OifHIFOverexpression of HIF-1 T i th R l M d ll Att t d S ...pli/Presentation/EB2010/EB2010-HIF...
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O i f HIF 1 T i th R lM d ll Att t d S lt S iti H t i i D hl S R t Overexpression of HIF-1α Transgene in the Renal Medulla Attenuated Salt Sensitive Hypertension in Dahl S Rats Overexpression of HIF-1α Transgene in the Renal Medulla Attenuated Salt Sensitive Hypertension in Dahl S Rats Overexpression of HIF 1α Transgene in the Renal Medulla Attenuated Salt Sensitive Hypertension in Dahl S Rats Qing Zhu Zhengchao Wang Min Xia Pin Lan Li Fan Zhang Ningjun Li Qing Zhu, Zhengchao Wang, Min Xia, Pin-Lan Li, Fan Zhang, Ningjun Li D f Ph l dT i l M di lC ll f Vi ii Vi ii C lhUi i Ri h d VA 23298 Department of Pharmacology and Toxicology Medical College of Virginia Virginia Commonwealth University Richmond VA23298 Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298 In vivo transfection of HIF-1α expression plasmids into the renal medulla ABSTRACT ABSTRACT In vivo transfection of HIF-1α expression plasmids into the renal medulla. A 15 ABSTRACT ABSTRACT Animals were uninephroctemized one week before the experiment A medullary A HIF-1α 15 Luciferase ABSTRACT ABSTRACT Animals were uninephroctemized one week before the experiment. A medullary e HIF 1α Luciferase ABSTRACT ABSTRACT interstitial catheter (tapped tip 4 5mm) was implanted into the remaining left kidney ce 12 HIF 1 interstitial catheter (tapped tip, 4-5mm) was implanted into the remaining left kidney nc ) β Actin 12 HIF-1α and anchored into place on the kidney surface with Vetbond Tissue Adhesive A la 4h β-Actin 200 W h i l h th t HIF 1 di t d ti ti i th and anchored into place on the kidney surface with Vetbond Tissue Adhesive. A al 24 200 HIF 1α 9 We have previously shown that HIF-1α-mediated gene activation in the mixture of HIF 1α expression plasmid (50μg) in 600 μL i i j tPEI transfection ba /2 LS HS HS HIF 1 HS C Cl HIF-1α 9 mixture of HIF-1α expression plasmid (50μg) in 600 μL in vivo-jetPEI transfection m kg/ LS HS HS+HIF-1α HS+CoCl 2 L if * renal medulla in response to high salt intake (HS) plays an important role t if d it th l d ll t d f 20 l/ i Aft if i um /k 2 160 Luciferase ) 4 6 renal medulla in response to high salt intake (HS) plays an important role reagent was infused into the renal medulla at a speed of 20 μl/min. After infusion, iu e/ * el 160 wt) * 4 6 in the control of salt sensitivity of blood pressure The present study was th th t t d bl kd b i f ft ti ith V tb d Ti od ol * ve n) kw * 3 in 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 mo * * ev tin k 3 3 to test whether impairment of the renal medullary HIF 1α pathway is Adh i L if l id d t l Th iti f th th t y s mm * * le Act 120 /g 3 3 to test whether impairment of the renal medullary HIF-1α pathway is Adhesive. Luciferase plasmids were used as control. The position of the catheters ly (m * n A 120 n/ 3 3 ibl f th lt iti h t i i D hl S (SS) t B fi d h d f i A l d d (S i 2000 ai ( ei β-A min 2 0 responsible 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 β 80 /m * 2 0 ih ) di l li d h kid ih 1 l d 10% D ro to 80 μl/ 2 Western blot analysis, HS significantly increased renal medullary HIF-1α Rich-Mar) was directly applied onto the kidneys with a 1-MHz ultrasound at 10% p n t (μ 2 Ctrl HS1 HS2 HS3 Western blot analysis, HS significantly increased renal medullary HIF 1α Rich Mar) was directly applied onto the kidneys with a 1 MHz ultrasound at 10% α on V ( 1 Ctrl HS1 HS2 HS3 levels 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 1α tio 40 .V 1 levels 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 40 U 1 e renal medullary HIF 1α Renal medullary HIF 1α levels were middle and at the end of the infusion IF R U 1 35 L if ce renal medullary HIF-1α. Renal medullary HIF-1α levels were middle and at the end of the infusion. H (R 0 35 Luciferase an d i DS t b ith t f ti f HIF 1 i H 0 0 la overexpressed in DS rats by either transfection of HIF-1α expression Ch i it i f t il bl d i i t M t il 0 30 HIF-1α al Chronic monitoring of arterial blood pressure in conscious rats. Mean arterial 30 HIF 1α b ) plasmid or chronic infusion of CoCl 2 into the renal medulla which was (MAP) d i tl t bl d di t 25 m g) plasmid or chronic infusion of CoCl 2 into the renal medulla, which was pressure (MAP) was measured using a telemetry blood pressure recording system s 14 * B 25 um kg accompanied by increased expressions of local anti hypertensive genes (D t Si It ti l) At th d f h i MAP di th kid ls 14 B 20 diu e/ accompanied by increased expressions of local anti-hypertensive genes, (Data Sciences International). At the end of chronic MAP recording, the kidneys ve 12 20 od ole nitric o ide s nthase 2 and heme o genase 1 H pertension ind ced b ll d f h d i f HIF 1 l l 40 ev 12 so mo nitric oxide synthase-2 and heme oxygenase-1. Hypertension induced by were collected for the detection of HIF-1α level. 40 HIF 1 * le 10 15 e s mm 2 k HS i ifi l d i d ih HIF 1 HIF-1α * A 10 * 15 ve m 2-week HS was significantly attenuated in rats treated with HIF-1α wt) NA 8 * 10 tiv (m * Measurement of pressure natriuresis in response to the elevations of renal Luciferase w RN 8 10 at * plasmid or CoCl 2 The mean arterial pressure in these DS rats was 114 7 Measurement of pressure natriuresis in response to the elevations of renal 30 kw mR 6 5 ula * plasmid or CoCl 2 . The mean arterial pressure in these DS rats was 114.7 perfusion pressure (RPP) In additional groups of rats one week after HIF 1α gene 30 g m 6 5 mu ± 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-1α gene 30 n/g -1 4 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 min O- 4 0 Cu ith HIF 1 l id d 135 ± 2 8 HS ith C Cl ti l transfection, rats were surgically prepared for measurement of pressure natriuresis m HO 2 0 C with HIF-1α plasmid and 135 ± 2.8 on HS with CoCl 2 , respectively . d ib d i l e/m H 2 Ctrl HS1 HS2 HS3 2 Th l h b l f l d ll HIF as described previously . 20 ole * 0 Ctrl HS1 HS2 HS3 These results suggest that an abnormal response of renal medullary HIF- 20 mo 0 m C s 1α to HS in SS rats may represent a novel mechanism mediating salt- Measurement of urinary sodium excretion in response to acute sodium loading (μ Fi 5 Eff t f l d ll HIF 1 t f ti di C 15 ls 1α 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-1α transfection on sodium 15 * ve sensitive hypertension and that induction of HIF 1α expression in the Additional animals as above were surgically prepared for measurement of arterial 10 .V bl f hi h l l d Th hi h l id d ii l bl ev sensitive hypertension and that induction of HIF-1α expression in the Additional animals as above were surgically prepared for measurement of arterial 10 a. balance after high salt load. The high salt-induced positive salt balance le 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 10 * 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-1α-transfected rats compared to the 10 * NA ii h i ( NIH HL89563) sample collections a 5% body weight isotonic saline load was administered was significantly attenuated in HIF 1α 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-1α transgene mR sensitive hypertension (support: NIH grant HL89563). intravenously and three 10 minute samples were collected over 30 minutes and then 0 control rats, indicating that overexpression of HIF-1α transgene m intravenously 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 1α in 5 2 3 more 10 minute postcontrol samples were taken 60 80 100 120 140 160 180 attenuated sodium retention. These data suggest that defect of HIF-1α in 5 X-2 3 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 dli OX RPP (mmHg) the renal medulla may be responsible for the impaired renal salt handling O BACKGROUND BACKGROUND RPP (mmHg) i D hl S HS1 2 d 3 d HS * P 0 05 l ( 3) 0 C BACKGROUND BACKGROUND 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) 0 C BACKGROUND BACKGROUND Measurement of Daily Sodium Balance. Additional animals as above were housed LS HS HS+HIF1a HS+CoCl2 LS HS HS+HIF1α HS+CoCl in metabolic cages and daily indexes of sodium balance were computed by LS HS HS+HIF1a HS+CoCl2 LS HS HS+HIF1α HS+CoCl 2 Fig re 3 Effect of renal med llar HIF 1α gene tranfection into in metabolic cages and daily indexes of sodium balance were computed by Figure 3. Effect of renal medullary HIF-1α gene tranfection into subtracting urinary sodium excretion from total sodium intake After 1 day of Figure 2 Effect of HIF-1α gene transfection into the renal medulla on th l d ll ti i Th i fl d W h i l h th t h i id ibl f t (HIF) 1 subtracting urinary sodium excretion from total sodium intake. After 1 day of Figure 2. Effect of HIF-1α gene transfection into the renal medulla on 170 HIF-1a HS HIF 1α HS the renal medulla on pressure natriuresis. The urine flow and We have previously shown that hypoxia-inducible factor (HIF)-1α, a control measurements the animals were switched from tap water to 1% NaCl water * levels of its target genes in Dahl S rats A: Western blot analysis showing HIF-1a HS HIF-1α HS t i ti f t i b d tl d i th l d ll d hi h control measurements, the animals were switched from tap water to 1% NaCl water, * * levels of its target genes in Dahl S rats. A: Western blot analysis showing 160 Luci HS Ctrl HS sodium excretion rate in response to the elevation of renal perfusion transcription factor, is abundantly expressed in the renal medulla and high and experimental measurements were continued for 3 days * * the overexpression of HIF 1α in the renal medulla by either transfection of 160 L i LS Ctrl LS sodium excretion rate in response to the elevation of renal perfusion lt itk lt l d ll HIF 1 l l I t hi h and experimental measurements were continued for 3 days. * * the overexpression of HIF-1α in the renal medulla by either transfection of Luci LS Ctrl LS pressure (RPP) were significantly reinforced in HIF-1α-transfected salt intake upregulates renal medullary HIF-1α levels. In response to high * * HIF 1α expression plasmid or chronic infusion of CoCl into the renal 150 CoCl2 HS CoCl 2 HS pressure (RPP) were significantly reinforced in HIF-1α-transfected l h ll HIF 1 di h i i f ii * HIF-1α expression plasmid or chronic infusion of CoCl 2 into the renal 150 CoCl2 HS CoCl 2 HS group compared with the control group indicating that overexpression salt challenge, HIF-1α mediates the activation of oxygen-sensitive genes Western blot analysis of HIF-1α levels. Nuclear extraction from renal medullary * medulla B and C: The mRNA levels of HIF 1α target genes heme 140 g) group compared with the control group, indicating that overexpression h ii id h l 2 d h 1 i Western blot analysis of HIF 1α levels. Nuclear extraction from renal medullary * * medulla. B and C: The mRNA levels of HIF-1α target genes heme 140 Hg f HIF 1 t i d l d ll f ti Th dt such as nitric oxide synthase, cyclooxygenase-2 and heme oxygenase-1 in tissue was subjected to Western blot using anti-HIF-1α antibody (monoclonal * (HO) 1 d l (COX) 2 i th l d ll b Q * mH of HIF-1α transgene improved renal medullary function. These data such as nitric oxide synthase, cyclooxygenase 2 and heme oxygenase 1 in tissue was subjected to Western blot using anti HIF 1α antibody (monoclonal, oxygenase (HO)-1 and cyclooxygenase (COX)-2 in the renal medulla by Q- 130 m h df f HIF 1 i h l d ll b ibl the renal medulla, which plays an important role in the control of renal Novus) RT PCR l i Th lt d t td fl i f 130 mm suggest that defect of HIF-1α in the renal medulla may be responsible the renal medulla, which plays an important role in the control of renal Novus) RT PCR analysis. These results demonstrated a successful overexpression of (m mdeullary functions and salt sensitivity of arterial pressure HIF 1 d ti ti f it t t i th l d ll i D hl S t 120 P for the impaired renal medullary function in Dahl S rats. * P<0.05 vs. mdeullary functions and salt sensitivity of arterial pressure. HIF-1α and activation of its target genes in the renal medulla in Dahl S rats. 120 AP for the impaired renal medullary function in Dahl S rats. P 0.05 vs. * P 0 05 h LS d HS ( 3) MA control rats (n=3) It has been reported that there is a defect in nitric oxide synthase (NOS)-2 * P < 0.05 vs. the LS and HS. (n = 3) 110 M control rats. (n 3) It has been reported that there is a defect in nitric oxide synthase (NOS) 2, one of the HIF-1α target genes and that the activations of NOS-2 by a RESULTS RESULTS 100 one of the HIF 1α target genes, and that the activations of NOS 2 by a RESULTS RESULTS 100 high-salt diet and angiotensin II are diminished in the renal medulla in RESULTS RESULTS high-salt diet and angiotensin II are diminished in the renal medulla in 90 Dahl salt-sensitive hypertensive rats indicating a possible impairment in 90 2 1 3 5 7 9 11 13 15 Dahl salt-sensitive hypertensive rats, indicating a possible impairment in 1 3 5 7 9 11 13 15 -2 1 3 5 7 9 11 13 15 renal medullary HIF 1α in this rat strain Days on high salt diet HIF 1α renal medullary HIF-1α in this rat strain. 20 * A kDa Days on high salt diet 80 HIF-1α 20 HIF-1α * A kDa 80 f The present study was to test the hypothesis that an impairment of the HIF 1α * HIF-1α - 110 Luciferase The present study was to test the hypothesis that an impairment of the Luciferase - 110 Luciferase renal medullary HIF 1α pathway is responsible for the salt sensitive Luciferase ) Figure 6 Effect of renal medullary HIF-1α gene transfection on * β Actin 46 renal medullary HIF-1α pathway is responsible for the salt sensitive 16 wt) Figure 6. Effect of renal medullary HIF-1α gene transfection on * β- Actin - 46 60 hypertension in Dahl S rats t) 16 kw arterial blood pressure in Dahl S rats The mean arterial pressure * 60 hypertension in Dahl S rats. wt k arterial blood pressure in Dahl S rats. The mean arterial pressure kw /g (MAP) was significantly increased after 2-week high salt intake LS HS LS HS We transfected HIF 1α plasmids into the renal medulla and then detect the g 12 n/ (MAP) was significantly increased after 2-week high salt intake. SS13BN SS We transfected HIF-1α plasmids into the renal medulla and then detect the n/g 12 min However high slat-induced hypertension was significantly attenuated in SS13BN SS 40 renal sodi m e cretion and arterial press re after high salt challenge Or in /m However, high slat-induced hypertension was significantly attenuated in B 40 renal sodium excretion and arterial pressure after high salt challenge. Our m e/ rats treated with HIF 1α gene transfection or renal medullary infusion of B 30 S S 40 dt h d th t f l d ll HIF 1 l l td l/m 8 ole rats treated with HIF-1α gene transfection or renal medullary infusion of B 3.0 LS HS data showed that recovery of renal medullary HIF-1α levels promoted μ 8 mo CoCl a HIF 1α inducer indicating that the defect of HIF 1α pathway di ti d tt td h t i ft di l di i V ( 8 μm CoCl 2 , a HIF-1α inducer, indicating that the defect of HIF-1α pathway 24 y sodium excretion and attenuated hypertension after sodium loading in V (μ in the renal medulla may be responsible for the salt sensitive 2.4 sit n) 20 D hl S t U. V ( in the renal medulla may be responsible for the salt-sensitive ns tin 20 Dahl S rats. U 4 .V hypertension in Dahl S rats * P< 0 05 vs other groups (n = 4 5) te Act 4 Na hypertension in Dahl S rats. . * P< 0.05 vs. other groups. (n = 4-5) 18 Int - A UN 1.8 d I β - U ed to 0 METHODS METHODS 0 12 iz o t * 0 METHODS METHODS 0 1.2 al tio C1 C2 S1 S2 S3 P1 P2 P3 METHODS METHODS m Rat C1 C2 S1 S2 S3 P1 P2 P3 C C1 C2 S1 S2 S3 P1 P2 P3 or ( R Conclusion Conclusion C1 C2 S1 S2 S3 P1 P2 P3 0.6 No ( Conclusion Conclusion Animals Male Dahl S and SS 13BN rats (Charles River Laboratories 0.6 N Conclusion Conclusion Animals. Male Dahl S and SS-13BN rats (Charles River Laboratories, Wil i t MA ) 300 350 d SS 13BN t i i 00 Wilmington, MA ), 300-350 g, were used. SS-13BN rat is a consomic 0.0 SS13BN SS Decreased Decreased level level and and abnormal abnormal response response to to high high salt salt intake intake Fi 4 Eff t f l d ll HIF 1 t f ti i di ti i t t di l di i D hl S t subcolony of Dahl S rats with substitution of chromosome 13 from SS13BN SS Decreased Decreased level level and and abnormal abnormal response response to to high high salt salt intake intake Figure 4. Effects of renal medullary HIF-1α gene transfection on urinary sodium excretion in response to acute sodium loading in Dahl S rats. subcolony of Dahl S rats with substitution of chromosome 13 from Fi 1 C i f hi h lt id d h i HIF 1 l l i th l in in renal renal medullary medullary HIF HIF 1α α may may represent represent a novel novel Brown Norway (BN) rats This rat strain exhibits minimal differences in Figure 1. Comparison of high salt-induced changes in HIF-1α levels in the renal in in renal renal medullary medullary HIF HIF-1α α may may represent represent a novel novel To further evaluate the impact of renal medullary HIF-1α transfection on renal salt handling, we examined the sodium excretion after acute sodium Brown Norway (BN) rats. This rat strain exhibits minimal differences in d ll bt D hl lt iti d SS13BN t A R i l mechanism mechanism mediating mediating salt salt sensitive sensitive hypertension hypertension in in DS DS rats rats To further evaluate the impact of renal medullary HIF 1α transfection on renal salt handling, we examined the sodium excretion after acute sodium genotype from Dahl S rats (1 95% in differences) which is much smaller medulla between Dahl salt-sensitive and SS13BN rats. A, Representative gel mechanism mechanism mediating mediating salt salt-sensitive sensitive hypertension hypertension in in DS DS rats rats. loading Acute sodium loading dramatically increased urine volume (U·V) and urinary sodium excretion (U ·V) These increases in U·V and U ·V genotype from Dahl S rats (1.95% in differences), which is much smaller loading. Acute sodium loading dramatically increased urine volume (U V) and urinary sodium excretion (U Na V). These increases in UV and U Na V th th diff bt D hl S t d th l d documents of Western blot analyses B Summarized intensities of the HIF-1α blots Id ti Id ti f HIF HIF 1 i i th th l d ll d ll ld ld were considerably enhanced in HIF 1α transfected rats These results demonstrated that overexpression of HIF 1α transgene in the renal medulla than the differences between Dahl S rats and other commonly used documents of Western blot analyses. B, Summarized intensities of the HIF 1α blots. Induction Induction of of HIF HIF-1α α expression expression in in the the renal renal medulla medulla could could were considerably enhanced in HIF-1α-transfected rats. These results demonstrated that overexpression of HIF-1α transgene in the renal medulla High salt induced a significant increase in HIF-1α in SS-13BN rats which was not b th ti th ti h f th th t t t t t t f lt lt k bl i d th bilit f th kid t t di l d i D hl S t Th dt dditi ll t th t d d l "control" strains: BN 77%, ACI 57%, Sprague-Dawley 52%, Dahl-R High salt induced a significant increase in HIF-1α in SS-13BN rats, which was not be be a therapeutic therapeutic approach approach for for the the treatment treatment of of salt salt- remarkably improved the capability of the kidneys to remove extra sodium load in Dahl S rats. These data additionally suggest that decreased renal control strains: BN 77%, ACI 57%, Sprague Dawley 52%, Dahl R observed in Dahl S rats indicating there was a defect of HIF 1α pathway in Dahl S iti iti h t i h t i d ll HIF 1 b ibl f th i i d l ti f di ti i D hl S t * P 0 05 t l t ( 3) 30% Therefore SS-13BN rat is considered as one of the best control observed in Dahl S rats, indicating there was a defect of HIF-1α pathway in Dahl S Fig 4 sensitive sensitive hypertension hypertension. medullary HIF-1α may be responsible for the impaired regulation of sodium excretion in Dahl S rats. * P < 0.05 vs. control rats. (n =3) 30%. Therefore, SS-13BN rat is considered as one of the best control t *P<0 05 th ( 6) LS l lt HS hi h lt SS D hl lt iti Fig 4 (normotensive) rat strains for Dahl S rats rats. *P<0.05 vs others (n=6). LS, low salt; HS, high salt; SS, Dahl salt-sensitive. (normotensive) rat strains for Dahl S rats.
Transcript of OifHIFOverexpression of HIF-1 T i th R l M d ll Att t d S ...pli/Presentation/EB2010/EB2010-HIF...
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-1α Transgene in the Renal Medulla Attenuated Salt Sensitive Hypertension in Dahl S RatsOverexpression of HIF-1α Transgene in the Renal Medulla Attenuated Salt Sensitive Hypertension in Dahl S RatsOverexpression of HIF 1α 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-1α expression plasmids into the renal medulla
ABSTRACTABSTRACTIn vivo transfection of HIF-1α expression plasmids into the renal medulla. A 15ABSTRACTABSTRACT Animals were uninephroctemized one week before the experiment A medullary A HIF-1α 15 LuciferaseABSTRACTABSTRACT Animals were uninephroctemized one week before the experiment. A medullary e
HIF 1αLuciferaseABSTRACTABSTRACT
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was significantly attenuated in HIF 1α 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-1α transgenemRsensitive hypertension (support: NIH grant HL89563).
intravenously and three 10 minute samples were collected over 30 minutes and then 0 control rats, indicating that overexpression of HIF-1α transgenemintravenously and three 10-minute samples were collected over 30 minutes, and then60 80 100 120 140 160 180 attenuated sodium retention These data suggest that defect of HIF 1α in52
3 more 10 minute postcontrol samples were taken 60 80 100 120 140 160 180 attenuated sodium retention. These data suggest that defect of HIF-1α 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 handlingO
BACKGROUNDBACKGROUND RPP (mmHg) y p p gi 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 Measurement of Daily Sodium Balance. Additional animals as above were housed a S ats. S , a d 3 days o S. 0.05 vs. co t o . ( 3)
LS HS HS+HIF1a HS+CoCl2LS HS HS+HIF1α HS+CoClin metabolic cages and daily indexes of sodium balance were computed by LS HS HS+HIF1a HS+CoCl2LS HS HS+HIF1α HS+CoCl2Fig re 3 Effect of renal med llar HIF 1α gene tranfection into
in metabolic cages and daily indexes of sodium balance were computed byFigure 3. Effect of renal medullary HIF-1α gene tranfection intosubtracting urinary sodium excretion from total sodium intake After 1 day of Figure 2 Effect of HIF-1α gene transfection into the renal medulla on g y gth l d ll t i i Th i fl dW h i l h th t h i i d ibl f t (HIF) 1
subtracting urinary sodium excretion from total sodium intake. After 1 day of Figure 2. Effect of HIF-1α gene transfection into the renal medulla on170 HIF-1a HSHIF 1α HSthe renal medulla on pressure natriuresis. The urine flow andWe have previously shown that hypoxia-inducible factor (HIF)-1α, a control measurements the animals were switched from tap water to 1% NaCl water *levels of its target genes in Dahl S rats A: Western blot analysis showing HIF-1a HSHIF-1α HSpp y yp ( ) ,
t i ti f t i b d tl d i th l d ll d hi hcontrol measurements, the animals were switched from tap water to 1% NaCl water,
* *levels of its target genes in Dahl S rats. A: Western blot analysis showing160 Luci HSCtrl HSsodium excretion rate in response to the elevation of renal perfusiontranscription factor, is abundantly expressed in the renal medulla and high and experimental measurements were continued for 3 days * *the overexpression of HIF 1α in the renal medulla by either transfection of 160
L i LSCtrl LSsodium excretion rate in response to the elevation of renal perfusionp , y p g
lt i t k l t l d ll HIF 1 l l I t hi hand experimental measurements were continued for 3 days.
**the overexpression of HIF-1α in the renal medulla by either transfection of
Luci LS Ctrl LSpressure (RPP) were significantly reinforced in HIF-1α-transfectedsalt intake upregulates renal medullary HIF-1α levels. In response to high * *HIF 1α expression plasmid or chronic infusion of CoCl into the renal 150 CoCl2 HSCoCl2 HSpressure (RPP) were significantly reinforced in HIF-1α-transfectedp g y p g
l h ll HIF 1 di h i i f i i*HIF-1α expression plasmid or chronic infusion of CoCl2 into the renal 150 CoCl2 HSCoCl2 HS
group compared with the control group indicating that overexpressionsalt challenge, HIF-1α mediates the activation of oxygen-sensitive genes Western blot analysis of HIF-1α levels. Nuclear extraction from renal medullary *medulla B and C: The mRNA levels of HIF 1α target genes heme140g)
group compared with the control group, indicating that overexpressiong , yg gh i i id h l 2 d h 1 i
Western blot analysis of HIF 1α levels. Nuclear extraction from renal medullary**medulla. B and C: The mRNA levels of HIF-1α target genes heme
140Hg
f HIF 1 t i d l d ll f ti Th d tsuch as nitric oxide synthase, cyclooxygenase-2 and heme oxygenase-1 in tissue was subjected to Western blot using anti-HIF-1α antibody (monoclonal *g g
(HO) 1 d l (COX) 2 i th l d ll b Q *
mHof HIF-1α transgene improved renal medullary function. These datasuch as nitric oxide synthase, cyclooxygenase 2 and heme oxygenase 1 in tissue was subjected to Western blot using anti HIF 1α antibody (monoclonal, oxygenase (HO)-1 and cyclooxygenase (COX)-2 in the renal medulla by Q-
130m
g p yh d f f HIF 1 i h l d ll b iblthe renal medulla, which plays an important role in the control of renal Novus)
yg ( ) y yg ( ) y QRT PCR l i Th lt d t t d f l i f 130m
msuggest that defect of HIF-1α in the renal medulla may be responsiblethe renal medulla, which plays an important role in the control of renal Novus) RT PCR analysis. These results demonstrated a successful overexpression of
(mgg y pmdeullary functions and salt sensitivity of arterial pressure
y pHIF 1 d ti ti f it t t i th l d ll i D hl S t 120P for the impaired renal medullary function in Dahl S rats. * P<0.05 vs.mdeullary functions and salt sensitivity of arterial pressure. HIF-1α and activation of its target genes in the renal medulla in Dahl S rats. 120
APfor the impaired renal medullary function in Dahl S rats. P 0.05 vs.g g
* P 0 05 h LS d HS ( 3) MAcontrol rats (n=3)It has been reported that there is a defect in nitric oxide synthase (NOS)-2 * P < 0.05 vs. the LS and HS. (n = 3)
110Mcontrol rats. (n 3)It has been reported that there is a defect in nitric oxide synthase (NOS) 2, ( )one of the HIF-1α target genes and that the activations of NOS-2 by a RESULTSRESULTS 100one of the HIF 1α target genes, and that the activations of NOS 2 by a RESULTSRESULTS 100high-salt diet and angiotensin II are diminished in the renal medulla in RESULTSRESULTShigh-salt diet and angiotensin II are diminished in the renal medulla in
90Dahl salt-sensitive hypertensive rats indicating a possible impairment in 902 1 3 5 7 9 11 13 15
Dahl salt-sensitive hypertensive rats, indicating a possible impairment in1 3 5 7 9 11 13 15-2 1 3 5 7 9 11 13 15renal medullary HIF 1α in this rat strain Days on high salt dietHIF 1α
renal medullary HIF-1α in this rat strain.20 *A kDa Days on high salt diet80 HIF-1α 20 HIF-1α *A kDa
80fThe present study was to test the hypothesis that an impairment of the
HIF 1α*HIF-1α -110 LuciferaseThe present study was to test the hypothesis that an impairment of the
Luciferase-110 Luciferase
renal medullary HIF 1α pathway is responsible for the salt sensitive Luciferase) Figure 6 Effect of renal medullary HIF-1α gene transfection on*β Actin 46renal medullary HIF-1α pathway is responsible for the salt sensitive 16wt) Figure 6. Effect of renal medullary HIF-1α gene transfection on
*β-Actin -46
60hypertension in Dahl S rats t) 16
kw arterial blood pressure in Dahl S rats The mean arterial pressure*60hypertension in Dahl S rats. wt k arterial blood pressure in Dahl S rats. The mean arterial pressure
kw /g
(MAP) was significantly increased after 2-week high salt intakeLS HS LS HSWe transfected HIF 1α plasmids into the renal medulla and then detect the g 12n/ (MAP) was significantly increased after 2-week high salt intake.
SS13BN SSWe transfected HIF-1α plasmids into the renal medulla and then detect the n/g 12
min However high slat-induced hypertension was significantly attenuated inSS13BN SS
40renal sodi m e cretion and arterial press re after high salt challenge O r in /m However, high slat-induced hypertension was significantly attenuated inB 40renal sodium excretion and arterial pressure after high salt challenge. Our m e/ rats treated with HIF 1α gene transfection or renal medullary infusion ofB 3 0 S S
40p g gd t h d th t f l d ll HIF 1 l l t d l/m 8ol
e rats treated with HIF-1α gene transfection or renal medullary infusion ofB 3.0 LS HSdata showed that recovery of renal medullary HIF-1α levels promoted μ 8mo CoCl a HIF 1α inducer indicating that the defect of HIF 1α pathwayy y p
di ti d tt t d h t i ft di l di i V ( 8
μm
CoCl2, a HIF-1α inducer, indicating that the defect of HIF-1α pathway2 4y sodium excretion and attenuated hypertension after sodium loading in V (μ in the renal medulla may be responsible for the salt sensitive2.4si
tn) 20
yp gD hl S t U
.
V ( in the renal medulla may be responsible for the salt-sensitivens tin 20Dahl S rats. U 4.V hypertension in Dahl S rats * P< 0 05 vs other groups (n = 4 5)te A
ct 4
Na hypertension in Dahl S rats. . * P< 0.05 vs. other groups. (n = 4-5)1 8Int
-A
UN1.8
d I
β-
Ued to
0METHODSMETHODS 01 2iz o t
* 0METHODSMETHODS 01.2al tio
C1 C2 S1 S2 S3 P1 P2 P3METHODSMETHODS 0
m Rat
C1 C2 S1 S2 S3 P1 P2 P3CCC1 C2 S1 S2 S3 P1 P2 P3or (R C C S S S3 3ConclusionConclusionC1 C2 S1 S2 S3 P1 P2 P30.6N
o ( ConclusionConclusionAnimals Male Dahl S and SS 13BN rats (Charles River Laboratories0.6N ConclusionConclusionAnimals. Male Dahl S and SS-13BN rats (Charles River Laboratories,
Wil i t MA ) 300 350 d SS 13BN t i i 0 0Wilmington, MA ), 300-350 g, were used. SS-13BN rat is a consomic 0.0g , ), g,SS13BN SS
DecreasedDecreased levellevel andand abnormalabnormal responseresponse toto highhigh saltsalt intakeintakeFi 4 Eff t f l d ll HIF 1 t f ti i di ti i t t di l di i D hl S tsubcolony of Dahl S rats with substitution of chromosome 13 from SS13BN SSDecreasedDecreased levellevel andand abnormalabnormal responseresponse toto highhigh saltsalt intakeintakeFigure 4. Effects of renal medullary HIF-1α gene transfection on urinary sodium excretion in response to acute sodium loading in Dahl S rats.subcolony of Dahl S rats with substitution of chromosome 13 from
Fi 1 C i f hi h lt i d d h i HIF 1 l l i th lpp gg
inin renalrenal medullarymedullary HIFHIF 11αα maymay representrepresent aa novelnovelg y g y p g
Brown Norway (BN) rats This rat strain exhibits minimal differences in Figure 1. Comparison of high salt-induced changes in HIF-1α levels in the renal inin renalrenal medullarymedullary HIFHIF--11αα maymay representrepresent aa novelnovelTo further evaluate the impact of renal medullary HIF-1α transfection on renal salt handling, we examined the sodium excretion after acute sodiumBrown Norway (BN) rats. This rat strain exhibits minimal differences in g p g gd ll b t D hl lt iti d SS13BN t A R i l
yy yy ppmechanismmechanism mediatingmediating saltsalt sensitivesensitive hypertensionhypertension inin DSDS ratsrats
To further evaluate the impact of renal medullary HIF 1α transfection on renal salt handling, we examined the sodium excretion after acute sodiumgenotype from Dahl S rats (1 95% in differences) which is much smaller medulla between Dahl salt-sensitive and SS13BN rats. A, Representative gel mechanismmechanism mediatingmediating saltsalt--sensitivesensitive hypertensionhypertension inin DSDS ratsrats..loading Acute sodium loading dramatically increased urine volume (U·V) and urinary sodium excretion (U ·V) These increases in U·V and U ·Vgenotype from Dahl S rats (1.95% in differences), which is much smaller , p g gg ypyploading. Acute sodium loading dramatically increased urine volume (U V) and urinary sodium excretion (UNa V). These increases in U V and UNa Vth th diff b t D hl S t d th l d documents of Western blot analyses B Summarized intensities of the HIF-1α blots
I d tiI d ti ff HIFHIF 11 ii ii thth ll d lld ll ldldwere considerably enhanced in HIF 1α transfected rats These results demonstrated that overexpression of HIF 1α transgene in the renal medullathan the differences between Dahl S rats and other commonly used documents of Western blot analyses. B, Summarized intensities of the HIF 1α blots.InductionInduction ofof HIFHIF--11αα expressionexpression inin thethe renalrenal medullamedulla couldcouldwere considerably enhanced in HIF-1α-transfected rats. These results demonstrated that overexpression of HIF-1α transgene in the renal medullay
High salt induced a significant increase in HIF-1α in SS-13BN rats which was not ppbb th tith ti hh ff thth t t tt t t ff ltltk bl i d th bilit f th kid t t di l d i D hl S t Th d t dditi ll t th t d d l"control" strains: BN 77%, ACI 57%, Sprague-Dawley 52%, Dahl-R High salt induced a significant increase in HIF-1α in SS-13BN rats, which was notbebe aa therapeutictherapeutic approachapproach forfor thethe treatmenttreatment ofof saltsalt--remarkably improved the capability of the kidneys to remove extra sodium load in Dahl S rats. These data additionally suggest that decreased renalcontrol strains: BN 77%, ACI 57%, Sprague Dawley 52%, Dahl R
observed in Dahl S rats indicating there was a defect of HIF 1α pathway in Dahl S pp ppppitiiti h t ih t i
y p p y y y ggd ll HIF 1 b ibl f th i i d l ti f di ti i D hl S t * P 0 05 t l t ( 3)30% Therefore SS-13BN rat is considered as one of the best control observed in Dahl S rats, indicating there was a defect of HIF-1α pathway in Dahl SFig 4 sensitivesensitive hypertensionhypertension..medullary HIF-1α may be responsible for the impaired regulation of sodium excretion in Dahl S rats. * P < 0.05 vs. control rats. (n =3)30%. Therefore, SS-13BN rat is considered as one of the best control
t *P<0 05 th ( 6) LS l lt HS hi h lt SS D hl lt itiFig 4 ypypy y p p g ( )
(normotensive) rat strains for Dahl S rats rats. *P<0.05 vs others (n=6). LS, low salt; HS, high salt; SS, Dahl salt-sensitive.(normotensive) rat strains for Dahl S rats. ( ) , ; , g ; ,
