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An intrinsic phosphate (Pi) transport defect in the proximal tubule (PT) presumably underlies X-linked hypophosphatemic rickets. We recently reported normal Pi transport in the S1 segment of the Hyp mouse PT. Whether Pi wasting results from an abnormality in the S2 or S3 segment remains unknown. Thus, we compared Pi transport in S2 and S3 immortalized cells from transgenic (simian virus 40) normal and Hyp mice. These cells display biochemical features of PT cells, including alkaline phosphatase- and hormone- stimulated cAMP activity as well as gluconeogenesis. Moreover, kinetic studies in S2 cells reveal a similar Km[0.26 +/- 0.03 (+/-SEM) vs. 0.22 +/- 0.03 mM] and maximum velocity (Vmax; 5.5 +/- 0.66 vs. 5.9 +/- 0.72 nmol/mg x 5 min) in normal and Hyp mice, respectively. Km and Vmax were also similar in cells from the S3 segment; however, the Vmax values in S3 cells in normal and Hyp mice (2.8 +/- 0.45 and 3.0 +/- 0.56 nmol/mg x 5 min) were reduced in both animal models compared to those in S2 cells (P < 0.001), whereas the Km values in S3 cells from normal and Hyp mice (0.10 +/- 0.02 and 0.11 +/- 0.04 mM) were increased relative to those in S2 cells (P < 0.001). These data indicate that Pi transport throughout the PT of Hyp mice is intrinsically normal. Such observations exclude the presence of a nascent defect in renal Pi transport in the kidneys of Hyp mice and support the hypothesis that a humoral abnormality underlies X-linked hypophosphatemic rickets.
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PMID:Normal phosphate transport in cells from the S2 and S3 segments of Hyp-mouse proximal renal tubules. 860 7

Isolated microperfused rabbit renal proximal tubule S2 segments, if incubated in conventional substrate containing HCO3- Ringer solution, exhibit lower cell membrane potentials (Vb) and elevated intracellular Na+ concentrations ([Na]i) compared to rat tubules in vivo. Assuming that these and other differences reflect insufficient metabolic and/or hormonal stimulation of the cells, we have used microelectrode techniques to test whether improving substrate supply and applying norepinephrine (NE, to compensate for the missing nerve supply) reverts Vb and [Na]i to values observed in vivo. Application of D-glucose (5.5 mmol/l) and additional application of pyruvate, lactate, or L-alanine (each 10 mmol/l), or bathing the tubules in Dulbecco's modified Eagle's tissue culture medium (DMEM) significantly increased Vb and, whenever tested, reduced [Na]i as compared to substrate-free or D-glucose-containing control solution and these effects could be prevented - as tested in the case of pyruvate - by inhibition of the Na/K pump with ouabain. However, high concentrations of acetate, beta-hydroxybutyrate, or L-glutamine had no significant effect. The largest effect was obtained with joint application of DMEM and NE (10 micromol/l) which increased Vb from -42.8 +/- 1.3 mV (SEM) to -55.3 +/- 2.5 mV (n = 11). Interestingly we noticed that under the latter conditions the Vb response to bath application of 1 mmol/l amiloride virtually disappeared, i.e. it changed from a depolarization of +14.6 +/- 1.4 mV (in D-glucose Ringer solution) to +0.6 +/- 0.7 mV (in DMEM plus NE) (n = 8), with some tubules showing even a small hyperpolarization. The latter implies partial restoration of the in vivo behaviour, since in experiments on rat proximal tubules in vivo amiloride regularly hyperpolarized the cells (by -3.4 +/- 0.76 mV, n = 5). Obviously under conventional in vitro conditions an amiloride-inhibitable K+ conductance is activated which is inactive in vivo and also inactivates under improved conditions in vitro. In agreement with observations reported in the subsequent publication our results demonstrate that isolated proximal tubules undergo functional alterations which may be largely prevented by improved metabolic and stimulatory incubation conditions.
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PMID:Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. I. Change of amiloride-inhibitable K+ conductance. 921 2

In the preceding publication we reported that some transport properties of proximal tubules perfused in vitro differ from those of tubules perfused in vivo, and that the in vivo function can be largely recuperated by improved metabolic substrate supply and stimulation with norepinephrine (NE). Since we have previously observed that the basolateral Na-HCO3-cotransporter operates with an overall stoichiometric ratio of q approximately 3 HCO3- :1 Na+ in vivo, but with q approximately 2 HCO3- :1 Na+ in vitro and that it responds differently in both cases to acetazolamide (ACZ), we have now tested whether the cotransporter can regain its in vivo function in vitro if the incubation conditions are improved. Cell membrane potentials (Vb) and cell pH (pHi) were measured with microelectrodes and microfluorimetric techniques on isolated S2 segments of rabbit proximal tubule and the instantaneous Vb response to a 2:1 reduction of bath HCO3- or Na+ concentration was determined. (DeltaVb)HCO3 and (deltaVb)Na averaged 13.1 +/- 0.9 mV (SEM) and 6.9 +/- 0.5 mV in D-glucose-containing control HCO3-Ringer solution and decreased respectively to 10.1 +/- 0.5 mV and 3.8 +/- 0.2 mV (n = 8) after incubation in tissue culture medium and NE (10(-5) mmol/l). These data imply that q increased from 1.9 to 2.7. Concomitantly the tubules became susceptible to ACZ (1 mmol/l), which reduced (deltaVb)HCO3 in control conditions only to 94.6 +/- 1.2% but under improved incubation conditions to 64.5 +/- 2.4%. As verified in voltage divider measurements the latter reduction was not caused by activation of a basolateral K+ conductance. The results indicate that improved incubation conditions can at least partially revert cotransport function towards that of the in vivo state. The effect of ACZ may be explained if in the improved state 1 CO32- + 1 HCO3- + 1 Na+ are cotransported, in which case inhibition of carbonic anhydrase (CA) may cause a CO32-/pH disequilibrium to develop in the basal labyrinth which impedes the cotransport. Under conventional incubation conditions, however, when only 2 HCO3- + 1 Na+ are cotransported no such disequilibrium should develop irrespective of whether CA is active or inhibited.
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PMID:Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. II. Change of Na-HCO3 cotransport stoichiometry and of response to acetazolamide. 921 3

The kidney bears the brunt of the demands of a tropical climate for water and electrolyte homeostasis. We hypothesised that a tropical climate may cause adaptive changes in the entire organism leading to altered renal function in our subjects. Hence renal function data for residents of a temperate climate may not be applicable to tropical residents. We therefore sought to elucidate renal function in subjects residing in a tropical climate. We used lithium clearance, CLi, a non-invasive tool for assessing proximal tubular function in humans, and endogenous creatinine clearance, CCr, to estimate proximal tubular function and glomerular function, respectively, in our subjects. We did this in order to establish whether or not nephron function in our subjects differs from that for residents of a temperate climate. Nineteen male and 12 female Ghanaian subjects aged between 15 and 48 years were studied. The estimated GCr was 117.3 +/- 6.6 ml/min for male subjects and 97 +/- 6.4 ml/min for female subjects. CLi was 20.3 +/- 1.6 ml/min for male and 19.1 +/- 0.4 ml/min for female subjects, respectively. The estimated absolute reabsorption rate of fluid of proximal tubules was 97.0 +/- 6.0 ml/min for males and 78.1 +/- 6.0 ml/min for females. The percentage proximal fluid reabsorption for male and female subjects was 81.2 +/- 1.4 and 79.5 +/- 1.6, respectively. The differences between male and female values (mean +/- SEM) were not statistically significant. The data suggest that the proximal tubule in residents of a tropical climate may reabsorb more fluid compared to that in residents of a temperate climate. Our values for proximal tubular reabsorption are higher than those reported for residents of a temperature climate. Our estimate of glomerular filtration, however, is similar to published data for Caucasians. The difference in proximal tubular function may reflect possible renal adaptation to a hot, humid climate. We conclude that renal function of tropical residents differs from that of residents of a temperate climate. This difference may be due to renal adaptation to the hot, tropical climate.
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PMID:A study of nephron function in normal tropical residents using the creatinine and lithium clearances. 933 74

The stability of single nephron glomerular filtration rate (SNGFR) is assured by specific mechanisms such as the tubulo-glomerular feedback system and autoregulation. Studies on renal physiology rely heavily on the measurements of SNGFR, which are feasible only in animals. The measurement of SNGFR by collection of total tubular fluid may be influenced by the fall in intratubular hydrostatic pressure that may reflect the negative pressure applied to the sampling pipette. This effect may become more important with shortening of the distance between the sampling site and the Bowman space. We analysed this putative effect by performing collections of total tubular fluid from the late proximal (LP), and then from the early proximal (EP) segment of the same nephrons. In 128 paired collections LP-SNGFR averaged 35 (SEM 2) nl x min(-1), and was no different from the paired mean EP-SNGFR of 37 (SEM 2) nl x min(-1), P > 0.179. Then EP- and LP- SNGFR were significantly correlated (r = 0.77, P < 0.001). As expected, the respective paired means of absolute and percentage reabsorptions, and those of collection rates were significantly different. The average SNGFR computed from each LP and EP paired measurement was significantly correlated with the simultaneously measured kidney glomerular filtration rate, GFR (r = 0.60, P < 0.0001). The ratio of GFR to SNGFR indicated the expected number of glomeruli. These data would indicate that the sampling site does not influence the measurement of SNGFR in the proximal tubule when the total fluid collection technique is correctly performed. They also exclude a time-dependent activation of the macula densa capable of upregulating SNGFR within the interval elapsing between the beginning of LP and the completion of EP collections, which in our study averaged 4.4 (SEM 0.1) min.
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PMID:Consistency of nephron filtration measurements by collection of total tubular fluid from different proximal sites. 936 77

To determine the effects of circulating noradrenaline on fetal renal function noradrenaline was infused intravenously into 7 chronically catheterised fetal sheep (127-138 days) at a dose (1 microgram/kg/min) which resulted in plasma levels similar to those which occur during hypoxia. Fetal mean arterial pressure increased by approximately 14 mmHg (p < 0.001) and haematocrit rose (p < 0.005). Glomerular filtration rate rose from 3.85 +/- 0.47 (SEM) to 4.70 +/- 0.50 ml/min (p < 0.05) during the first hour and fractional reabsorption of sodium by the proximal tubule fell (p < 0.05) during the second hour. Urine flow rate increased from 0.61 +/- 0.13 to 1.18 +/- 0.24 ml/min (p < 0.001) and osmolar excretion increased from 78 +/- 15 to 153 +/- 36 mu osm/min (p < 0.005). By contrast lung liquid flow fell (p < 0.05), but the increase in urine flow was much greater than the decline in lung liquid. These findings suggest that during hypoxia, noradrenaline may play an important role in the maintenance of urine flow and consequently amniotic fluid volume and, as suggested by others, in the distribution of fluid between the vascular and interstitial compartments.
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PMID:Effects of intravenous infusions of noradrenaline on renal function in chronically catheterised fetal sheep. 955 Nov 92

Epifluorescence microscopy was used to study peritubular transport of the fluorescent mycotoxin ochratoxin A (OTA) into single proximal tubule segments of the rabbit. Initial rates of OTA uptake into S2 segments were saturable and adequately described by Michaelis-Menten kinetics, with an apparent Km of 2.2+/-0.3 microM (SEM). Several lines of evidence indicated that peritubular uptake of OTA in S2 segments was effectively limited to the "classical" organic anion transporter. First, 5 mM p-aminohippurate (PAH) cis-inhibited the uptake of 1 microM OTA into tubules by 96%. Kinetic analysis of the inhibition of OTA uptake by PAH (100 microM to 5 mM) yielded an apparent Ki of 164 microM, similar to the 100 to 200 microM range of Km values previously reported for the peritubular uptake of PAH. Second, efflux of OTA from tubules was trans-stimulated 3.2-fold by the presence of 2.5 mM PAH in the uptake medium. Third, 100 microM alpha-ketoglutarate (alphaKG) trans-stimulated the uptake rate of 1 microM OTA by 1.8-fold. Fourth, besides PAH, other organic anions effectively cis-inhibited the uptake of 1 microM OTA into tubules (inhibitor, % inhibition): 1.5 mM alphaKG, 80%; 1 mM probenecid, 100%; 1 mM piroxicam, 100%; 1 mM octanoate, 100%. In contrast, 1.5 mM tetraethylammonium, an organic cation, blocked uptake of 1 microM OTA by only 7%. The inhibition of OTA uptake into S1 and S3 segments of the proximal tubule was qualitatively similar: 5 mM PAH cis-inhibited the uptake of 1 microM OTA by approximately 95% in both S1 and S3 segments. Thus, peritubular OTA uptake into all segments of the proximal tubule appears to be dominated by its interaction with the classical organic anion transporter. The high-affinity and relatively high capacity of this pathway for OTA suggest that peritubular uptake may be a significant avenue for the entry of this toxin into proximal tubule cells.
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PMID:Peritubular transport of ochratoxin A by single rabbit renal proximal tubules. 980 82

Renin secretion can be stimulated by ATP via purinergic P2Y receptors. ATP is a cotransmitter with norepinephrine and is released from the cytosol during cell damage. Such release could account for the de novo renin expression seen in the proximal tubule in renal disease and in myocardial infarct borders. Whereas most P2Y purinoceptor subtypes utilize phosphoinositide signal-transduction pathways, the effector mechanisms of the subtype P2Y(11) also involve increases in cAMP, a well-known renin secretagogue and stimulus to renin production. The present study tested the effect of ATP on human renin gene (REN) promoter activity and the role of P2Y(11). By means of reverse transcriptase-polymerase chain reaction, we found that renin-expressing Calu-6 cells express P2Y(11) mRNA. Expression was also detected in the brain, kidney, testis, muscle, liver, and spleen. We made a novel cell line (Calu-6/P2Y11) in which P2Y(11) cDNA, under the control of a strong promoter, was stably integrated into genomic DNA. These cells produced P2Y(11) mRNA during culture. Treatment of Calu-6/P2Y11 cells with 1 mmol/L ATP caused a 3-fold increase in renin mRNA and protein over 36 hours. Transient transfection of Calu-6/P2Y11 cells with constructs containing 896 bp of human REN 5'-flanking DNA linked to the luciferase reporter gene led to a 5.8+/-0.6-fold increase (mean+/-SEM) in reporter activity in response to ATP (P=0.0015). In contrast, UTP produced only a 1.4+/-0.1-fold increase (P=0.016). For ADP, it was 1.7+/-0.1-fold (P=0.011). The response profile was ATP>ADP>AMP=adenosine=0, consistent with a P2Y(11) effect. Mutation of the cAMP response element (CRE) located at -222 in the REN promoter DNA abolished the effect of ATP. Furthermore, ATP induced a rapid, time-dependent increase in the phosphorylation of CRE binding protein (CREB) and activating transcription factor-1. These data implicate a cAMP pathway in mediation of the P2Y(11) effect. In conclusion, we have made a novel cell line that overexpresses the P2Y(11) purinoceptor. Stimulation of these cells by ATP activates a cAMP signal-transduction pathway that phosphorylates CREB and stimulates renin promoter activity via the CRE at -222. The data raise the possibility of a contribution of ATP/P2Y(11) effects to sympathetic stimulation of renin, as well as to responses in renin seen after tissue damage, such as in kidney disease and myocardial infarction.
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PMID:Capacity for purinergic control of renin promoter via P2Y(11) receptor and cAMP pathways. 1111 31

Plasma sodium (Na+) concentration, i.e. natraemia, results from body tonicity equilibrium. During exercise, a change in body tonicity can result from an imbalance between intake and loss of Na+, potassium (K+) and water (H2O) due to renal and/or extra-renal mechanisms. Whether exercise-induced changes in kidney function could be responsible for such an imbalance was studied by measuring glomerular filtration rate (creatinine clearance), proximal tubule activity (lithium clearance) and renal handling of Na+ and K+ at rest and during exercise. Since hyponatraemia during or after exercise has been reported, we also investigated whether a water load could be appropriately excreted during exercise. Ten young men pedalled on a cycle ergometer at 60% of maximal oxygen uptake for 45 min with (HE, hydrated exercise) or without (DHE, dehydrated exercise) a supply of water. In both conditions, creatinine, lithium, and electrolyte (Na+ + K+) clearances decreased and natraemia did not change. The DHE induced a loss of body mass (-1.29%), decreased diuresis and large extra-renal water loss [mean (SEM)] [880 (73) ml]. The HE led to no loss in body mass, increased diuresis and lower extrarenal water loss [680 (48) ml]. Electrolyte-free water excretion, negative for DHE, represented 60% of diuresis during HE. Thus the kidney, by increasing electrolyte reabsorption mainly in the proximal tubule, and appropriately excreting a water load, seems efficacious in regulating extracellular fluid volume and body tonicity and so not responsible for the imbalance between (Na+ + K+)/H2O intake and loss. Therefore, extra-renal changes could be the main causes of exercise-induced tonicity imbalances which could ultimately lead to dysnatraemia.
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PMID:Renal handling of salt and water in humans during exercise with or without hydration. 1199 Jul 26

Cyclosporine A (CsA), a neutral, highly hydrophobic cyclic peptide with 11 amino acids, is currently the most widely used immunosuppressive drug for preventing graft rejection and autoimmune diseases. Despite its efficacy, the use of CsA is limited by severe side effects, mainly nephrotoxicity and arterial hypertension. Single cell microfluorimetry was used to evaluate the role of CsA on Ca(2+) signaling pathway in intact cells of the porcine proximal tubule-like cell line LLC-PK1; the assay of the in vitro activity of the plasma membrane Ca(2+) pump (PMCA) was carried out through the preparation and isolation of membranes. The addition of CsA to incubation medium at doses ranging from 0.1 to 2 microM did not change the basal level of intracellular calcium ([Ca(2+)](i)), whereas it affected the [Ca(2+)](i) response to thapsigargin (TG), a powerful inhibitor of microsomal Ca(2+) pump. In control studies, 5 microM TG produced a biphasic response: [Ca(2+)](i) peaked with a 60-s lag, and it then declined to a plateau of elevated [Ca(2+)](i), which remains above basal. However, it became evident that CsA strengthened the Ca(2+) response to TG because the addition of 5 microM TG to cells exposed to 400 nM CsA did not affect the peak response to TG, but it markedly affected the subsequent sustained phase ([Ca(2+)](i) = 156 +/- 4.84 versus 130 +/- 3.28 nmol, mean +/- SEM, n = 6, P < 0.001). In membrane preparations, 200 nM CsA brought about, in the presence of 10 microM calmodulin (CaM), a significant decrease of plasma membrane Ca(2+) pump (PMCA) activity (46.96 +/- 0.26 versus 53.48 +/- 1.96 nmol x mg of protein(-1) x min(-1), n = 6, P < 0.02), a value similar to that obtained in the presence of equimolar amounts of cyclosporine H (CsH), a non-immunosuppressive analogue of CsA. These findings suggest that in this cell line CsA affects the Ca(2+) export pathway through the reduction of the PMCA activity with consequent amplification and strengthening of [Ca(2+)](i) response after exposure to agents that trigger intracellular Ca(2+) release. The increased cell sensitivity during Ca(2+) signaling events ensuing from the impairment of this "defense system" may be regarded as one of the basic mechanisms involved in the development of the side effects induced by CsA.
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PMID:Cyclosporine A amplifies Ca2+ signaling pathway in LLC-PK1 cells through the inhibition of plasma membrane Ca2+ pump. 1276 Dec 43


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