Contribution and Expression of Organic Cation Transporters and Aquaporin Water Channels in Renal Cancer.

The body homeostasis is maintained mainly by the function of the kidneys, which regulate salt and water balance and excretion of metabolism waste products and xenobiotics. This important renal function is determined by the action of many transport systems, which are specifically expressed in the different parts of the nephron, the functional unit of the kidneys. These transport systems are involved, for example, in the reabsorption of sodium, glucose, and other important solutes and peptides from the primary urine. They are also important in the reabsorption of water and thereby production of a concentrated urine. However, several studies have shown the importance of transport systems for different tumor entities. Transport systems, for example, contributed to the proliferation and migration of cancer cells and thereby on tumor progression. They could also serve as drug transporters that could enable drug resistance by outward transport of, for example, chemotherapeutic agents and other drugs. Although many renal transporters have been characterized in detail with respect to the significance for proper kidney function, their role in renal cancer progression is less known. Here, we describe the types of renal cancer and review the studies that analyzed the role of organic cation transporters of the SLC22-family and of the aquaporin water channel family in kidney tumors.

Genetic deletion of the nuclear factor of activated T cells 5 in collecting duct principal cells causes nephrogenic diabetes insipidus.

Water homeostasis is tightly regulated by the kidneys via the process of urine concentration. During reduced water intake, the antidiuretic hormone arginine vasopressin (AVP) binds to the vasopressin receptor type II (V2R) in the kidney to enhance countercurrent multiplication and medullary osmolality, and increase water reabsorption via aquaporin-2 (AQP2) water channels. The importance of this AVP, V2R, and AQP2 axis is highlighted by low urine osmolality and polyuria in people with various water balance disorders, including nephrogenic diabetes insipidus (NDI). ELF5 and nuclear factor of activated T cells 5 (NFAT5) are two transcription factors proposed to regulate Aqp2 expression, but their role is poorly defined. Here we generated two novel mouse lines with principal cell (PC)-specific deletion of ELF5 or NFAT5 and phenotyped them in respect to renal water handling. ELF5-deficient mice (ELF5PC-KO ) had a very mild phenotype, with no clear differences in AQP2 abundance, and mild differences in renal water handling and maximal urinary concentrating capacity. In contrast, NFAT5 (NFAT5PC-KO ) mice had significantly higher water intake and their 24 h urine volume was almost 10-fold greater than controls. After challenging with dDAVP or 8 h water restriction, NFAT5PC-KO mice were unable to concentrate their urine, demonstrating that they suffer from NDI. The abundance of AQP2, other AQPs, and the urea transporter UT-A1 were greatly decreased in NFAT5PC-KO mice. In conclusion, NFAT5 is a major regulator of not only Aqp2 gene transcription, but also other genes important for water homeostasis and its absence leads to the development of NDI.

Site-specific methylation of 18S ribosomal RNA by SNORD42A is required for acute myeloid leukemia cell proliferation.

Noncoding RNAs, including small nucleolar RNAs (snoRNAs), play important roles in leukemogenesis, but the relevant mechanisms remain incompletely understood. We performed snoRNA-focused CRISPR-Cas9 knockout library screenings that targeted the entire snoRNAnome and corresponding host genes. The C/D box containing SNORD42A was identified as an essential modulator for acute myeloid leukemia (AML) cell survival and proliferation in multiple human leukemia cell lines. In line, SNORD42A was consistently expressed at higher levels in primary AML patient samples than in CD34+ progenitors, monocytes, and granulocytes. Functionally, knockout of SNORD42A reduced colony formation capability and inhibited proliferation. The SNORD42A acts as a C/D box snoRNA and directs 2'-O-methylation at uridine 116 of 18S ribosomal RNA (rRNA). Deletion of SNORD42A decreased 18S-U116 2'-O-methylation, which was associated with a specific decrease in the translation of ribosomal proteins. In line, the cell size of SNORD42A deletion carrying leukemia cells was decreased. Taken together, these findings establish that high-level expression of SNORD42A with concomitant U116 18S rRNA 2'-O-methylation is essential for leukemia cell growth and survival.

Getting a Notch closer to renal dysfunction: activated Notch suppresses expression of the adaptor protein Disabled-2 in tubular epithelial cells.

Reactivation of Notch signaling in kidneys of animal models and patients with chronic kidney disease (CKD) has been shown to contribute to epithelial injury and fibrosis development. Here, we investigated the mechanisms of Notch-induced injury in renal epithelial cells. We performed genome-wide transcriptome analysis to identify Notch target genes using an in vitro system of cultured tubular epithelial cells expressing the intracellular domain of Notch1. One of the top downregulated genes was Disabled-2 ( Dab2). With the use of Drosophila nephrocytes as a model system, we found that Dab (the Drosophila homolog of Dab2) knockdown resulted in a significant filtration defect, indicating that loss of Dab2 plays a functional role in kidney disease development. We showed that Dab2 expression in cultured tubular epithelial cells is involved in endocytic regulation and that it also protects cells from TGF-ß-induced epithelial-to-mesenchymal transition. In vivo correlation studies indicated its additional role in renal ischemia-induced injury. Together, these data suggest that Dab2 plays a versatile role in the kidney and may impact on acute and CKDs.-Schütte-Nütgen, K., Edeling, M., Mendl, G., Krahn, M. P., Edemir, B., Weide, T., Kremerskothen, J., Michgehl, U., Pavenstädt, H. Getting a Notch closer to renal dysfunction: activated Notch suppresses expression of the adaptor protein Disabled-2 in tubular epithelial cells.

Identification of Prognostic Organic Cation and Anion Transporters in Different Cancer Entities by In Silico Analysis.

The information derived from next generation sequencing technology allows the identification of deregulated genes, gene mutations, epigenetic modifications, and other genomic events that are associated with a given tumor entity. Its combination with clinical data allows the prediction of patients' survival with a specific gene expression pattern. Organic anion transporters and organic cation transporters are important proteins that transport a variety of substances across membranes. They are also able to transport drugs that are used for the treatment of cancer and could be used to improve treatment. In this study, we have made use of publicly available data to analyze if the expression of organic anion transporters or organic cation transporters have a prognostic value for a given tumor entity. The expression of most organic cation transporters is prognostic favorable. Within the organic anion transporters, the ratio between favorable and unfavorable organic anion transporters is nearly equal for most tumor entities and only in liver cancer is the number of unfavorable genes two times higher compared to favorable genes. Within the favorable genes, UNC13B, and SFXN2 cover nine cancer types and in the same way, SLC2A1, PLS3, SLC16A1, and SLC16A3 within the unfavorable set of genes and could serve as novel target structures.

Claudin 19 Is Regulated by Extracellular Osmolality in Rat Kidney Inner Medullary Collecting Duct Cells.

The inner medullary collecting duct (IMCD) is subject to severe changes in ambient osmolality and must either allow water transport or be able to seal the lumen against a very high osmotic pressure. We postulate that the tight junction protein claudin-19 is expressed in IMCD and that it takes part in epithelial adaptation to changing osmolality at different functional states. Presence of claudin-19 in rat IMCD was investigated by Western blotting and immunofluorescence. Primary cell culture of rat IMCD cells on permeable filter supports was performed under different osmotic culture conditions and after stimulation by antidiuretic hormone (AVP). Electrogenic transepithelial transport properties were measured in Ussing chambers. IMCD cells cultivated at 300 mosm/kg showed high transepithelial resistance, a cation selective paracellular pathway and claudin-19 was mainly located in the tight junction. Treatment by AVP increased cation selectivity but did not alter transepithelial resistance or claudin-19 subcellular localization. In contrast, IMCD cells cultivated at 900 mosm/kg had low transepithelial resistance, anion selectivity, and claudin-19 was relocated from the tight junctions to intracellular vesicles. The data shows osmolality-dependent transformation of IMCD epithelium from tight and sodium-transporting to leaky, with claudin-19 expression in the tight junction associated to tightness and cation selectivity under low osmolality.

The kidney-specific expression of genes can be modulated by the extracellular osmolality.

With this study, we wanted to prove the hypothesis that the unique extracellular osmolality within the renal medulla modulates a specific gene expression pattern. The physiologic functions of the kidneys are mediated by the segment-specific expression of key proteins. So far, we have limited knowledge about the mechanisms that control this gene expression pattern. The hyperosmolality in the renal medullary interstitium is of major importance as a driving force for urine concentration. We made use of primarily cultured rat renal inner medullary collecting-duct cells and microarray analysis to identify genes affected by the environmental osmolality of the culture medium. We identified hundreds of genes that were either induced or repressed in expression by hyperosmolality in a time- and osmolality-dependent fashion. Further analysis demonstrated that many of them, physiologically, showed a kidney- and even collecting-duct-specific expression, including secreted proteins, kinases, and transcription factors. On the other hand, we identified factors, down-regulated in expression, that have a diuretic effect. In conclusion, the kidney is the only organ that has such a hyperosmotic environment, and study provides an excellent method for controlling tissue-specific gene expression.-Schulze Blasum, B., Schröter, R., Neugebauer, U., Hofschröer, V., Pavenstädt, H., Ciarimboli, G., Schlatter E., Edemir, B. The kidney-specific expression of genes can be modulated by the extracellular osmolality.

Putative role of the H(+)/sucrose symporter SLC45A3 as an osmolyte transporter in the kidney.

The solute carrier family 45 a3 member (SLC45A3), known also as prostein, has been implicated with prostate cancer and the regulation of lipid metabolism in oligodendrocytes. Recently, we expressed SLC45A3 in yeast cells and characterised it as a proton-coupled sucrose symporter. However, the physiological functions of SLC45A3 were still unknown. Here, we report that SLC45A3 occurs in the kidney and is highly expressed in the medullary collecting duct (IMCD), a part of the kidney responsible for final urine concentration and faced to hyperosmotic environment. Moreover, messenger RNA (mRNA) expression of endogenous SLC45A3 in rat IMCD cells as well as in NRK52E cells increased up to four-fold under hyperosmotic conditions at 600 mOsmol/kg. Using NRK52E cells as an experimental model, we investigated the proton-coupled sugar transport and found that the uptake of sucrose or glucose was enhanced by hyperosmolarity. Down-regulation of expression by small interfering RNA (siRNA) decreased the osmotically inducible part of sucrose uptake and confirmed the involvement of SLC45A3 in this process. Furthermore, we observed an up to four-fold elevation of sucrose uptake triggered by hyperosmolarity across the apical membrane of NRK52E cells, while uptake across the basolateral membrane was not affected. Due to this finding, we conclude that SLC45A3 may occur at the luminal side of kidney epithelial cells and thus may take up solutes from the tubular fluid. Altogether, we show that SLC45A3 is a novel sugar transporter in kidney and hypothesise that the disaccharide sucrose, and probably the monosaccharides glucose and fructose, may serve as compatible osmolytes in urine.

Rho GAP myosin IXa is a regulator of kidney tubule function.

Mammalian class IX myosin Myo9a is a single-headed, actin-dependent motor protein with Rho GTPase-activating protein activity that negatively regulates Rho GTPase signaling. Myo9a is abundantly expressed in ciliated epithelial cells of several organs. In mice, genetic deletion of Myo9a leads to the formation of hydrocephalus. Whether Myo9a also has essential functions in the epithelia of other organs of the body has not been explored. In the present study, we report that Myo9a-deficient mice develop bilateral renal disease, characterized by dilation of proximal tubules, calyceal dilation, and thinning of the parenchyma and fibrosis. These structural changes are accompanied by polyuria (with normal vasopressin levels) and low-molecular-weight proteinuria. Immunohistochemistry revealed that Myo9a is localized to the circumferential F-actin belt of proximal tubule cells. In kidneys lacking Myo9a, the multiligand binding receptor megalin and its ligand albumin accumulated at the luminal surface of Myo9a-deficient proximal tubular cells, suggesting that endocytosis is dysregulated. In addition, we found, surprisingly, that levels of murine diaphanous-related formin-1, a Rho effector, were decreased in Myo9a-deficient kidneys as well as in Myo9a knockdown LLC-PK1 cells. In summary, deletion of the Rho GTPase-activating protein Myo9a in mice causes proximal tubular dilation and fibrosis, and we speculate that downregulation of murine diaphanous-related formin-1 and impaired protein reabsorption contribute to the pathophysiology.

Mouse organic cation transporter 1 determines properties and regulation of basolateral organic cation transport in renal proximal tubules.

The proximal tubule of mouse kidney expresses mouse organic cation transporter 1 (mOCT1), mOCT2, and much less mOCT3. Therefore, mOCT-mediated transport across the basolateral membrane of proximal tubules reflects properties of at least mOCT1 and mOCT2. Here, we unraveled substrate affinities and modulation of transport activity by acute regulation by protein kinases on mOCT1 and mOCT2 separately and compared these findings with those from isolated proximal tubules of male and female mOCT2−/− mice. These data are also compared to our recent reports on isolated tubules from wild-type and mOCT1/2 double knockout (mOCT1/2−/−) mice. OCT-mediated transport in proximal tubules of mOCT2−/− mice was only 20 % lower compared to those isolated from wild-type mice. While mOCT1 was regulated by all five pathways examined [protein kinase A (PKA), protein kinase C (PKC), p56lck, phosphoinositide 3-kinase (PI3K), and calmodulin (CaM)], mOCT2 activity was modulated by PKA, p56lck, and CaM only, however, in the same direction. As mOCT-mediated transport across the basolateral membrane of mOCT2−/− mice expressing only mOCT1 and to a small amount mOCT3 was identical to that observed for tubules isolated from wild-type mice and to that observed for human embryonic kidney 293 (HEK293) cells stably expressing mOCT1, mOCT1 represents the relevant paralog for OCT-dependent organic cation transport in the mouse kidney. Gender does not play a major role in expression and activity of renal OCT-mediated transport in the mouse. Properties of mouse OCT considerably differ from those of rat or human origin, and thus, observations made in these rodents cannot directly be transferred to the human situation

The organic cation transporter 3 (OCT3) as molecular target of psychotropic drugs: transport characteristics and acute regulation of cloned murine OCT3.

The organic cation transporter 3 (OCT3) is a widely expressed transporter for endogenous and exogenous organic cations. Of particular interest is OCT3 expression and function in the brain, where it plays a role in serotonin clearance and influences mood and behavior. Protein kinase signaling mediates rapid modulation of cerebral processes, but little is known about acute regulation of OCT3 by protein kinases. Therefore, we cloned mouse OCT3 (mOCT3) and generated a human embryonic kidney cell line stably expressing the transporter to study transport characteristics, acute regulation by protein kinases, and interaction with psychotropic drugs. Uptake measurement was performed using the fluorescent cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+), 1 µM) as a substrate. The translational value of these findings was determined by comparing results obtained with cloned mouse and human OCT3. mOCT3-mediated transport is membrane potential dependent and pH independent. ASP(+) uptake by mOCT3 and human OCT3 (hOCT3) was efficiently inhibited by 1-methyl-4-phenylpyridinium, tetrapentylammonium (TPA(+)), corticosterone, serotonin, and histamine and by the drugs ketamine, fluoxetine, and diazepam. The half maximal inhibitory concentrations of mOCT3 and hOCT3 for TPA(+), serotonin, diazepam, and ketamine are significantly different. Diazepam is a non-transported inhibitor. Furthermore, the activities of mOCT3 and hOCT3 are acutely regulated by the p56 (lck) tyrosine kinase by decreasing their V max. Studies with freshly isolated renal proximal tubules from mOCT1/2(-/-) mice, in which mOCT3 is the only OCT present, confirmed this regulation pathway. Only the activity of hOCT3 is regulated by calmodulin. These findings suggest that even though many transport properties of mOCT3 and hOCT3 are similar, there are also species-specific aspects of OCT3 function.

Kidney transplantation down-regulates expression of organic cation transporters, which translocate ß-blockers and fluoroquinolones.

Kidney transplanted patients are often treated with immunosuppressive, antihypertensive, and antibiotic drugs such as cyclosporine A (CsA), ß-blockers, and fluoroquinolones, respectively. Organic cation transporters (OCT) expressed in the basolateral membrane of proximal tubules represent an important drug excretion route. In this work, the renal expression of OCT after syngeneic and allogeneic kidney transplantation in rats with or without CsA immunosuppression was studied. Moreover, the interactions of CsA, ß-blockers (pindolol/atenolol), and fluoroquinolones (ofloxacin/norfloxacin) with rOCT1, rOCT2, hOCT1, and hOCT2 in stably transfected HEK293-cells were studied. Kidney transplantation was associated with reduced expression of rOCT1, while rOCT2 showed only reduced expression after allogeneic transplantation. All drugs interacted subtype- and species-dependently with OCT. However, only atenolol, pindolol, and ofloxacin were transported by hOCT2, the main OCT in human kidneys. While CsA is not an OCT substrate, it exerts a short-term effect on OCT activity, changing their affinity for some substrates. In conclusion, appropriate drug dosing in transplanted patients is difficult partly because OCT are down-regulated and because concomitant CsA treatment may influence the affinity of the transporters. Moreover, drug-drug competition at the transporter can also alter drug excretion rate.

SAM-Competitive EZH2-Inhibitors Induce Platinum Resistance by EZH2-Independent Induction of ABC-Transporters.

T-cell lymphomas are heterogeneous and rare lymphatic malignancies with unfavorable prognosis. Consequently, new therapeutic strategies are needed. The enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and responsible for lysine 27 trimethylation of histone 3. EZH2 is overexpressed in several tumor entities including T-cell neoplasms leading to epigenetic and consecutive oncogenic dysregulation. Thus, pharmacological EZH2 inhibition is a promising target and its clinical evaluation in T-cell lymphomas shows favorable results. We have investigated EZH2 expression in two cohorts of T-cell lymphomas by mRNA-profiling and immunohistochemistry, both revealing overexpression to have a negative impact on patients' prognosis. Furthermore, we have evaluated EZH2 inhibition in a panel of leukemia and lymphoma cell lines with a focus on T-cell lymphomas characterized for canonical EZH2 signaling components. The cell lines were treated with the inhibitors GSK126 or EPZ6438 that inhibit EZH2 specifically by competitive binding at the S-adenosylmethionine (SAM) binding site in combination with the common second-line chemotherapeutic oxaliplatin. The change in cytotoxic effects under pharmacological EZH2 inhibition was evaluated revealing a drastic increase in oxaliplatin resistance after 72 h and longer periods of combinational incubation. This outcome was independent of cell type but associated to reduced intracellular platinum. Pharmacological EZH2 inhibition revealed increased expression in SRE binding proteins, SREBP1/2 and ATP binding cassette subfamily G transporters ABCG1/2. The latter are associated with chemotherapy resistance due to increased platinum efflux. Knockdown experiments revealed that this was independent of the EZH2 functional state. The EZH2 inhibition effect on oxaliplatin resistance and efflux was reduced by additional inhibition of the regulated target proteins. In conclusion, pharmacological EZH2 inhibition is not suitable in combination with the common chemotherapeutic oxaliplatin in T-cell lymphomas revealing an EZH2-independent off-target effect.

Interaction between bradykinin and natriuretic peptides via RGS protein activation in HEK-293 cells.

In this study, the interaction of natriuretic peptides (NP) and bradykinin (BK) signaling pathways was identified by measuring membrane potential (V(m)) and intracellular Ca(2+) using the patch-clamp technique and flow cytometry in HEK-293 cells. BK and NP receptor mRNA was identified using RT-PCR. BK (100 nM) depolarized cells activating bradykinin receptor type 2 (B(2)R) and Ca(2+)-dependent Cl(-) channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 µM). The BK-induced Ca(2+) signal was blocked by the B(2)R inhibitor HOE 140. [Des-Arg(9)]-bradykinin, an activator of B(1)R, had no effect on intracellular Ca(2+). NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K(+) channels. ANP, urodilatin, BNP [binding to natriuretic peptide receptor (NPR)-A] and 8-bromo-(8-Br)-cGMP inhibited the BK-induced depolarization while CNP (binding to NPR-Bi) failed to do so. The inhibitory effect on BK-triggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca(2+) signaling was completely blocked by the phospholipase C (PLC) inhibitor U-73122 (10 nM). The inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 50 µM) completely inhibited the BK-induced Ca(2+) signaling. UTP, another activator of the PLC-mediated Ca(2+) signaling pathway, was blocked by U-73122 as well but not by 8-Br-cGMP, indicating an intermediate regulatory step for NP via PKG in BK signaling such as regulators of G-protein signaling (RGS) proteins. When RGS proteins were inhibited by CCG-63802 in the presence of BK and 8-Br-cGMP, cells started to depolarize again. In conclusion, as natural antagonists of the B(2)R signaling pathway, NP may also positively interact in pathological conditions caused by BK.

The nuclear factor of activated T cells 5 (NFAT5) contributes to the renal corticomedullary differences in gene expression.

The corticomedullary osmotic gradient between renal cortex and medulla induces a specific spatial gene expression pattern. The factors that controls these differences are not fully addressed. Adaptation to hypertonic environment is mediated by the actions of the nuclear factor of activated T-cells 5 (NFAT5). NFAT5 induces the expression of genes that lead to intracellular accumulation of organic osmolytes. However, a systematical analysis of the NFAT5-dependent gene expression in the kidneys was missing. We used primary cultivated inner medullary collecting duct (IMCD) cells from control and NFAT5 deficient mice as well as renal cortex and inner medulla from principal cell specific NFAT5 deficient mice for gene expression profiling. In primary NFAT5 deficient IMCD cells, hyperosmolality induced changes in gene expression were abolished. The majority of the hyperosmolality induced transcripts in primary IMCD culture were determined to have the greatest expression in the inner medulla. Loss of NFAT5 altered the expression of more than 3000 genes in the renal cortex and more than 5000 genes in the inner medulla. Gene enrichment analysis indicated that loss of NFAT5 is associated with renal inflammation and increased expression of kidney injury marker genes, like lipocalin-2 or kidney injury molecule-1. In conclusion we show that NFAT5 is a master regulator of gene expression in the kidney collecting duct and in vivo loss of NFAT function induces a kidney injury like phenotype.

Impact of cytotoxic agents or apoptosis stimulants on αklotho in MDCK, NRK-52E and HK2 kidney cells.

αKlotho is a transmembrane protein acting as a co-receptor for FGF23, a bone hormone regulating renal phosphate and vitamin D metabolism. αKlotho expression is controlled by PPARγ. Soluble αklotho (sKL) regulates cellular signaling impacting stress resistance and death. αKlotho deficiency causes early onset of aging-associated diseases while its overexpression markedly increases lifespan. Cellular stress due to cytotoxic therapeutics or apoptosis induction through caspase activation or serum deficiency may result in cell death. Owing to αklotho's role in cellular stress and aging, this study explored the effect of cytotoxic agents or apoptosis stimulants on cellular αklotho expression. Experiments were performed in renal MDCK, NRK-52E and HK-2 cells. Gene expression was determined by qRT-PCR, sKL by ELISA, apoptosis and necrosis by annexin V binding and a fluorescent DNA dye, and cell viability by MTT assay. Cytostatic drugs cisplatin, paclitaxel, and doxorubicin as well as apoptosis induction with caspase 3 activator PAC-1 and serum deprivation induced αklotho and PPARG gene expression while decreasing viability and proliferation and inducing apoptosis of MDCK and NRK-52E cells to a variable extent. PPARγ antagonism attenuated up-regulation of αklotho in MDCK cells. In HK-2 cells, αklotho gene expression and sKL protein were down-regulated by chemotherapeutics. SKL serum levels in patients following chemotherapy were not significantly changed. In summary, potentially fatal stress results in up-regulation of αKlotho gene expression in MDCK and NRK-52E cells and down-regulation in HK-2 cells. These results indicate that different renal cell lines may exhibit completely different regulation of αklotho.

Mechanisms of cell polarity and aquaporin sorting in the nephron.

The kidneys participate in whole-body homeostasis, regulating acid-base balance, electrolyte concentrations, extracellular fluid volume, and regulation of blood pressure. Many of the kidney's functions are accomplished by relatively simple mechanisms of filtration, reabsorption, and secretion, which take place in the nephron. The kidneys generate 140-180 l of primary urine per day, while reabsorbing a large percentage, allowing for only the excretion of approximately 2 l of urine. Within the nephron, the majority of the filtered water and solutes are reabsorbed. This is mainly facilitated by specialized transporters and channels which are localized at different segments of the nephron and asymmetrically localized within the polarized epithelial cells. The asymmetric localization of these transporters and channels is essential for the physiological tasks of the renal tissues. One family of these proteins are the water-permeable aquaporins which are selectively expressed in cells along the nephron and localized at different compartments. Here, we discuss potential molecular links between mechanisms involved in the establishment of cell polarity and the members of the aquaporin family. In the first part of this review, we will focus on aspects of apical cell polarity. In the second part, we will review the motifs identified so far that are involved in aquaporin sorting and point out potential molecular links.

Different effects of CsA and FK506 on aquaporin-2 abundance in rat primary cultured collecting duct cells.

Calcineurin (Cn) inhibitors (CnI) such as cyclosporine A (CsA) and FK506 are nephrotoxic immunosuppressant drugs, which decrease tubular function. Here, we examined the direct effect of CnI on aquaporin-2 (AQP2) expression in rat primary cultured inner medullary collecting duct cells. CsA (0.5-5 µM) but not FK 506 (0.01-1 µM) decreased expression of AQP2 protein and messenger RNA (mRNA) in a concentration and time dependent manner, without affecting mRNA stability. This effect was observed despite similar inhibition of Cn activity by both CnI, thereby suggesting that the CsA-dependent decrease in AQP2 expression was Cn independent. Another inhibitor of cyclophilin A, the primary intracellular target of CsA, had no effect on AQP2 expression. In order to investigate the mechanism of decreased AQP2 transcription, we studied activation status of two suggested transcriptional regulators of AQP2, cAMP-responsive element binding protein (CREB), and tonicity enhancer binding protein (TonEBP). Localization of TonEBP, as well as TonEBP-mediated gene transcription, was not affected by CsA. Phosphorylation of CREB at an activating phosphorylation site (S133) was decreased by CsA, but not by FK506. However, both CnI did not affect cellular cAMP levels. We show that CsA decreases transcription of AQP2, a process that is in part independent of Cn or cyclophilin A and suggests dependence on decreased activity of CREB.

Properties and regulation of organic cation transport in freshly isolated mouse proximal tubules analyzed with a fluorescence reader-based method.

The main elimination site of organic cations (OCs) is the renal proximal tubule (PT). OC transporters (OCT) accept endogenous and exogenous substances and xenobiotics. As transgenic mouse models are increasingly used in translational medicine, functional properties with special focus on regulation of OCT of isolated mouse PTs were studied with a new fluorescence reader-based method, which allows studying larger numbers of tubules per kidney. OC transport across the basolateral membrane of PTs from male mice was measured as initial uptake of the fluorescent dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP). A microtiter plate fluorescence reader was used to semi-automatically analyze OC transport in freshly isolated tubules. Relative mRNA expression of OCT1/OCT2/OCT3 in PTs was 1/0.3/0.01 and did not vary from S1 to S3 segments. ASP was transported by PTs with a K (m) of 6 µM. It was inhibited by TEA, TPA, or cimetidine (IC(50)=5, 19, or 53 µM, respectively). Angiotensin II stimulated ASP uptake (+63%), while stimulation of PKC reduced (-37%) OC transport. Inhibition of p56(lck) tyrosine kinase (-60%), of PI3K (-36%), of Ca(2+)/calmodulin (-25%), or of PKA (-33%) reduced OC transport. In PTs from OCT1/2(-/-) mice ASP uptake was reduced to ~20%. Using this fluorescence reader-based method, we report substrate specificities and a complex pattern of acute regulation of OC transport in isolated mouse PTs. Compared to isolated human PTs or rat and human OCT isoforms expressed in HEK293-cells, OC transport across the basolateral membrane of freshly isolated mouse PTs shows similarities but also specific differences.