in HSPG was observed in the basement membrane of the tubular epithelium. The decreased HSPG in the glomerulus may reflect the potentially disrupted anion/neutral barrier for glomerular filtration, which would encourage the accumulation of stone solutes. The drop in HSPG staining intensity in the basement membrane of the mucosa of ureter/renal pelvis may suggest the tendency of adhesion of crystal to urothelial surfaces. Based on these immunological data, it appears that HSPG plays a modulatory role in the pathogenesis of this disease. [ABSTRACT FROM AUTHOR]

116 normocalcemic and 8 primary hyperparathyroid (PHPT) patients with calcium (Ca) nephrolithiasis and 10 normal controls underwent 1 g of oral Ca tolerance test following 4 days of Ca restricted diet (400 mg/day). On the basis of urinary Ca/creatinine (Cr) ratio obtained by the test, the 116 patients with normocalcemic nephrolithiasis were divided into 3 groups (normocalciuric nephrolithiasis; NN, absorptive hypercalciuria; AH, renal hypercalciuria; RH) according to our criteria which were slightly modified from Pak et al. Changes in urinary Ca/Cr ratio, and those in serum Ca and phosphorus (P), tubular maximum reabsorption of phosphate/glomerular filtration rate (TmPO/GFR), nephrogenous adenosine 3′,5′-monophosphate (NcAMP) and plasma immunoreactive parathyroid hormone (iPTH) were determined. As a result, the 116 patients were divided into 82NN, 13AH and 21RH. In general, a rise in serum Ca and fall in NcAMP were seen first, followed by rises in urinary Ca/Cr ratio, serum P and TmPO/GFR although the changes were small. The group PHPT showed abnormality in the changes of TmPO/GFR, NcAMP and plasma iPTH. The former one decreased constantly during the test and the latter two did not fall to within the normal range, suggesting parathyroid autonomy or abnormal suppressibility. Regarding the normal controls, all the changes were smallest among the 5 groups and clear parathyroid suppression was not observed while it was seen in the groups NN, AH and RH. In conclusion, oral Ca tolerance test is useful not only to separate NN, AH and RH, but also for the diagnosis of PHPT by demonstrating parathyroid autonomy or abnormal suppressibility assessed by NcAMP and/or TmPO/GFR. [ABSTRACT FROM AUTHOR]

Previous studies have shown a significant decrease of heparin sulfate proteoglycan (HSPG) in the basement membrane of the glomerulus and the mucosa of the ureter/renal pelvis in patients with calcium nephrolithiasis. In this study, we looked at the localization of another influential proteoglycan, chondroitin sulfate (CSPG), using similar study groups by indirect immunofluorescence staining. Microscopic images were digitized and image analysis was used to quantitate the staining intensity of CSPG present in the basement membrane of the nephron. Our data showed significant loss of CSPG in the Bowman's capsule and the basement membrane of the mucosa of the ureter/renal pelvis using Mann-Whitney U-Wilcoxon Rank Sum W test with P-values of 0.0043 and 0.0041, respectively. However, absence of staining was noted in the basement membrane of the glomerulus and no significant change in the basement membrane of the tubular epithelium was observed. In conclusion, our results showed changes in the localization of CSPG in the basement membrane of the nephron, accompanied with HSPG, which may contribute to the pathological condition of calcium nephrolithiasis. [ABSTRACT FROM AUTHOR]

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.