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  • O i f HIF 1 T i th R l M d ll Att t d S lt S iti H t i i D hl S R tOverexpression of HIF-1 Transgene in the Renal Medulla Attenuated Salt Sensitive Hypertension in Dahl S RatsOverexpression of HIF-1 Transgene in the Renal Medulla Attenuated Salt Sensitive Hypertension in Dahl S RatsOverexpression of HIF 1 Transgene in the Renal Medulla Attenuated Salt Sensitive Hypertension in Dahl S RatsQing Zhu Zhengchao Wang Min Xia Pin Lan Li Fan Zhang Ningjun LiQing Zhu, Zhengchao Wang, Min Xia, Pin-Lan Li, Fan Zhang, Ningjun LiQ g , g g, , , g, gj

    D f Ph l d T i l M di l C ll f Vi i i Vi i i C l h U i i Ri h d VA 23298Department of Pharmacology and Toxicology Medical College of Virginia Virginia Commonwealth University Richmond VA 23298Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298p gy gy, g g , g y, ,

    In vivo transfection of HIF-1 expression plasmids into the renal medulla

    ABSTRACTABSTRACTIn vivo transfection of HIF-1 expression plasmids into the renal medulla. A 15ABSTRACTABSTRACT Animals were uninephroctemized one week before the experiment A medullary A HIF-1 15 LuciferaseABSTRACTABSTRACT Animals were uninephroctemized one week before the experiment. A medullary e HIF 1 LuciferaseABSTRACTABSTRACTinterstitial catheter (tapped tip 4 5mm) was implanted into the remaining left kidney c

    e

    12 HIF 1interstitial catheter (tapped tip, 4-5mm) was implanted into the remaining left kidney nc ) Actin 12 HIF-1and anchored into place on the kidney surface with Vetbond Tissue Adhesive A la 4h-Actin 200

    W h i l h th t HIF 1 di t d ti ti i thand anchored into place on the kidney surface with Vetbond Tissue Adhesive. A al 24200 HIF 1

    9We have previously shown that HIF-1-mediated gene activation in the mixture of HIF 1 expression plasmid (50g) in 600 L i i j tPEI transfection ba /2LS HS HS HIF 1 HS C Cl

    HIF-19p y g mixture of HIF-1 expression plasmid (50g) in 600 L in vivo-jetPEI transfection m

    kg

    /LS HS HS+HIF-1 HS+CoCl2 L if *renal medulla in response to high salt intake (HS) plays an important rolep p ( g) j

    t i f d i t th l d ll t d f 20 l/ i Aft i f i um /k2 160 Luciferase)

    4 6renal medulla in response to high salt intake (HS) plays an important role reagent was infused into the renal medulla at a speed of 20 l/min. After infusion, iu e/*e

    l 160wt)

    *4 6in the control of salt sensitivity of blood pressure The present study wasg p ,

    th th t t d bl k d b i f f t ti ith V tb d Ti od ol*ve n) kw *

    3in the control of salt sensitivity of blood pressure. The present study was the catheter was cut and blocked by a piece of fat tissue with Vetbond Tissue so m

    o

    * *ev tin k3

    3to test whether impairment of the renal medullary HIF 1 pathway isy p

    Adh i L if l id d t l Th iti f th th t y s mm * *le Act 120/g

    3 3to test whether impairment of the renal medullary HIF-1 pathway is Adhesive. Luciferase plasmids were used as control. The position of the catheters ly (m*n A 120n/3 3

    ibl f th lt iti h t i i D hl S (SS) t Bp p

    fi d h d f i A l d d (S i 2000 ai (ei -A

    min

    2 0responsible for the salt sensitive hypertension in Dahl S (SS) rats. By was confirmed at the end of experiments. An ultrasound transducer (Sonitron 2000, Da

    ote

    o

    80/m *2 0p yp ( ) y w s co ed e e d o e pe e s. u sou d sduce (So o 000,i h ) di l li d h kid i h 1 l d 10%

    Dro to 80l/

    20

    Western blot analysis, HS significantly increased renal medullary HIF-1 Rich-Mar) was directly applied onto the kidneys with a 1-MHz ultrasound at 10% p n t (2 Ctrl HS1 HS2 HS3

    Western blot analysis, HS significantly increased renal medullary HIF 1 Rich Mar) was directly applied onto the kidneys with a 1 MHz ultrasound at 10%

    on V (

    1Ctrl HS1 HS2 HS3levels in control (SS13BN) rats but not in DS rats indicating a defect in power output, for a total of 60 s with 30-s intervals on each side of the kidney in the 1 tio 40.V1levels in control (SS13BN) rats but not in DS rats, indicating a defect in power output, for a total of 60 s with 30 s intervals on each side of the kidney in the F-

    1 at 40U

    1 erenal medullary HIF 1 Renal medullary HIF 1 levels were middle and at the end of the infusion IF R U1

    35 L ifcerenal medullary HIF-1. Renal medullary HIF-1 levels were middle and at the end of the infusion. H (R

    0 35 Luciferasean

    y yd i DS t b ith t f ti f HIF 1 i

    H

    00 laoverexpressed in DS rats by either transfection of HIF-1 expression Ch i it i f t i l bl d i i t M t i l0

    30 HIF-1alp y p Chronic monitoring of arterial blood pressure in conscious rats. Mean arterial 30 HIF 1b )plasmid or chronic infusion of CoCl2 into the renal medulla which was

    g f p(MAP) d i t l t bl d di t 25m

    g)

    plasmid or chronic infusion of CoCl2 into the renal medulla, which was pressure (MAP) was measured using a telemetry blood pressure recording system s 14 *B 25um kgaccompanied by increased expressions of local anti hypertensive genesp ( ) g y p g y(D t S i I t ti l) At th d f h i MAP di th kid l

    s 14B20d

    iu e/accompanied by increased expressions of local anti-hypertensive genes, (Data Sciences International). At the end of chronic MAP recording, the kidneys ve 12 20od ole

    nitric o ide s nthase 2 and heme o genase 1 H pertension ind ced b( ) g, y

    ll d f h d i f HIF 1 l l 40ev 12

    so monitric oxide synthase-2 and heme oxygenase-1. Hypertension induced by were collected for the detection of HIF-1 level. 40 HIF 1 *

    le 10 15e s

    mm

    y yg yp y2 k HS i ifi l d i d i h HIF 1

    we e co ec ed o e de ec o o eve . HIF-1 *A 10 * 15ve m2-week HS was significantly attenuated in rats treated with HIF-1 wt)NA

    8 *10ti

    v (m *g y

    Measurement of pressure natriuresis in response to the elevations of renal LuciferasewRN 8

    10at *plasmid or CoCl2 The mean arterial pressure in these DS rats was 114 7 Measurement of pressure natriuresis in response to the elevations of renal 30uc e asekwm

    R 65u

    la *plasmid or CoCl2. The mean arterial pressure in these DS rats was 114.7 perfusion pressure (RPP) In additional groups of rats one week after HIF 1 gene 30g

    m 65m

    u

    2 7 mmHg on low salt intake 157 6 5 on HS 133 2 1 on HS perfusion pressure (RPP). In additional groups of rats, one week after HIF-1 gene30

    n/g

    -1 4 5

    um 2.7 mmHg on low salt intake, 157 6.5 on HS, 133 2.1 on HS transfection rats were surgically prepared for measurement of pressure natriuresis minO- 4

    0Cu

    ith HIF 1 l id d 135 2 8 HS ith C Cl ti ltransfection, rats were surgically prepared for measurement of pressure natriuresis mH

    O

    2 0Cwith HIF-1 plasmid and 135 2.8 on HS with CoCl2, respectively.g y p p p

    d ib d i l e/mH 2

    Ctrl HS1 HS2 HS3p 2, p y

    Th l h b l f l d ll HIFas described previously. 20ol

    e *0 Ctrl HS1 HS2 HS3These results suggest that an abnormal response of renal medullary HIF- p y 20mo0These results suggest that an abnormal response of renal medullary HIF mC s1 to HS in SS rats may represent a novel mechanism mediating salt- Measurement of urinary sodium excretion in response to acute sodium loading ( Fi 5 Eff t f l d ll HIF 1 t f ti diC 15ls1 to HS in SS rats may represent a novel mechanism mediating salt Measurement of urinary sodium excretion in response to acute sodium loading.

    V ( Figure 5. Effect of renal medullary HIF-1 transfection on sodium15 *vesensitive hypertension and that induction of HIF 1 expression in the Additional animals as above were surgically prepared for measurement of arterial 10.V

    g yb l f hi h l l d Th hi h l i d d i i l b le

    vsensitive hypertension and that induction of HIF-1 expression in the Additional animals as above were surgically prepared for measurement of arterial 10a. balance after high salt load. The high salt-induced positive salt balancele

    l d ll ld b th ti h f th t t t f lt pressure and urine flow After 2 hour equilibration and two 10 minute control period UN

    b ce e g s o d. e g sa duced pos ve sa ba a ce10 *A

    renal medulla could be a therapeutic approach for the treatment of salt- pressure and urine flow. After 2-hour equilibration and two 10-minute control period U was significantly attenuated in HIF-1-transfected rats compared to the10 *NAp pp

    i i h i ( NIH HL89563) sample collections a 5% body weight isotonic saline load was administeredwas significantly attenuated in HIF 1 transfected rats compared to the

    RN

    sensitive hypertension (support: NIH grant HL89563). sample collections, a 5% body weight isotonic saline load was administered 0 control rats indicating that overexpression of HIF-1 transgenemRsensitive hypertension (support: NIH grant HL89563).

    intravenously and three 10 minute samples were collected over 30 minutes and then 0control rats, indicating that overexpression of HIF-1 transgenemintravenously and three 10-minute samples were collected over 30 minutes, and then

    60 80 100 120 140 160 180 attenuated sodium retention These data suggest that defect of HIF 1 in52

    3 more 10 minute postcontrol samples were taken 60 80 100 120 140 160 180attenuated sodium retention. These data suggest that defect of HIF-1 in5

    X-23 more 10-minute postcontrol samples were taken. 60 80 100 120 140 160 180

    th l d ll b ibl f th i i d l lt h dliOXp p

    RPP (mmHg) the renal medulla may be responsible for the impaired renal salt handlingOBACKGROUNDBACKGROUND RPP (mmHg)y p p g

    i D hl S HS1 2 d 3 d HS * P 0 05 l ( 3)0CBACKGROUNDBACKGROUND Measurement of Daily Sodium Balance Additional animals as above were housed in Dahl S rats. HS1, 2 and 3 = days on HS. * P< 0.05 vs. control. (n =3)0CBACKGROUNDBACKGROUND