Adaptation of Kidney Medulla to Hypertonicity Function of

Adaptation of Kidney Medulla to Hypertonicity : Function of Ton. EBP/NFAT 5 transcriptional activator 분당서울대학교병원 내과 나기영

- Introduction - Hypertonic stress - Role of Ton. EBP in protection against hypertonicity - Ton. EBP as a differentiation factor of renal medulla - Structure of Ton. EBP - Ton. EBP regulation by hypertonicity in MDCK cells - Ton. EBP regulation in rat kidney - Loss-of-function study of Ton. EBP using RNAi - Ton. EBP function in non-renal tissues - Future directions

Functions of kidney 1. Excretion of the waste products of metabolism 2. Water and electrolyte balance by water reabsorption and urinary concentration 3. Acid-base balance 4. Erythropoietin – RBC production 5. Calcium, phosphate and bone metabolism 6. Hormone secretion (renin, angiotensin II, PG, NO. . )

Outer Medulla Cortex Outer Stripe Inner Medulla 1 = Renal corpuscle (Bowman’s capsule & glomerulus) 2 = Proximal convoluted tubule (PCT) 3 = Proximal straight tubule 4 = Descending thin limb 5 = Ascending thin limb 6 = Thick ascending limb of Henle loop (TAL) 7 = Macula densa located within the final portion of the TAL 8 = Distal convoluted tubule (DCT) 9 = Connecting tubule 9* = Connecting tubule of the juxtamedullary nephron that forms an arcade 10 = Cortical collecting duct 11 = Outer medullary collecting duct 12 = Inner medullary collecting duct

Medullary hyperosmolality Driving force of water reabsorption and urinary concentration

Solutes in the papilla of rat kidney Organic Osmolytes Compatible Osmolytes (Beck et al, Kid Int 25: 397 -403, 1984)

Role of transporters of compatible osmolytes in cell volume regulation H 2 O Compatible osmolytes 70 m. M K+ 140 m. M Compatible osmolytes 280 m. M K+ K+ Cl- Minutes Hours, days Cl- Seconds 140 m. M K+ KCl 280 m. M K+ K+ Hours, days H 2 O Minutes Seconds 70 m. M K+ 140 m. M Compatible osmolytes 140 m. M K+ 280 m. M Compatible osmolytes Myo-inositol Betaine Taurine H 2 O 140 m. M Na. Cl 280 m. M Na. Cl (Kwon and Handler, Curr Opin Cell Biol 7: 465 -7, 1995)

Why is hypertonicity (high salt) stressful to cells? hypertonicity (high salt) cell ionic strength Perturbation of protein cell death double stranded DNA breaks ATM, ATR DNA-PK p 53 GADD 45/153 Cell cycle arrest DNA repair

Compatible osmolytes in renal medulla myo-inositol - Na+/myo-inositol cotransporter (SMIT) betaine - Na+/Cl-/betaine cotransporter (BGT 1) taurine - Na+/Cl-/taurine cotransporter sorbitol - aldose reductase (AR) glycerophosphorylcholine

Role of transcription in cellular accumulation of compatible osmolytes glucose sorbitol AR Myo-inositol SMIT betaine BGT 1 Ton. EBP Hypertonicity

Ton. EBP : tonicity-responsive enhancer binding protein (NFAT 5 : nuclear factor of activated T cell 5) Hypertonicity Q Ton. EBP Q Q Q SMIT, BGT 1, AR Ton. E

Role of Ton. EBP in protection against high salt and high urea in the renal inner medulla Hypertonicity SMIT, BGT 1, AR Ton. EBP UT-A compatible osmolytes HSP 70 High urea protection from high ionic strength protection from urea Cell death

AQP 1(-/-) mice - Compromised hyperosmolality of renal medulla - Countercurrent multiplier knockout mice - Expression of UT-A and AQP 4 : ↓ - Expression of AQP 3 and ENa. C-γ : normal Hypertonicity : local signal of differentiation of renal medulla Ton. EBP : differentiation factor of renal medulla

Structure of Rel family transcription factor AD 1 Ton. EBP MD 2 MD 1 RHD Q AD NFAT NF B AD 3 AD 2 RHD p 50 RHD p 65 RHD AD CBP/p 300 AD: activation domain, MD: modulation domain, RHD: Rel homology domain NFқB: nuclear factor қ chain transcription in B cells, CBP/p 300: co-activator (Lee et al, J Biol Chem 48: 47571 -7, 2003)

Crystal structure of RHD of Ton. EBP (Stroud et al, Nature Struct Biol 9: 90 -94, 2002)

Hypertonicity signaling to Ton. EBP Hypertonicity phosphorylation nuclear translocation transactivation induction Q Ton. EBP Q Q Q SMIT, BGT 1, AR UT-A, HSP 70 Ton. E

Bidirectional nature of Ton. EBP Effect of hypertonicity Effect of hypotonicity (Woo et al, Am J Physiol Renal Physiol 278: F 1006 -12, 2000)

Nucleocytoplasmic trafficking of Ton. EBP in ambient tonicity 130 335 500 (mosmol/kg. H 2 O) (Lee et al, Biochem Biophys Res Commun 294: 968 -75, 2002)

- Induction : maximum in 12 hr - Nuclear translocation : complete in 30 min - Phosphorylation : 30 min and on Changes in Ton. EBP have been studied focusing on early changes Long-term regulation of Ton. EBP mimicking kidney in situ - Confluent monolayer of MDCK cells - Long-term exposure to hypertonicity in combination with urea - Effects of betaine loading

Long-term Regulation of Ton. EBP in MDCK cells Isotonic 440 m. M Na. Cl 600 m. M Urea 5 m. M Betaine

Ton. EBP distribution in rat kidneys

Ton. EBP distribution in rat kidneys water loaded dehydrated Inner stripe of outer medulla Initial portion of inner medulla Uosm: 591 mosm/kg 3, 613 mosm/kg (Cha et al J Am Soc Nephrol 12: 2221 -30, 2001)

Gene silencing by RNA interference (Stevenson M, Nat Rev Immunol 3: 851 -8, 2003)

Small interfering (si. RNA) duplexes targeted for human Ton. EBP

Silencing of Ton. EBP in He. La cells

Dose-dependence of Ton. EBP silencing

Silencing of Ton. EBP by si. RNA duplexes is transitory. HSC 70 9 R 56 inv H 9 R I 56 inv 9 R H 56 inv I 9 R H 14 day 56 9 inv R 7 day 56 inv 9 R I 56 56 inv Ton. EBP inv H 9 R I 3 day 56 9 inv R 2 day

Relative luciferase activity Ton. EBP silencing reduces Ton. E-driven transcription. 100 80 60 40 20 0 inv 569 R isotonic inv 569 R hypertonic 0 inv 279 R isotonic inv 279 R hypertonic

Ton. EBP silencing reduces m. RNA expression of AR and SMIT AR m. RNA SMIT m. RNA Calcyclin m. RNA G 3 PDH m. RNA 56 9 R hypertonic inv 56 inv 9 R isotonic

Ton. EBP expression in mouse embryo (Maouyo et al, Am J Renal Physiol 282: F 802 -9, 2002)

Ton. EBP function in non-renal tissues Ton. EBP in thymus - induction by T cell receptor activation - required for T cell proliferation/survival - stimulates TNF and lymphotoxin AR in diabetic nephropathy - diffuse positive reactions for AR in glomerular mesangium Ton. EBP in cancer - In epithelia, Ton. EBP is induced by 6 4 integrin and promotes carcinoma invasion.

Future directions - Signaling pathway to Ton. EBP - Local signals for the changes in nucleocytoplasmic distribution of renal medullary Ton. EBP Development of cell culture model mimicking Ton. EBP regulation of kidney in situ Biological functions of Ton. EBP in non-renal tissues Loss-of-function study of Ton. EBP using RNA interference