Expression of SLC2A9 Isoforms in the Kidney and Their Localization in Polarized Epithelial Cells

Background

Many genome-wide association studies pointed out that SLC2A9 gene, which encodes a voltage-driven urate transporter, SLC2A9/GLUT9 (a.k.a. URATv1), as one of the most influential genes for serum urate levels. SLC2A9 is reported to encode two splice variants: SLC2A9-S (512 amino acids) and SLC2A9-L (540 amino acids), only difference being at their N-termini. We investigated isoform-specific localization of SLC2A9 in the human kidney and role of N-terminal amino acids in differential sorting in vitro.

Methodology/Principal Findings

Isoform specific antibodies against SLC2A9 were developed and human kidney sections were stained. SLC2A9-S was expressed in the apical side of the collecting duct while SLC2A9-L was expressed in the basolateral side of the proximal tubule. GFP fused SLC2A9s were expressed in MDCK cells and intracellular localization was observed. SLC2A9-S was expressed at both apical and basolateral membranes, whereas SLC2A9-L was expressed only at the basolateral membrane. Although SLC2A9-L has a putative di-leucine motif at 33th and 34th leucine, deletion of the motif or replacement of leucine did not affect its subcellular localization. When up to 16 amino acids were removed from the N-terminal of SLC2A9-S or when up to 25 amino acids were removed from the N-terminal of SLC2A9-L, there was no change in their sorting. Deletion of 20 amino acids from SLC2A9-S was not expressed in the cell. More than 30 amino acids deletion from SLC2A9-L resulted in expression at both apical and basolateral membranes as well as in the lysosome. When amino acids from 25th and 30th were changed to alanine in SLC2A9-L, expression pattern was the same as wild-type.

Conclusions/Significance

SLC2A9-L was expressed in the basolateral membrane of kidney proximal tubules in humans and this isoform is likely to responsible for urate reabsorption. N-terminal amino acids unique to each isoform played an important role in protein stability and trafficking.     

Molecular Cloning, Water Channel Activity and Tissue Specific Expression of Two Isoforms of Radish Vacuolar Aquaporin1

A major membrane intrinsic protein (VM23) in vacuoles of radish(Raphanus) tap root was investigated. The cDNAs for two isoformsof VM23,      

Isoforms of SLC26A6 mediate anion transport and have functional PDZ interaction domains

The solute carrier gene family SLC26consists of tissue-specific anion exchanger genes, three of themassociated with distinct human recessive disorders. By a genome-drivenapproach, several new SLC26 family members have been identified,including a kidney- and pancreas-specific gene, SLC26A6. We report thefunctional characterization of SLC26A6 and two new alternativelyspliced variants, named SLC26A6c and SLC26A6d. Immunofluorescencestudies on transiently transfected cells indicated membranelocalization and indicated that both NH₂- and COOH-terminaltails of the SLC26A6 variants are located intracellularly, suggesting atopology with an even number of transmembrane domains. Functionalexpression of the three proteins in Xenopus oocytesdemonstrated Cl and SO transportactivity. In addition, the transport of SO andCl was inhibited by DIDS and HCO. We demonstrated also that the COOH terminus of SLC26A6 binds to the firstand second PDZ domains of the Na⁺/H⁺ exchanger(NHE)3 kinase A regulatory protein (E3KARP) and NHE3 regulatory factor(NHERF) proteins in vitro. Truncation of the last three amino acids(TRL) of SLC26A6 abrogated the interaction but did not affect transportfunction. These results demonstrate that SLC26A6 and its two splicevariants can function as anion transporters linked to PDZ-interactionpathways. Our results support the general concept of microdomainorganization for ion transport and suggest a mechanism for cysticfibrosis transmembrane regulator (CFTR)-mediated SLC26A6 upregulationin pancreatic duct cells.

     

SLC37A1 and SLC37A2 are phosphate-linked, glucose-6-phosphate antiporters

Blood glucose homeostasis between meals depends upon production of glucose within the endoplasmic reticulum (ER) of the liver and kidney by hydrolysis of glucose-6-phosphate (G6P) into glucose and phosphate (P(i)). This reaction depends on coupling the G6P transporter (G6PT) with glucose-6-phosphatase-α (G6Pase-α). Only one G6PT, also known as SLC37A4, has been characterized, and it acts as a P(i)-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:P(i) exchangers, have not been characterized functionally. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-α. G6PT- and mock-proteoliposomes are used as positive and negative controls, respectively. We show that SLC37A1 and SLC37A2 are ER-associated, P(i)-linked antiporters, that can transport G6P. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-α. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis.     

Expression and analysis of two novel rat organic cation transporter homologs, SLC22A17 and SLC22A23

The organic cation transporter (OCT, SLC22) family is a family of polyspecific transmembrane proteins that are responsible for the uptake or excretion of many cationic drugs, toxins, and endogenous metabolites in a variety of tissues. Many of the OCTs have been previously characterized, but there are a number of orphan genes whose functions remain unknown. In this study, two novel rat SLC22 genes, SLC22A17 (BOCT1) and SLC22A23 (BOCT2), were cloned and characterized. Northern blot analysis showed that BOCT1 and BOCT2 mRNA was expressed in a wide variety of tissues. BOCT1 was strongly expressed in brain, primary neurons and brain endothelial cells, with highest expression in choroid plexus. BOCT2 was also abundantly expressed in brain, as well as in liver. To characterize the products of these genes, BOCT1 cDNA was isolated from a rat blood-brain barrier cDNA library, and BOCT2 cDNA was isolated from rat brain capillary and from cultured neurons using PCR techniques. Plasmids expressing BOCT1 and BOCT2 were transfected into HEK-293 cells, as were control cDNAs for OCT1 and OCTN2. Recombinant cell surface protein was verified by western blot and fluorescence microscopy. Transport activity of BOCT1 and BOCT2 was evaluated using radioisotope uptake assays. The OCT1- and OCTN2-expressing cells transported the canonical substrates, 1-methyl-4-phenyl-pyridinium (MPP(+)) and carnitine, respectively. However, BOCT1 and BOCT2-expressing cells did not show transport activity for these substrates or a number of other SLC22 substrates. These novel family members have a nonconserved amino terminus, relative to other OCTs, that may preclude typical SLC22 transport function.     

The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep

Male Kazak sheep and Xinjiang fine wool sheep, six for each different age group (days 2, 30, 60, 90 and 120), were used in the present study to investigate the tissue distribution and developmental changes of ghrelin mRNA expression in abomasum; however, there was no 120-day-old Kazak sheep. After measurement of body weight, the tissues such as hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle were sampled. And the total RNA of different tissues was extracted to determine the abundance of ghrelin mRNA by RT-PCR and real-time PCR. The results showed that (1) for both breeds, body weight among different ages was significantly different (P<0.05). And from day 30 to 90, the body weight of Kazak was significantly higher than that of Xinjiang (P<0.01); (2) Ghrelin mRNA existed in all the above tissues and was significantly higher in the abomasum than in other tissues (P<0.05); (3) the temporal patterns of abomasum ghrelin mRNA expression in Kazak and Xinjiang were similar. From day 2 to 60 in Kazak and 2 to 90 in Xinjiang, there was a steady increase in the ghrelin mRNA level. By day 60 in Kazak and day 90 in Xinjiang, the level reached a plateau and remained steady. These results also demonstrated that from birth to day 90, ghrelin mRNA level was significantly higher in Kazak than in Xinjiang (P<0.01).     

Renalase's Expression and Distribution in Renal Tissue and Cells

To study renalase''s expression and distribution in renal tissues and cells, renalase coded DNA vaccine was constructed, and anti-renalase monoclonal antibodies were produced using DNA immunization and hybridoma technique, followed by further investigation with immunological testing and western blotting to detect the expression and distribution of renalase among the renal tissue and cells. Anti-renalase monoclonal antibodies were successfully prepared by using DNA immunization technique. Further studies with anti-renalase monoclonal antibody showed that renalase expressed in glomeruli, tubule, mesangial cells, podocytes, renal tubule epithelial cells and its cells supernatant. Renalase is wildly expressed in kidney, including glomeruli, tubule, mesangial cells, podocytes and tubule epithelial cells, and may be secreted by tubule epithelial cells primarily.     

Tissue Distribution,Gender- and Genotype-Dependent Expression of Autophagy-Related Genes in Avian Species

As a result of the genetic selection of broiler (meat-type breeders) chickens for enhanced growth rate and lower feed conversion ratio, it has become necessary to restrict feed intake. When broilers are fed ad libitum, they would become obese and suffer from several health-related problems. A vital adaptation to starvation is autophagy, a self-eating mechanism for recycling cellular constituents. The autophagy pathway has witnessed dramatic growth in the last few years and extensively studied in yeast and mammals however, there is a paucity of information in avian (non-mammalian) species. Here we characterized several genes involved in autophagosome initiation and elongation in Red Jungle fowl (Gallus gallus) and Japanese quail (coturnix coturnix Japonica). Both complexes are ubiquitously expressed in chicken and quail tissues (liver, leg and breast muscle, brain, gizzard, intestine, heart, lung, kidney, adipose tissue, ovary and testis). Alignment analysis showed high similarity (50.7 to 91.5%) between chicken autophagy-related genes and their mammalian orthologs. Phylogenetic analysis demonstrated that the evolutionary relationship between autophagy genes is consistent with the consensus view of vertebrate evolution. Interestingly, the expression of autophagy-related genes is tissue- and gender- dependent. Furthermore, using two experimental male quail lines divergently selected over 40 generations for low (resistant, R) or high (sensitive, S) stress response, we found that the expression of most studied genes are higher in R compared to S line. Together our results indicate that the autophagy pathway is a key molecular signature exhibited gender specific differences and likely plays an important role in response to stress in avian species.     

Increased Expression of SLC2A9 Decreases Urate Excretion From the Kidney

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.