• Journal of Life Science
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• v.26 no.3
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• pp.331-337
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• 2016

Although fibroblast growth factor 23 (FGF23) is exclusively produced in osteoblasts and osteocytes, its main target is the kidney, where it decreases phosphate reabsorption by suppressing Na-phosphate cotransporters. Independently of its action on phosphate homeostasis, FGF23 also inhibits bone formation in vivo. In a calvarial osteoblastic cell model, FGF23 was shown to negatively affect extracellular matrix mineralization. This study investigated whether FGF23 had similar effects on osteoblast maturation, including differentiation and mineralization of bone marrow-derived mesenchymal stem cells (MSCs). D1 MSCs were cultured in an osteogenic medium containing β-glycerophosphate, ascorbic acid, and dexamethazone. Osteoblastic differentiation was evaluated by alkaline phosphatase (Alp) staining, and matrix mineralization was evaluated by alizarin red staining and calcium deposition. The expression of differentiation-stimulating genes Runx2, Alp, and osteocalcin and mineralization-inhibiting genes Enpp1 and Ank was analyzed using semiquantitative RT-PCR. Supraphysiological doses of FGF23 did not stimulate proliferation or osteoblastic differentiation of MSCs. Matrix mineralization 1, 2, and 3 weeks after the FGF23 treatment did not vary between control and FGF23 groups, although time-dependent enhancement of mineralization was obvious. Calcium deposition was also unchanged after the FGF23 treatment. mRNA expression levels of differentiation- and mineralization-related genes were also similar between the groups. Despite these negative findings, FGF23 signaling through FGF receptors seemed to function normally, with phosphorylation of the Erk protein more evident in the FGF23 group than in controls. These findings suggest that unlike calvarial osteoblasts, FGF23 is not likely to affect osteoblastic differentiation and mineralization of MSCs.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.2
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• pp.91-95
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• 2012

The role of the kidney in combating metabolic acidosis has been a subject of considerable interest for many years. The present study was aimed to determine whether there is an altered regulation of renal acid base transporters in acute and chronic acid loading. Male Sprague-Dawley rats were used. Metabolic acidosis was induced by administration of $NH_4Cl$ for 2 days (acute) and for 7days (chronic). The serum and urinary pH and bicarbonate were measured. The protein expression of renal acid base transporters [type 3 $Na^+/H^+$ exchanger (NHE3), type 1 $Na^+/{HCO_3}^-$ cotransporter (NBC1), Na-$K^+$ ATPase, $H^+$-ATPase, anion exchanger-1 (AE-1)] was measured by semiquantitative immunoblotting. Serum bicarbonate and pH were decreased in acute acid loading rats compared with controls. Accordingly, urinary pH decreased. The protein expression of NHE3, $H^+$-ATPase, AE-1 and NBC1 was not changed. In chronic acid loading rats, serum bicarbonate and pH were not changed, while urinary pH was decreased compared with controls. The protein expression of NHE3, $H^+$-ATPase was increased in the renal cortex of chronic acid loading rats. These results suggest that unaltered expression of acid transporters combined with acute acid loading may contribute to the development of acidosis. The subsequent increased expression of NHE3, $H^+$-ATPase in the kidney may play a role in promoting acid excretion in the later stage of acid loading, which counteract the development of metabolic acidosis.

• YAKHAK HOEJI
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• v.40 no.6
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• pp.705-712
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• 1996

We have previously shown that determination of glucose uptake using ${\alpha}$-methylglucose(${\alpha}$-MG) is very sensitive and rapid parameter for the assessment of loss of cellular fu nction in renal cell line($LLC-PK_1$). The present study was designed to elucidate the mechanism of inhibition of ${\alpha}$-MG uptake and the intracellular site of toxic action of cisplatin(CIS). $LLC-PK_1$ cells were exposed to various concentrations(5 ${\mu}$M-l00 ${\mu}$M) of CIS for 5 hrs or 24 hrs and ${\alpha}$-MG uptake was determined. Mitochondrial function was evaluated by measuring intracellular ATP content and MTT reduction. The activities of marker enzymes for the basolateral membrane(Na$^+$-K$^+$ ATPase) and brush border membrane (alkaline phosphatase: ALP) were also measured. CIS treatment significantly inhibited the ${\alpha}$-MG uptake in a time- and dose-dependent manner above 25 ${\mu}$M for 5 hrs. Intracellular ATP content and MTT reduction were affected by 24 hr-treatment of 50 ${\mu}$M CIS. The activities of Na$^+$-K$^+$ ATPase and ALP were significantly decreased at 10 ${\mu}$M and 5 ${\mu}$M of CIS for 24 hrs, respectively. The incubation with CIS for 5 hrs had no effects on the intracellular ATP content, MTT reduction and the activities of marker enzymes up to 100 ${\mu}$M. These results partly indicate that inhibition of ${\alpha}$-MG uptake by CIS may not be attributed to the disturbance of mitochondrial function or inhibition of the activity of Na$^+$-K$^+$ ATPase and can be resulted from direct effect of CIS on the Na$^+$/glucose cotransporter in brush border membrane. This study shows that additional mechanistic information, indicating the intracellular site of nephrotoxic action, can be gained by coupling the ${\alpha}$-MG uptake and ATP content or the activity of Na$^+$-K$^+$ ATPase.

• Korean Journal of Veterinary Research
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• v.36 no.3
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• pp.551-563
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• 1996

This study investigated the properties of primary cultured proximal tubule cells in hormonally defined(insulin, transferrin, and hydrocortisone), serum-free medium or 10% serum-supplemented medium. The growth rate of the primary cultured proximal tubule cells was lower in the hormonally defined, serum-free medium than in the 10% serum- supplemented medium(p < 0.05), while the activities of brush border marker enzymes, alkaline phosphatase(AP), leucine aminopeptidase(LAP), and y-glutamyl transpeptidase(${\gamma}$-GTP) were increased(p < 0.05). The activities of these enzymes, however, decreased with the lapse of incubation time to 50-70% after 6 days culture compared to those of the freshly-prepared proximal tubules. The enzymatic activities of the primary cultured proximal tubul cells on 6, 9, 12, and 15 days of culture were significantly increased in the hormonally defined, serum-free medium compared to the 10% serum-supplemented medium(p < 0.05). The functional differentiation of the primary culture was examined by observing multicellular domes of the confluent monolayer, which is indicative of transepithelial solute transport. The dome formation by the proximal tubule cultures occurred at a higher frequency in the hormonally defined, serum-free medium than in the 10% serum-supplemented medium(p < 0.05). Upon electron microscopic examination, an increased density of the brush border was observed in the hormonally defined, serum-free medium compared to the cells grown in 10% serum-supplemented medium. The activities of $Na^+$glucose cotransporter($^{14}C$-a-MG uptake), $Na^+$phosphate cotransportere($^{32}P$ uptake) and $Na^+$ transporter($^{22}Na^+$ uptake) in the brush border membrane, and of $Na^+/K^+$-ATPase($^{86}Rb$ uptake) in the basolateral membrane were significantly stimulated in the hormonally defined, serum-free medium than in 10% serum-supplemented medium(p < 0.05). In conclusion, the primary cultured proximal tubule cells grown in the hormonally defined, serum-free medium demonstrated a slower growth rate, but the functions of cell were enhanced.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.4
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• pp.521-527
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• 1998

An important property of the intestine is the ability to secrete fluid. The intestinal secretion is regulated by a number of substances including vasoactive intestinal peptide (VIP), ATP and different inflammatory mediators. One of the most important secretagogues is adenosine during inflammation. However, the controversy concerning the underlying mechanism of adenosine-stimulated $Cl^-$ secretion in intestinal epithelial cells still continues. To investigate the effect of adenosine on $Cl^-$ secretion and its underlying mechanism in the rabbit colon mucosa, we measured short circuit current ($I_{SC}$) under automatic voltage clamp with DVC-1000 in a modified Ussing chamber. Adenosine, when added to the basolateral side of the muocsa, increased $I_{SC}$ in a dose-dependent manner. The adenosine-stimulated $I_{SC}$ response was abolished when $Cl^-$ in the bath solution was replaced completely with gluconate. In addition, the $I_{SC}$ response was inhibited by a basolateral Na-K-Cl cotransporter blocker, bumetanide, and by apical $Cl^-$ channel blockers, dephenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), glibenclamide. Amiloride, an epithelial $Na^+$ channel blocker, and 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS), a $Ca^{2+}-activated$ $Cl^-$ channel blocker, had no effect. In the mucosa pre-stimulated with forskolin, adenosine did not show any additive effect, whereas carbachol resulted in a synergistic potentiation of the $I_{SC}$ response. The adenosine response was inhibited by 10 ${\mu}M$ H-89, an inhibitor of protein kinase A. These results suggest that the adenosine-stimulated $I_{SC}$ response is mediated by basolateral to apical $Cl^-$ secretion through a cAMP-dependent $Cl^-$ channel. The rank order of potencies of adenosine receptor agonists was $5'-(N-ethylcarboxamino)adenosine(NECA)＞N^6-(R-phenylisopropyl)adenosine(R-$ PIA)＞2-[p-(2-carbonylethyl)-phenyl-ethylamino]-5'-N-ethylcarboxaminoadenosine(CGS21680). From the above results, it can be concluded that adenosine interacts with the $A_{2b}$ adenosine receptor in the rabbit colon mucosa and a cAMP-dependent signalling mechanism underlies the stimulation of $Cl^-$ secretion.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.4
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• pp.493-501
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• 1998

The most significant direct role of estrogen in vivo is its ability to elicit receptor-mediated cellular proliferation in mammalian target tissues. However, the mechanism by which exogenously added estrogen causes the neoplastic transformation of renal cortical cells is yet to be uncovered. The present study was designed to evaluate interaction of $17{\beta}-estradiol\;(E_2)$ with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) on proliferation and $P_i$ uptake in primary cultured rabbit renal proximal tubular cells in phenol red-free, hormonally defined-medium. $[^3H]-thymidine$ incorporation increased markedly by about 133% and 141% more in the presence of $10^{-9}\;and\;10^{-6}\;M\;E_2$, respectively, than that of control. Cell count was 162% and 143% greater in the presence of $10^{-9}\;and\;10^{-6}\;M\;E_2$ , respectively, compared with control. Among all time points examined, there was an increase in $[^3H]-thymidine$ incorporation in the presence of $10^{-9}\;M\;E_2$ at day 9 or 13, respectively. However, $E_2$ ($10^{-9}\;M$) significantly drove up cell count to 160% of that of control at day 13, while it had a slight but statistically insignificant effect at day 9. $E_2-induced$ stimulation of $[^3H]-thymidine$ incorporation was completely reversed by $E_2$ antagonists (progesterone or tamoxifen). $E_2$ ($10^{-9}\;M$) or EGF ($10^{-8}\;M$) significantly stimulated $[^3H]-thymidine$ incorporation by 144% and 154% of control. $E_2$ plus EGF was synergistic on $[^3H]-thymidine$ incorporation (204% of control), while $E_2$ plus IGF-I showed a slight but no significant synergistic effect. Cell number also displayed similar pattern. $E_2$ ($10^{-9}\;M$) significantly stimulated $P_i$ uptake to 134% of control. $E_2$-induced stimulation of $P_i$ uptake was partially reversed by $E_2$ antagonists. EGF or IGF-I ($10^{-8}\;M$) significantly also increased $P_i$ uptake to 132% or 129% of control. $E_2$ plus EGF had synergistic effect on $P_i$ uptake, while $E_2$ plus IGF-I did not. In conclusion, $E_2$ may act not only directly interaction with its receptors but also indirectly as a modulator of EGF in proliferation and $P_i$ uptake of primary cultured rabbit renal proximal tubular cells.

• Animal Bioscience
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• v.35 no.12
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• pp.1921-1928
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• 2022

Objective: This research aimed to evaluate the effects of age on growth, tibia development, and intestinal calcium (Ca) and phosphorus (P) transporter gene expressions in broiler chickens. Methods: A total of 224 male Arbor Acres broilers were fed with nutrient-adequate diets and reared in eight cages (28 broilers per cage). Eight broilers (one broiler per cage) were selected and killed at 5, 10, 15, 20, 25, 30, 35, and 40 days of age, respectively. Results: Body weight continuously increased with age of broiler chickens from 5 to 40 days. The bone weight, ash weight, diameter, and length of the tibia also increased with broiler age. By contrast, the tibia ash, Ca, and P percentages quadratically changed with age (p<0.001), and the highest values of mineral contents were observed at 20, 25, and 25 days of age, respectively. The mRNA abundances of calcium-binding protein 28-kDa (CaBP-D28k), sodium-calcium exchanger 1 (NCX1), and plasma membrane ATPase 1b (PMCA1b) increased from 5 to 25 days and then decreased up to 40 days. Similar results were noted in the mRNA abundances of IIb sodium-phosphate cotransporter (NaPi-IIb), inorganic phosphate transporter 1 (PiT-1), inorganic phosphate transporter 2 (PiT-2), nuclear vitamin D receptor (nVDR), and membrane vitamin D receptor (mVDR). The mRNA abundances of Ca and P transporters and VDRs were the highest at 25 days of age. Conclusion: These data indicate that age quadratically affects intestinal Ca and P transporter gene expression and mineral absorption capacity in broiler chickens.

• Journal of the korean academy of Pediatric Dentistry
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• v.25 no.2
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• pp.352-367
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• 1998

Intracellular pH (pHi) plays an important role in the regulation of cellular processes by influencing the acitivity of various enzymes in cells. Therefore, almost every type of mammalian cell possesses an ability to regulate its pHi. One of the most prominent mechanisms in the regulation of pHi is $Na^+/H^+$ exchanger. This exchanger has been known to be activated when cells are stimulated by the binding of agonist to the muscarinic receptors. Therefore, the aims of this study were to compare the rates of $H^+$ extrusion through $Na^+/H^+$ exchanger before and during muscarinic stimulation and to investigate the possible existence of $HCO_3^-$ transporter which is responsible for the continuous supply of $HCO_3^-$ ion to saliva. Acinar cells were isolated from the rat mandibular salivary glands and loaded with pH-sensitive fluoroprobe, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), for 30min at room temperature. Cells were attached onto the coverglass in the perfusion chamber and the changes in pHi were measured on the iverted microscope using spectrofluorometer. 1. By switching the perfusate from $HCO_3^-$-free to $HCO_3^-$-buffered solution, pHi decreased by $0.39{\pm}0.02$ pH units followed by a slow increase at an initial rate of $0.04{\pm}0.007$ pH units/min. The rate of pHi increase was reduced to $0.01{\pm}0.002$ pH units/min by the simultaneous addition of 1 mM amiloride and $100{\mu}M$ DIDS. 2. An addition and removal of $NH_4^+$ caused a decrease in pHi which was followed by an increase in pHi. The increase of pHi was almost completely blocked by 1mM amiloride in $HCO_3^-$-free perfusate which implied that the pHi increase was entired dependent on the activation of $Na^+/H^+$ exchanger in $HCO_3^-$-free condition. 3. An addition of $10{\mu}M$ carbachol increased the initial rate of pHi recovery from $0.16{\pm}0.01$ pH units/min to $0.28{\pm}0.03pH$ units/min. 4. The initial rate of pHi decrease induced by 1mM amiloride was also increased by the exposure of the acinar cells to $10{\mu}M$ carbachol ($0.06{\pm}0.008pH$ unit/min) compared with that obtained before carbachol stimulation ($0.03{\pm}0.004pH$ unit/min). 5. The intracellular buffering capacity ${\beta}1$ was $14.31{\pm}1.82$ at pHi 7.2-7.4 and ${\beta}1$ increased as pHi decreased. 6. The rate of $H^+$ extrusion through $Na^+/H^+$ exchanger was greatly enhanced by the stimulation of the cells with $10{\mu}M$ carbachol and there was an alkaline shift in the activity of the exchanger. 7. An intrusion mechanism of $HCO_3^-$ was identified in rat mandibular salivary acinar cells. Taken all together, I observed 3-fold increase in $Na^+/H^+$ exchanger by the stimulation of the acinar cells with $10{\mu}M$ carbachol at pH 7.25. In addition, I have found an additional mechanism for the regulation of pHi which transported $HCO_3^-$ into the cells.

• Journal of Animal Science and Technology
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• v.65 no.2
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• pp.336-350
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• 2023

Two experiments were conducted in this research. Experiment 1 investigated the spatial expression characteristics of calcium (Ca) and phosphorus (P) transporters in the duodenum, jejunum, and ileum of 21-day-old broilers provided with adequate nutrient feed. Experiment 2 evaluated the effects of dietary vitamin D₃ (VD₃) concentration (0, 125, 250, 500, 1,000, and 2,000 IU/kg) on growth performance, bone development, and gene expression levels of intestinal Ca and P transporters in 1-21-day-old broilers provided with the negative control diet without supplemental VD₃. Results in experiment 1 showed that the mRNA levels of calcium-binding protein 28-kDa (CaBP-D28k), sodium-calcium exchanger 1 (NCX1), plasma membrane calcium ATPase 1b (PMCA1b), and IIb sodium-phosphate cotransporter (NaPi-IIb) were the highest in the broiler duodenum. By contrast, the mRNA levels of inorganic phosphate transporter 1 (PiT-1) and 2 (PiT-2) were the highest in the ileum. Results in experiment 2 showed that adding 125 IU/kg VD₃ increased body weight gain (BWG), feed intake (FI), bone weight, and percentage and weight of Ca and P in the tibia and femur of 1-21-day-old broilers compared with the negative control diet (p < 0.05). The rise in dietary VD₃ levels from 125 to 1,000 IU/kg further increased the BWG, FI, and weights of the bone, ash, Ca, and P (p < 0.05). No difference in growth rate and leg bone quality was noted in the broilers provided with 1,000 and 2,000 IU/kg VD₃ (p > 0.05). Supplementation with 125-2,000 IU/kg VD₃ increased the mRNA abundances of intestinal Ca and P transporters to varying degrees. The mRNA level of CaBP-D28k increased by 536, 1,161, and 28 folds in the duodenum, jejunum, and ileum, respectively, after adding 1,000 IU/kg VD₃. The mRNA levels of other Ca and P transporters (PMCA1b, NCX1, NaPi-IIb, PiT-1, and PiT-2) increased by 0.57-1.74 folds by adding 1,000-2,000 IU/kg VD₃. These data suggest that intestinal Ca and P transporters are mainly expressed in the duodenum of broilers. Moreover, the addition of VD₃ stimulates the two mineral transporter transcription in broiler intestines.