Third Circular - dnh-smc.com

Third Circular 2015 Kitasato-Yale Fluid Symposium: Molecular Control of Cellular and Epithelial Function

Date: Monday March 2, 2015 (Fixed) Place: Room 1603, Kitasato University Sch of Pharmacy, Bldg. No.1, Shirokane, 5-9-1, Minato-ku, Tokyo 108-8641, Japan http://www.kitasato-u.ac.jp/pharm/en/access/ Organizers: Katsumasa Kawahara, Sagamihara

Shuichi Hirono, Tokyo Secretary: Tomoni Oshima, Sagamihara Supported by Kitasato U Grad Sch Med Sci

Co-supported by Kitasato U Sch Med and Kitasato University

Invited speakers: Peter S. Aronson, Yale U Sch Med, New Haven, USA Michael J. Caplan, Yale U Sch Med, New Haven, USA Contact us at: [email protected] Dept. of Cell & Mol Physiology, Kitasato U Grad Sch Med Sci (KUGSMS) and Dept of Physiology, Kitasato U Sch Med (KUSM), Kitasato 1-15-1, Minami-ku,

Sagamihara 252-0374

Participants and Program (Fixed) Registration, Monday March 2, 2015, 12:00- Opening, 12:55 13:00-14:20 Session I: Revisiting Kidney Function and Disease

Chairpersons: Naohiko Anzai, Dokkyo Med Sch, Mibu and

Yoshiro Sohma, Keio U Sch Med, Tokyo

13:00-13:20 Acid-base imbalance in mice lacking vasopressin V1a receptor (V1aR): A role of V1aR in the kidney collecting duct.

Yukiko Yasuoka, Kitasato U Sch Med, Sagamihara

13:20-13:40 Roles of delayed rectifier K+-channels (Kv1.3) in T lymphocytes and their therapeutic implications for chronic kidney disease

Itsuro Kazama, Tohoku U Sch Med, Sendai

13:40-14:00 Altered renal magnesium handling in diabetic nephropathy and interstitial impairment

Hajime Hasegawa, Saitama Med Sch, Kawagoe

14:00-14:30 Erythropoietin production by the nephron Hiroshi Nonoguchi, Kitasato Medical Center, Kitamoto

14:30-14:40 Tea & Coffee Break

14:40-16:00 Session II: New Technology & Abnormal Kidney Function Chairpersons: Yoshio Takei, Tokyo U AORI, Kashiwa and

Itsuro Kazama, Tohoku U Sch Med, Sendai

14:40-15:00 Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA Junji Suzuki, Tokyo U Grad Sch Med, Tokyo

15:00-15:20 Two novel direct observations in membrane transports: Transepithelial water diffusion and single channel molecules Yoshiro Sohma, Keio U Sch Med, Tokyo

15:20-16:00 Novel protein trafficking and signaling pathways in kidney tubule cells Michael J Caplan, Yale U Sch Med, New Haven, USA

16:00-16:20 Tea & Coffee Break 16:20-18:00 Session III: Body Fluid Control by Kidney

Chairpersons: Hiroshi Nonoguchi, Kitasato Medical Center,

Kitamoto and Hitoshi Endou, J-Phama Co., Ltd., Yokohama 16:20-16:50 What can be learned from fishes for sulfate regulation by the

kidney: Environmental sensing and transport mechanisms in eels Yoshio Takei, Tokyo U AORI, Kashiwa

16:50-17:20 Renal tubular transport of organic anions and urate Naohiko Anzai, Dokkyo Med Sch, Mibu

17:20-18:00 Anion Transporters: Roles in Renal Salt and Oxalate Excretion

Peter S Aronson, Yale U Sch Med, New Haven, USA

18:00- 18:05 Concluding Remarks

Katsumasa Kawahara, Kitasato U Grad Sch Med Sci, Sagamihara

18:40- Farewell Dinner at “Takehashi”, KKR Hotel Tokyo KKR Hotel Tokyo: http://www.kkr-hotel-tokyo.gr.jp/english/ Japanese Restaurant Takehashi: http://www.kkr-hotel-tokyo.gr.jp/restaurant/takehashi/

1. Acid-Base Imbalance in Mice Lacking Vasopressin V1a Receptor (V1aR): A Role of V1aR in the Kidney Collecting Duct

Yukiko Yasuoka1,2), Yuichi Sato3), Hiroshi Nonoguchi4), and

Katsumasa Kawahara1,2) 1Physiol, Kitasato U Sch Med (KUSM) and 2Cell & Mol Physiol, Kitasato U Grad

Sch Med Sci (KUGSMS), Sagamihara, Japan; 3Mol Diag, Kitasato U Grad Sch

Med Sci, Sagamihara, Japan, 4Internal Med, Kitasato U Med Center, Kitamoto,

Japan.

[email protected]

The kidney controls systemic acid-base balance by reabsorbing

bicarbonate from the glomerular filtrate and excreting acid/bicarbonate into the

urine. Recently, Nonoguchi and his colleagues demonstrated that in the rat

kidney collecting ducts (CDs), the levels of V1aR mRNA and protein expression

increased in parallel with metabolic acidosis1). Further, they showed the lower

urinary acidification in mice lacking V1aR (V1aR-/-)2). To study a role of V1aR in

the CD for the urinary acidification, we identified and quantified a segment/cell

specific localization and expression along the kidney nephron by using a highly

sensitive in situ hybridization with tyramide signal amplification (ISH-TSA)

method and a double staining immunohistochemistry (IHC) technique.

Results. First, in the MTALis and type A intercalated cell (IC-A) of OMCDis, the

level of V1aR mRNA expression was uniquely and significantly up-regulated

after NH4Cl-loading (free drinking of 2% sucrose containing 0.28 M NH4Cl

solution for 6 d). Second, V1aR-/- mice failed to maintain plasma pH within the

normal range under the conditions of normal and NH4Cl-drinking (6 d). Third, the

NH4Cl-induced hypertrophy occurred only in IC-A of OMCDis, but not in MTALis.

More interestingly, the acid-induced hypertrophy of the IC-A was significantly

attenuated in V1aR-/- mice.

In conclusion, vasopressin-V1aR signal in the IC-A of OMCDis may be

essential for urinary acidification and for a systemic acid-base balance, at least

during acidosis.

OMCDis: inner stripe of the outer medullary collecting duct

MTALis: thick ascending limb of Henle’s loop in the inner stripe of outer medulla

References:

1. Tashima Y, Kohda Y, Nonoguchi H, Ikebe M, Machida K, Star RA, Tomita K.

Intranephron localization and regulation of the V1a vasopressin receptor during

chronic metabolic acidosis and dehydration in rats. Pflügers Arch. 2001; 442:

652-61.

2. Izumi Y1, Hori K, Nakayama Y, Kimura M, Hasuike Y, Nanami M, Kohda Y, Otaki Y,

Kuragano T, Obinata M, Kawahara K, Tanoue A, Tomita K, Nakanishi T, Nonoguchi

H. Aldosterone requires vasopressin V1a receptors on intercalated cells to mediate

acid-base homeostasis. J Am Soc Nephrol. 2011; 22: 673-80.

2. Roles of Delayed Rectifier K+-channels (Kv1.3) in T Lymphocytes and their Therapeutic Implications for Chronic Kidney Disease

Itsuro Kazama

Department of Physiology I, Tohoku University Graduate School of Medicine,

Seiryo-cho, Aoba-ku, Sendai, Miyagi, Japan

E-mail: [email protected]

T lymphocytes predominantly express delayed rectifier K+-channels (Kv1.3) in their plasma membranes. Patch-clamp studies revealed that the channels play crucial roles in facilitating calcium influx necessary to trigger the lymphocyte

activation and proliferation. In addition to selective channel inhibitors that have been developed, we recently showed physiological evidence that the commonly-used drugs, such as non-steroidal anti-inflammatory drugs,

antibiotics, anti-hypertensives and anti-cholesterol drugs, effectively suppress the channel currents in lymphocytes, and thus exert immunosuppressive effects.

Using experimental animal models, previous studies revealed the

pathological relevance between the expression of ion channels and the progression of renal diseases. As an extension, we recently demonstrated that the overexpression of lymphocyte Kv1.3-channels contributed to the progression

of chronic kidney disease (CKD) by promoting cellular proliferation and interstitial fibrosis. According to one of our patch-clamp studies, since benidipine, a long-acting 1,4-dihydropyridine Ca2+ channel blocker, was also highly potent

as a Kv1.3-channel inhibitor, it could exert therapeutic efficacy in the progression of CKD. Therefore, using a rat model with advanced stage chronic renal failure (advanced CRF), we examined the effects of benidipine on the histopathological

features of the kidneys, cellular proliferation of leukocytes and the cortical expression of pro-inflammatory cytokines. In the cortical interstitium of advanced CRF rat kidneys, benidipine significantly ameliorated the progression of renal

fibrosis without affecting glomerular injury. This drug also reduced the number of proliferating leukocytes with a significant decrease in the pro-inflammatory cytokine expression. Together with our in vitro evidence, the studies indicated

the therapeutic potency of Kv1.3-channel inhibitors, such as benidipine, in the treatment or the prevention of CKD.

3. Altered Renal Magnesium Handling in Diabetic Nephropathy and Interstitial Impairment.

Hajime Hasegawa, Taisuke Shimizu, Kaori Takayanagi, Takatsugu Iwashita,

Yosuke Tayama, Hiroaki Hara, Nobuyuki Onizawa, Naohiko Anzai, Akira Ikari,

Koichi Kanozawa, Akihiko Matsuda

Department of Nephrology and Hypertension, Saitama Medical Center,

Saitama Medical University, Kamoda, Kawagoe, Saitama, Japan


Background and Aim: Elevated urine Mg excretion and its correlation with

histological damage in tubulo-interstitial nephropathy (TIN) were reported. In addition, hypermagnesiuric hypomagnesemia in diabetic nephropathy (DMN) is a characteristic clinical feature of early diabetes and known to be highly involved

in the development of insulin resistance and coronary events. While the hypermagnesiuria is important in the clinical medicine in those disease settings, its underlying mechanisms have not been sufficiently studied yet. We

investigated the mechanisms of the altered expression profile of the renal magnesium transporting molecules in the rat models of TIN and DMN to clarify the underlying mechanisms to provoke the altered magnesium handling.

Method: For the study of the altered magnesium handling in TIN, we used unilateral ureter obstruction (UUO) rat model. The left kidney was sampled at day-0 (control), day-1 (early phase) and day-7 (late phase) after ligation of the

left ureter. For the study in DMN, obese T2DM model, OLETF and LETO as a control at the age of 16, 24 and 34 weeks old were used. In both studies, we assessed the expression of renal magnesium-transporting molecules and the

development of TIN by immunohistochemistry, RT-PCR, and immunoblotting. Results: In the TIN experiments, the gene expression of claudin-16, tight

junctional magnesium pathway in TAL, was decreased in the late phase but was

not in the early phase (100.2±2.9% at day-0, 90.3±6.3% at day-1, 36.4±1.6% at day-7), which were consistent with immunohistochemistry and confirmed by immunoblotting. However, the expression of TRPM6, a channel for magnesium

reabsorption in DCT, was reduced even in the early phase. The immunohistochemistry and gene expression of pro-inflammatory cytokines showed that tubulo-interstitial damage was not apparent in the early phase but

was significant in the late phase. In addition, density of peritubular capillaries was diminished in the late phase but not in the early phase. In the DMN experiments, urine magnesium excretion was increased at 24 and 34 wks-old of

OLETF, and hypomagnesemia was apparent in 34 wk-old in OLETF, but not in LETO (UMgV: 0.16±0.01 in 24-LETO, 0.28±0.01 in 24-OLETF, µg/min/BW). The gene expression of TRPM6 was down-regulated (85.5±5.6% in 34-LETO,

63.0±3.5% in 34-OLETF) concomitant with Na-Cl cotransporter (NCC) down-regulation, whereas the expression of claudin-16 was not different. The results of the semi-quantitative analysis of immunohistochemistry were

consistent with these findings (TRPM6, positive area: 0.49±0.04% in 16-LETO, 0.10±0.01% in 16-OLETF, 0.52±0.03% in 24-LETO, 0.10±0.01% in 24-OLETF, 0.48±0.02% in 34-LETO, 0.12±0.02% in 34-OLETF). The gene expressions of

fibrosis-related pro-inflammatory cytokines and the histological changes showed that the hypermagnesiuria-related molecular changes and the TIN developed independently.

Conclusion: In the well-known disease settings showing hypermagnesiuria, TIN and DMN, the altered magnesium handling would be caused by the different molecular mechanisms. The dysfunction of TAL caused by the down regulation

of claudin-16 would be principally involved in the hypermagnesiuria in TIN, whereas diminished magnesium reabsorption in DCT caused by the down regulation of TRPM6 might be a principal genesis for the hypermagnesiuria in

DMN.

4. Erythropoietin Production by the Nephron

Hiroshi Nonoguchi1), Yuichiro Izumi2), Yukiko Yasuoka3), and

Katsumasa Kawahara3). 1Internal Medicine, Kitasato University Medical Center, 2Dept of Physiology,

Kitasato University School of Medicine, and 3Dept of Nephrology, Kumamoto

University School of Medical Sciences, Japan

[email protected]

Erythropoietin (Epo) production has been shown to occur in the peritubular fibroblast (Renal Epo-Producing: REP cells) in vivo. However,

severe anemia (hematocrit <15%) is required to see the appearance of REP cells. We investigated Epo production by the nephron and intercalated cells using in situ hybridization and rat intercalated cell line (IN-IC cells).

C57Bl/6J mice with basal condition and 4-hr hypoxia, treatment were used for high sensitive in situ hybridization (ISH) of Epo mRNA and immunohistochemistry of prolyl hydroxylase 2 (PHD2). In ISH for Epo, double

staining technique was performed with either anti AQP3 or anti AE1 antibodies to distinguish IC of CD. Hypoxia-inducible factor 2a (HIF2a) expression was examined by ISH and RT-PCR of microdissected nephrons. Wild type (WT)

and V1aR KO mice and IN-IC cells were also used for Western blot of HIF1a and 2a, and PHD1 and 2. Epo mRNA was detected in the cortical nephrons but not in peritubular

cells in basal condition. Hypoxia slightly increased Epo mRNA expression in cortical nephrons, and remarkably in peritubular cells. PHD2 was detected in the nephron in basal condition while it was increased in peritubular cells but

not in the nephron by hypoxia. HIF2α expression was observed in whole nephron segments and hypoxia increased the expression in peritubular cells. In IN-IC cells, aldosterone as well as hypoxia increased Epo production

through the activation of HIF1α, 2α and PHD2 pathway. Cortical nephrons produce Epo in basal and hypoxic conditions, while REP cells produce EPo only in hypoxia. Aldosterone stimulates Epo

production in intercalated cells through the activation of HIF1α, 2α and PHD2 pathway.

5. Imaging Intraorganellar Ca2+ at Subcellular Resolution Using CEPIA

Junji Suzuki1, Kazunori Kanemaru1, Kuniaki Ishii2, Masamichi Ohkura3,

Yohei Okubo1, Masamitsu Iino1 1Dept. Pharmacol., Grad. Sch. Med., Univ. Tokyo, Tokyo, Japan

2Dept. Pharmacol., Grad. Sch. Med., Yamagata Univ., Yamagata, Japan 3Brain Sci. Inst., Saitama Univ., Saitama, Japan

Email: [email protected]

The endoplasmic reticulum (ER) and mitochondria accumulate Ca2+ within

their lumens to regulate numerous cell functions. However, determining the

dynamics of intraorganellar Ca2+ has proven to be difficult. We generated a family of genetically-encoded Ca2+ indicators, named calcium-measuring organelle-entrapped protein indicators (CEPIA), which can be utilized for

intra-organellar Ca2+ imaging. CEPIA, which emit green, red or blue/green fluorescence, are engineered to bind Ca2+ at intra-organellar Ca2+ concentrations. They can be targeted to different organelles and may be used

alongside other fluorescent molecular markers, expanding the range of cell functions that can be simultaneously analyzed. The spatiotemporal resolution of CEPIA makes it possible to resolve Ca2+ import into individual mitochondria

while simultaneously measuring ER and cytosolic Ca2+. We have used these imaging capabilities to reveal differential Ca2+ handling in individual mitochondria. Thus, CEPIA enable to study the physiological functions of intraorganellar Ca2+

dynamics.

6. Two Novel Direct Observations in Membrane Transports: Transepithelial Water Diffusion and Single Channel Molecules

Yoshiro Sohma

Department of Pharmacology, Keio University School of Medicine, Shinjuku,

Tokyo 160-8582, Japan


Recent great advances in the nonlinear optical science and the nanotechnology give us novel kinds of information in the biomedical science field. In this symposium, we will introduce our challenges for the application of two

emerging measurement techniques, Coherent Anti-stokes Raman Scattering (CARS) microcopy and High-Speed Atomic Force Microscopy (HS-AFM), to the epithelial transport physiology.

The CARS laser-scanning microscopy gives us H2O images in cells and tissues from the O-H stretch vibration-specific scattering. Applying this technique to 3D cysts formed by MDCK cells, we succeeded to make a direct observation

of H2O/D2O exchange process across the MDCK epithelium and determined the diffusional water permeability of luminal and basolateral membranes by analyzing the time-lapse image data with a computer simulation model.

The HS-AFM has enabled, for the first time, direct visualization of dynamic structural changes and dynamic interactions occurring in individual biological molecules in a solution, which has been impossible with other techniques. The

HS-AFM succeeded to image dynamic structural changes in membrane proteins including CFTR channels and also to visualize the binding/unbinding process of autoantibodies to human aquaporins.

We expect that these two techniques will take a new turn in the epithelial transport physiology.

7. Novel Protein Trafficking and Signaling Pathways in Kidney Tubule Cells

Valeria Padovano, Emily Stoops, Michael Hull, Nikolay Gresko, David Merrick,

Hannah Chapin, Michael J. Caplan,

Department of Cellular and Molecular Physiology, Yale University

School of Medicine,

Cedar Street, New Haven, Connecticut, USA


The fluid and electrolyte transport properties of the nephron are

determined largely by the inventories of ion transport proteins that occupy the apical and basolateral plasma membrane domains of renal tubular epithelial cells. These membrane domains are distinguished by markedly different protein

components that mediate the majority of their characteristic functions. It is the asymmetric apportioning of ion channels, transporters and pumps among these surfaces that determines the nature of the fluxes maintained by any epithelium.

Generation and maintenance of differentiated plasmalemmal domains requires the cell to possess machinery capable of discriminating among newly synthesized membrane proteins. Information embedded in aspects of these

proteins' architectures must serve as sorting signals which, when interpreted by the cellular sorting apparatus, specify their appropriate subcellular destinations. The mechanisms and structural correlates of this sorting function have been

subjects of intense research. To follow the post synthetic fate of newly synthesized membrane proteins in polarized renal epithelial cells, we have adapted a technique that permits

direct observation of temporally defined cohorts of proteins via the combination of fluorescence microscopy with pulse-chase labeling protocols. The 20-kDa SNAP-Tag is a modified version of the DNA repair protein alkylguanine-DNA

alkyltransferase, which cleaves para-substituted benzyl guanines (BGs) by covalently transferring the substituted benzyl group to its active thiol. The resulting thioether bond irreversibly prevents the reacted SNAP-Tag from

participating in any further labeling reactions. Fluorescent BG derivatives allow for the labeling and detection of SNAP-Tagged fusion proteins in either live or fixed cells. Through the combination of a “pre-pulse” blocking step with

non-fluorescent BG, followed by selective labeling of newly synthesized protein with fluorescent BG, we can follow a cohort of protein as it is synthesized and trafficked. Using this technique, we have determined that three proteins that

are destined for the basolateral plasma membrane (Na,K-ATPase, E-cadherin and VSV G protein) travel from the trans Golgi network to the basolateral membrane in separate vesicular carriers. We have also found that an apical

membrane protein, gp135, is delivered to the apical surface at the base of the primary cilium and then moves radially towards the cell periphery via a microtubule-dependent process.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) affects ~1 in 1,000 people and is the most common potentially lethal genetic disease. It leads to end stage renal disease in ~50% of affected individuals. ADPKD is caused by

mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2). PC1 is an enormous membrane protein that is predicted to span the bilayer 11 times, with a large (~3,000 amino acid) extracellular N terminal domain and a

short (~200 amino acid) C terminal domain that faces the cytoplasm. PC2 is a member of the TRP family of non-selective calcium-permeable cation channels. The PC1 and PC2 proteins participate in a number of signaling pathways and

exhibit complex patterns of subcellular localization. PC1 and PC2 interact with each other and with numerous other proteins that may modulate their trafficking properties or their involvement in signal transduction. Both PC1 and PC2

localize to the primary cilium where they appear to participate in mechanosensory or chemosensory processes. We have found that polycystin trafficking and function are regulated by oxygen-sensing pathways. We have

also found that the C-terminal tail of Polycystin-1 (PC1-CTT) is released by a

γ-secretase-mediated cleavage. The PC1-CTT translocates to the nucleus, where it is involved in the regulation of several transcriptional pathways that

modulate proliferation, apoptosis and cell fate determination. Loss of PC1 expression results in increased proliferation and apoptosis, while re-introduction of its C-terminal tail fragment into polycystin-1 null cells reestablishes normal

growth rate and apoptosis, and prevents cyst formation in three dimensional

cultures. Inhibition of γ-secretase activity impairs the growth and apoptosis suppressive effects produced by full length PC1 expression. Thus, the

cleavage and nuclear translocation of the PC1 protein’s cytoplasmic domain mark PC1 as a member of a growing collection of plasma membrane proteins

that are cleaved by γ-secretase and participate in direct signaling to the nucleus. We find that this cleavage mechanism is also relevant to PC1 functions in non-renal tissues. Bone-specific suppression of PC1 expression leads to profound defects in bone formation and mineralization. We find that the

PC1-CTT interacts with and stimulates a co-activating protein that plays a critical role in controlling the activity of the transcription factor that mediates bone development. Thus, polycystin proteins signal from the cilium to the nucleus to

modulate gene expression in a variety of cell types.

8. What can be Learned from Fishes for Sulfate Regulation by the Kidney: Environmental Sensing and Transport Mechanisms in Eels

Yoshio Takei and Taro Watanabe

Laboratory of Physiology, Atmosphere and Ocean Research Institute,

The University of Tokyo, Kashiwa, Chiba 277-8564, Japan


Although sulfate is the second most abundant anion in the body and plays important roles in various aspects of body functions, much less is known about its regulation compared with chloride regulation. Eels are unique in sulfate

regulation among teleosts in that they maintain plasma sulfate concentration 6 folds higher in sulfate-deficient fresh water (FW) than in sulfate-excess seawater (SW), which contains >30 mM of sulfate. This enigma can be explained by

drastic changes in renal sulfate regulation; from active absorption in FW to active secretion in SW (urine sulfate concentration of SW eels reaches >100 mM). We found that the chloride ions, not sulfate, in SW are responsible for the switching

of regulation; after transfer of eels from FW to SW, they take up sulfate via Na-Cl cotransporter (NCC) in the gills in response to increased environmental chloride ions, and the resultant increase in plasma chloride concentration triggers the

change. In fact, sulfate transporters (anion exchangers) in the renal proximal tubules, which is divided into two segments (P1 and P2) in eels, is replaced after SW transfer. When eels are in FW, apical Slc13a1 and basolateral Slc26a1 in

the P1 and P2 almost completely reabsorb sulfate in the filtered primary urine. After transfer to SW, Slc13a1 on the apical membrane of epithelial cells disappears in a few days and it was replaced by Slc26a6a and Slc26a6c at the

P1 and by Slc26a6b at the P2. Furthermore, Slc26a1 on the basolateral membrane remains at the P2 for sulfate uptake from plasma, but the transporter (s) responsible for uptake of sulfate at the basolateral side of the P2 segment

remain unknown. It is intriguing to examine whether or not the similar changes occur in mammals after intake of excess sulfate ions.

9. Renal Tubular Transport of Organic Anions and Urate

Naohiko Anzai, Motoshi Ouchi, Naoyuki Otani, Promsuk Jutabha

Department of Pharmacology and Toxicology, Dokkyo Medical University

School of medicine, Kitakobayashi, Mibu-machi, Shimotsuga, Tochigi, Japan


A variety of endogenous and exogenous substances that are harmful to the

body can be classified into organic anions and cations. Their elimination is therefore essential for the maintenance of homeostasis. Excretory organs such as the kidney, liver and intestine defend the body against the potentially harmful

effects of these compounds by biotransformation into less active metabolites and by the excretory transport process. Particularly in the kidney, drugs are eventually excreted into the urine, either in the unchanged form or as

biotransformation products. This renal excretion is closely related to the physiological events occurring in nephrons, i.e., filtration, secretion, and reabsorption. Transport systems responsible for renal tubular secretion of

exogenous (e.g. drugs) and endogenous (e.g. urate) substances have been divided into either organic anion or organic cation transport systems based on their preferential substrate selectivity. The process of secreting organic anions

and cations through the proximal tubular cells is achieved via unidirectional transcellular transport, which involves the uptake of organic ions into the cells from the blood across the basolateral membrane, followed by extrusion across

the brush-border membrane into the proximal tubular fluid. During the last decade, molecular cloning has identified several families of multispecific organic anion transporters, such as organic anion transporter (OAT), organic

anion-transporting polypeptide (OATP), sodium-phosphate transporter (NPT), and peptide transporter (PEPT). Additional findings have also suggested ATP-dependent organic ion transporters such as the multidrug

resistence-associated protein (MRP) to act as an as efflux pump. Here, we outline the present knowledge of organic anion transport in the kidney. We will discuss its pharmacological and pathophysiological aspects.

10. Anion Transporters: Roles in Renal Salt and Oxalate Excretion

Peter S. Aronson

Departments of Medicine and of Cellular and Molecular Physiology

Yale University School of Medicine

New Haven, CT, USA

Email: [email protected]

Most of the Na, Cl and HCO3 filtered by the kidney are reabsorbed in the proximal tubule. While much of the Cl reabsorption in this nephron segment is passive and paracellular, a potential mechanism to mediate transcellular Cl

reabsorption is apical membrane Cl-anion exchange: Cl-formate exchange, Cl-oxalate exchange, Cl-OH exchange and/or Cl-HCO3 exchange. Studies to identify the transporter(s) mediating apical membrane Cl-anion exchange in the

proximal tubule led to the characterization of SLC26A6. Functional expression of SLC26A6 in Xenopus oocytes demonstrated that the transporter is capable of mediating Cl-formate exchange, Cl-oxalate exchange, Cl-OH exchange and

Cl-HCO3 exchange. Comparison of wild-type and Slc26a6 null mice with respect to transport in renal brush border vesicles and microperfused tubules demonstrated that SLC26A6 primarily mediates Cl-oxalate exchange rather than

Cl-formate exchange in the proximal tubule in vivo. Unexpectedly, Slc26a6 null mice were observed to have a high

incidence of calcium oxalate urolithiasis that was attributable to hyperoxaluria.

Hyperoxaluria in Slc26a6 null mice was found to result from a defect in oxalate back-secretion in the intestine, thereby causing increased net absorption of ingested oxalate and elevated plasma oxalate concentration. Intestinal

absorption of oxalate was found to be predominantly passive and paracellular. Consequently, feeding a diet high in soluble oxalate (high oxalate, low Ca diet) led to increased absorption of oxalate, hyperoxalemia, hyperoxaluria and

development of renal failure due to crystal nephropathy. Mice fed the diet high in soluble oxalate demonstrated increased NALP3

expression in the kidney. Nalp3-null mice were completely protected from the

progressive renal failure and death that occurred in wild-type mice fed the diet high in soluble oxalate. Thus, progressive renal failure in oxalate nephropathy results primarily from NALP3-mediated inflammation rather than intratubular

obstruction by crystals. We suggest that oxalate may be a pro-inflammatory factor contributing to CKD progression.

List of Participants Members of the Kitasato-Yale Academic Exchange Program

K Kawahara, Dept. Cell & Mol Physiol, Kitasato Univ. Grad. Sch. Med. Sci. (KUGSMS) and Dept. Physiol., Kitasato Univ. Sch. Med. (KUSM) K Kamata, Dept. Nephrol., Kitasato Univ., Grad. Sch. Med. Sci.

H Sakamoto, Dept. Nephrol., Kitasato Univ. Grad. Sch. Med. Sci. S Hirono, Kitasato Univ. Sch. Pharm. Y Takei, Lab. Physiol., Atmosphere & Ocean Res. Inst., Univ. Tokyo

MJ Caplan, Dept. Cell. & Mol. Physiol., Yale Univ. Sch. Med. PS Aronson, Dept. Med. and Dept. Cell. & Mol. Physiol., Yale Univ. Sch. Med.

Speakers and Chairpersons

Y Yasuoka, Dept. Cell & Mol Physiol., Kitasato Univ. Grad. Sch. Med. Sci.

I Kazama, Dept. Physiol. I, Tohoku Univ. Grad. Sch. Med.

H Hasegawa, Dept. Nephrol. & Hypertension, Saitama Med. Cent., Saitama

Med. Univ.

H Nonoguchi, Div. Int. Med., Kitasato Univ. Med. Cent.

J Suzuki, Dept. Pharmacol., Grad. Sch. Med., Univ. Tokyo

Y Sohma, Dept. Physiol., Keio Univ. Sch. Med.

N Anzai, Dept. Pharmacol., Dokkyo Med. Univ.

H Endou, Prof., emeritus, Dept. Pharmacol., Kyorin Univ. Sch. Med.; President

& CEO, J-Phama Co., Ltd. <E-mail: [email protected]>

Secretary:

T Oshima, Dept. Physiol., Kitasato Univ. Sch. Med., Kitasato 1-15-1,

Minami-ku, Sagamihara 252-0374

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