NPT2a gene variation in calcium nephrolithiasis with renal phosphate leak.

A decrease in renal phosphate reabsorption with mild hypophosphatemia (phosphate leak) is found in some hypercalciuric stone-formers. The NPT2a gene encodes a sodium-phosphate cotransporter, located in the proximal tubule, responsible for reclaiming most of the filtered phosphate load in a rate-limiting manner. To determine whether genetic variation of the NPT2a gene is associated with phosphate leak and hypercalciuria in a cohort of 98 pedigrees with multiple hypercalciuric stone-formers, we sequenced the entire cDNA coding region of 28 probands, whose tubular reabsorption of phosphate normalized for the glomerular filtration rate (TmP/GFR) was 0.7 mmol/l or lower. We performed genotype/phenotype correlations for each genetic variant in the entire cohort and expressed NPT2a variant RNAs in Xenopus laevis oocytes to test for cotransporter functionality. We identified several variants in the coding region including an in-frame 21 bp deletion truncating the N-terminal cytoplasmic tail of the protein (91del7), as well as other single-nucleotide polymorphisms that were non-synonymous (A133V and H568Y) or synonymous. Levels of TmP/GFR and urine calcium excretion were similar in heterozygote carriers of NPT2a variants compared to the wild-type (wt) homozygotes. The transport activity of the H568Y mutants was identical to the wt, whereas the N-terminal-truncated version and the 91del7 and A133V mutants presented minor kinetic changes and a reduction in the expression level. Although genetic variants of NPT2a are not rare, they do not seem to be associated with clinically significant renal phosphate or calcium handling anomalies in a large cohort of hypercalciuric stone-forming pedigrees.

Functional studies of a chimeric protein containing portions of the Na(+)/glucose and Na(+)/myo-inositol cotransporters.

We obtained cDNA chimeras between Na/glucose cotransporter (SGLT1) and the homologous Na(+)/myo-inositol cotransporter (SMIT) by creating random chimeras in plasmids. Of 12 chimeras, two were functional when expressed in Xenopus laevis oocytes but, upon sequencing, only one of them (C1) produced an actual chimeric protein. In C1, the first 69 amino acids of SGLT1 were replaced by the corresponding 50 amino acids of SMIT. C1 transports the same sugars as does SGLT1. C1's affinity for all sugar substrates was systematically increased by a factor of 3.3+/-0.4 but the V(max) was diminished by a factor of 15-40. In contrast, the cotransport affinity for Na(+) was unchanged. The surface expression of C1 was one seventh that of SGLT1, which explains part of the reduced V(max) and implies a significant reduction in turnover rate. N-terminal truncated constructs of SGLT1 cDNA showed that deleting amino acids 2-14 does not affect cotransporter activity, but that the pentapeptide T(14)RPVET(19) is important for normal levels of SGLT1 current. The main result of a kinetic analysis of the systematic increase in apparent affinity for sugars, together with the intact Na apparent affinity, suggests enhanced access to the sugar binding site in C1.

Sodium leak pathway and substrate binding order in the Na+-glucose cotransporter.

The Na+-glucose cotransporter (SGLT1) expressed in Xenopus laevis oocytes was shown to generate a phlorizin-sensitive sodium leak in the absence of sugars. Using the current model for SGLT1, where the sodium leak was presumed to occur after two sodium ions are bound to the free carrier before glucose binding, a characteristic concentration constant (Kc) was introduced to describe the relative importance of the sodium leak versus Na+-glucose cotransport currents. Kc represents the glucose concentration at which the Na+-glucose cotransport current is equal to the sodium leak. As both the sodium leak and the Na+-glucose cotransport current are predicted to occur after the binding of two sodium ions, the model predicted that Kc should be sodium-independent. However, by using a two-microelectrode voltage-clamp technique, the observed Kc was shown to depend strongly on the external sodium concentration ([Na+]o): it was four times higher at 5 mM [Na+]o than at 20 mM [Na+]o. In addition, the magnitude of the sodium leak varied as a function of [Na+]o in a Michaelian fashion, and the sodium affinity constant for the sodium leak was 2-4 times lower than that for cotransport in the presence of low external glucose concentrations (50 or 100 microM), whereas the current model predicted a sigmoidal sodium dependence of the sodium leak and identical sodium affinities for the sodium leak and the Na+-glucose cotransport. These observations indicate that the sodium leak occurs after one sodium ion is associated with the carrier and agree with predictions from a model with the binding order sodium-glucose-sodium. This conclusion was also supported by experiments performed where protons replaced Na+ as a "driving cation."

Isolation of single mammalian proximal tubule cells: effects of hypotonic shocks on cell yield and function.

Nord et al. [Am J Physiol 1986; 250:F539-F550] proposed a method to give a high yield of proximal tubule cells by exposing a suspension of rabbit cortical tubules to a hypotonic shock in calcium-free media. The present study describes the effects of both amplitude and duration of the hypotonic treatment on some transport-related characteristics of individual cells as compared to the starting tubule suspension. The averaged cell yield increased by an order of magnitude when the osmolality of the hypotonic solution was varied in four steps from 200 (C200 cells) to 70 mosm/kg H2O (C70 cells) while the proportion of trypan blue-positive cells progressively decreased from 33% for C200 cells to 9.5% for C70 cells. An increase in duration of the hypotonic shock from 0.5 to 6 min did not change the cell yield of C200 cells while it significantly increased that of C70 cells by 61%. Basal and ouabain-sensitive oxygen consumption (QO2) increased by 57 and 155%, respectively, from C70 to C200 cells but was approximately one order of magnitude smaller than the QO2 measured for tubule suspension. Intracellular ATP content averaged 5.5 +/- 0.8 nmol/mg for the starting tubule suspension, 4.6 +/- 0.8 nmol/mg for C70 cells but only 1.3 +/- 0.1 nmol/mg for C200 cells. The maximal velocity for phloridzin-sensitive alpha-methyl glucose transport averaged 13.7 +/- 1.7 nmol min-1 mg-1 for C70 cells and only 6.3 +/- 1.3 nmol min-1 mg-1 for C200 cells which is approximately one order of magnitude smaller than what can be expected from a tubule presenting a good access to luminal membrane. We conclude from these results that, in the process of isolating individual cells from a polarized epithelium, membrane transport rates have decreased by one order of magnitude and this reduction is intensified by a large hypotonic shock. In comparison with C200 cells, the cells obtained with a large hypotonic shock give a high yield, a larger proportion of trypan blue-negative cells and their lower overall transport rate allows the cells to maintain a better electrochemical gradient for Na and a higher intracellular ATP level.