Overexpression of a HIF-4-prolyl hydroxylase Transgene in the …pli/Presentation/2009 EB...
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0 10 20 30 40 50 60 70 U•V ( μ l/min/g kwt) Ctrl PHD2 * * METHODS METHODS We have demonstrated that HIF prolyl-4-hydroxylase domain-containing proteins (PHDs), a family of enzymes that promote HIF-1a degradation, are abundantly present in the renal medulla and regulate sodium excretion through regulation of HIF-1a and its targeted genes, such as nitric oxide synthase, cyclooxygenase and heme oxygenase. The present study was designed to test the hypothesis that overexpression a PHD transgene in the renal medulla increases the salt sensitivity of blood pressure. Plasmids expressing PHD2, the predominant isoform of PHDs in the kidneys, were transfected into the renal medulla in uninephretomized rats. Three weeks after PHD2 transfection, the mRNA levels of PHD2 was increased by two folds in the renal medulla. Mean arterial pressure measured by a telemetry system was significantly raised in PHD2 transfected rats after high salt challenge compared with control animals (131.3 ± 6.7 vs. 110.7 ± 4.2 mmHg). There was no blood pressure change in PHD2 transfected rats that were maintained on the normal salt diet. Urinary sodium excretion in response to acute sodium loading or elevation of renal perfusion pressure were blunted by 30% and 50%, respectively, in PHD2 transfected animals. In conclusion, PHD2 in the renal medulla importantly participates in the regulation of renal medullary function and in the long- term control of arterial blood pressure. (Supported by NIH grant DK54927 and HL89563) BACKGROUND BACKGROUND ABSTRACT ABSTRACT Overexpression Overexpression of PHD2 of PHD2 transgene transgene prevented high prevented high salt salt - - induced suppression of PHD2 in the renal medulla. induced suppression of PHD2 in the renal medulla. Overexpression Overexpression of PHD2 of PHD2 transgene transgene decreased decreased HIF HIF - - 1 1 α α levels levels in the renal medulla. in the renal medulla. Overexpression Overexpression of PHD2 of PHD2 transgene transgene in the renal medulla in the renal medulla blunted diuretic and blunted diuretic and natriuretic natriuretic responses to the responses to the elevation of RPP and acute sodium loading. elevation of RPP and acute sodium loading. Overexpression Overexpression of PHD2 of PHD2 transgene transgene in the renal medulla in the renal medulla increased salt sensitivity of blood pressure. increased salt sensitivity of blood pressure. RESULTS RESULTS SUMMARY SUMMARY CONCLUSION CONCLUSION Renal Renal medullary medullary PHD senses the high salt intake PHD senses the high salt intake and mediates renal salt adaptation, thereby, and mediates renal salt adaptation, thereby, regulates salt sensitivity of blood pressure. regulates salt sensitivity of blood pressure. Figure 3. Effects of renal medullary PHD2 transfection on urinary sodium excretion in response to acute sodium loading. To further evaluate the impact of renal medullary PHD2 transfection on renal salt handling, we examined the sodium excretion after acute sodium loading. Acute sodium loading dramatically increased urine volume (U·V) and urinary sodium excretion (U Na ·V). These increases in U·V and U Na ·V were considerably attenuated in PHD2-treated rats. These results demonstrated that overexpression of PHD2 transgene in the renal medulla remarkably impaired the capability of the kidneys to remove extra sodium load. These data additionally suggest that renal medullary PHD2 is a crucial determinant in the regulation of sodium excretion. * indicates P < 0.05 vs. PHD2 rats. (n = 5) Figure 2. Effect of renal medullary PHD2 tranfection on pressure natriuresis. We compared diuretic and natriuretic responses to the elevation of renal perfusion pressure (RPP) between control animals and PHD2 transfected animals. Both the urine flow and urinary sodium excretion rates were remarkably increased in response to the elevation of RPP. However, these pressure diuretic and natriuretic responses were significantly blunted in PHD2 plasmids transfected group compared with the control group. These data indicate that overexpression of PHD2 transgene impaired renal medullary function. * P<0.05 vs. PHD2 rats. (n=5) Figure 1. Overexpression of PHD transgene increased PHD2 mRNA levels and decreased HIF-1α protein levels in the renal medulla. A: In the animals transfected with PHD2 plasmids, the PHD2 levels were increased by two folds compared with that in control animals. Although high salt (HS) intake significantly suppresses the expression of PHD2, the levels of PHD2 in PHD2-transfected animals were preserved after high slat diet to the same levels as that in control animals on a low salt diet. These results demonstrated a successful PHD2 gene transfection that prevented the decrease in PHD2 levels in the renal medulla after high salt intake. * P < 0.05 vs. low salt (LS) from control rats or PHD2 rats, respectively; # P< 0.05 vs. control on the same salt diet rats. n = 6. B: PHD transfection reduced HIF-1α levels in the renal medulla (low salt diet, n = 3). Overexpression of a HIF-4-prolyl hydroxylase Transgene in the Renal Medulla Increases the Salt Sensitivity of Arterial Blood Pressure Min Xia, Qing Zhu, Zhengchao Wang, Fan Zhang, Pin-Lan Li, Ningjun Li Department of Pharmacology and Toxicology, Medical College of Virginia,Virginia Commonwealth University, Richmond, VA, 23298 0 5 10 15 20 80 120 150 RPP (mmHg) U•V Na ( μ mol/min/g kwt) Ctrl PHD2 * * We have shown that Hypoxia-inducible factor-1α (HIF-1α), a transcription factor, is abundantly expressed in the renal medulla and plays an important role in the control of renal mdeullary functions and arterial pressure, through regulating many oxygen-sensitive genes such as nitric oxide synthase, cyclooxygenase-2 and heme oxygenase-1. High salt intake up-regulates HIF-1α and its target genes in the renal medulla. Inhibition of HIF-1α within the renal medulla reduces sodium excretion and increases salt sensitivity of arterial blood pressure. HIF prolyl-4-hydroxylases are enzymes that promote the degradation of HIF-1α by catalyzing the proline hydroxylation of HIF-1α, which is one of the most important pathways for the regulation of HIF-1α. Prolyl-4- hydroxylase domain-containing proteins (PHDs) are highly expressed in the renal medulla with PHD2 as the predominant isoform of PHDs. We have also shown that renal medullary PHDs importantly contribute to the control of renal sodium excretion through regulation of HIF-1α and its targeted genes, and high salt intake up-regulates HIF-1α through inhibition of PHD2 in the renal medulla. The present study was designed to test the hypothesis that overexpression of a PHD transgene in the renal medulla increases the salt sensitivity of blood pressure. We first transfected PHD2 plasmids into the renal medulla and then detect the renal sodium excretion and arterial pressure after high salt challenge. Our data shows renal medullary PHD2 is an important determinant in salt sensitivity of blood pressure. Animals. Experiments were performed on the male Sprague-Dawley rats, 300-350 g (Harlan, Madison, WI). All animal procedures were approved by the Institutional Animal Care and Use Committee of the Virginia Commonwealth University. In vivo transfection of PHD2 expression plasmids into the renal medulla. Animals were uninephroctemized one week before the experiment. A medullary interstitial catheter (tapped tip, 4-5mm) was implanted into the remaining left kidney and anchored into place on the kidney surface with Vetbond Tissue Adhesive. A mixture of PHD2 expression plasmid (50μg, a generous gift from Dr. Frank Lee, University of Pennsylvania) in 600 μL in vivo-jetPEI™ Transfection Reagent was infused into the renal medulla at a speed of 20 μl/min. After infusion, the catheter was cut and blocked by a piece of fat tissue with Vetbond Tissue Adhesive. Luciferase plasmids were used as control. The position of the catheters was confirmed at the end of experiments. Chronic monitoring of arterial blood pressure in conscious rats. Mean arterial pressure (MAP) was measured using a telemetry blood pressure recording system (Data Sciences International). Telemetry transmitter was implanted after the injection of PHD2 plasmids in the same surgery. The blood pressure signals was recorded through a remote receiver, which allowed rats to be housed unrestrainedly. After baseline MAP was recorded on 3 control days while the rats remained on the normal salt diet, animals were switched to high salt diet containing 4% NaCl (Dyets, Inc) and MAP was recorded for additional three weeks. At the end of chronic MAP recording, the kidneys were collected for the detection of PHD2 and HIF-1α levels. Measurement of pressure natriuresis in response to the elevations of RPP. One week after PHD2 transfection, rats were surgically prepared for measurement of renal perfusion pressure (RPP) and urine flow. After 1-h equilibration followed by two 20-min control collection periods, RPP was acutely increased to 160 mmHg by tying off the celiac and mesenteric arteries. The RPP was set to 160, 120 and 80 mmHg, respectively, by an adjustable aorta clamp above the renal arteries. At each RPP level, after a 10-min equilibration period, urine were collected for 10-min clearance period. The urine flow rate was determined gravimetrically, and sodium (Na + ) concentrations of urine samples were measured using a flame photometer (Buck Scientific). The urine flow and urinary Na + excretion were factored per gram kidney weight (kwt). Measurement of urinary sodium excretion in response to acute sodium loading. Animals, the same as above, were surgically prepared for measurement of arterial pressure and urine flow. After 1-hour equilibration and two 10-minute control period sample collections, a 5% body weight isotonic saline load was administered intravenously and three 10-minute samples were collected over 30 minutes, and then 3 more 10-minute postcontrol samples were taken. RNA extraction and quantitative RT-PCR analysis of PHD2 mRNA levels. Total RNA from renal tissues was extracted and then reverse-transcribed (RT). The RT products were amplified using a TaqMan® Gene Expression Assays kit (Applied Biosystems), with 18S ribosomal RNA as an endogenous control. The relative mRNA levels were calculated in accordance with the ΔΔCt method. The ΔCt value in PHD2 from the control animals was used as a reference to calculate ΔΔCt values for other samples. Relative mRNA levels were expressed by the values of 2 -ΔΔCt . Western blot analysis of HIF-1α levels. Nuclear extraction from renal medullary tissue was subjected to Western blot using anti-HIF-1α antibody (monoclonal, Novus) Figure 4. Effect of renal medullary PHD2 transfection on salt sensitivity of blood pressure. After the rats were challenged with a high salt diet for 3 weeks, the MAP were significantly increased in PHD2-treated rats compared with control rats. When PHD2-treated rats were maintained on normal salt diet, there was no increase in MAP. These data suggest that PHD2-mediated pathway the renal medulla importantly participates in high salt adaptation of the kidneys and consequently mediates salt sensitivity of arterial blood pressure. * P< 0.05 vs. other groups. (n = 5) 0.0 0.5 1.0 1.5 2.0 2.5 Control PHD2 Normslized intensity (ratio to b-actin) HIF-1α − β− actin − B A HS PHD HIF HS PHD HIF - - 1 1 α α U U Na Na NO NO CO CO PG PG BP BP maintained maintained HS PHD HS PHD BP BP X 90 100 110 120 130 Ctrl-HS PHD2-HS PHD2-LS MAP (mmHg) * 0 10 20 30 40 50 60 70 C1 C2 VE1 VE2 VE3 P1 P2 P3 U•V ( μ l/min/g kwt) Ctrl PHD2 * * 0 5 10 15 20 C1 C2 VE1 VE2 VE3 P1 P2 P3 U Na •V ( μ mol/min/g kwt) Ctrl PHD2 * * 0.0 1.0 2.0 3.0 4.0 5.0 LS HS PHD2 mRNA level Control PHD2 * * # #
Transcript of Overexpression of a HIF-4-prolyl hydroxylase Transgene in the …pli/Presentation/2009 EB...
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METHODSMETHODSWe have demonstrated that HIF prolyl-4-hydroxylase domain-containing proteins (PHDs), a family of enzymes that promote HIF-1a degradation, are abundantly present in the renal medulla and regulate sodium excretion through regulation of HIF-1a and its targeted genes, such as nitric oxide synthase, cyclooxygenase and heme oxygenase. The present study was designed to test the hypothesis that overexpression a PHD transgene in the renal medulla increases the salt sensitivity of blood pressure. Plasmids expressing PHD2, the predominant isoform of PHDs in the kidneys, were transfected into the renal medulla in uninephretomized rats. Three weeks after PHD2 transfection, the mRNA levels of PHD2 was increased by two folds in the renal medulla. Mean arterial pressure measured by a telemetry system was significantly raised in PHD2 transfected rats after high salt challenge compared with control animals (131.3 ± 6.7 vs. 110.7 ± 4.2 mmHg). There was no blood pressure change in PHD2 transfected rats that were maintained on the normal salt diet. Urinary sodium excretion in response to acute sodium loading or elevation of renal perfusion pressure were blunted by 30% and 50%, respectively, in PHD2 transfectedanimals. In conclusion, PHD2 in the renal medulla importantly participates in the regulation of renal medullary function and in the long-term control of arterial blood pressure. (Supported by NIH grantDK54927 and HL89563)
BACKGROUNDBACKGROUND
ABSTRACTABSTRACT
OverexpressionOverexpression of PHD2 of PHD2 transgenetransgene prevented highprevented high saltsalt--induced suppression of PHD2 in the renal medulla.induced suppression of PHD2 in the renal medulla.OverexpressionOverexpression of PHD2 of PHD2 transgenetransgene decreased decreased HIFHIF--11α α levels levels in the renal medulla. in the renal medulla. OverexpressionOverexpression of PHD2 of PHD2 transgenetransgene in the renal medulla in the renal medulla blunted diuretic and blunted diuretic and natriureticnatriuretic responses to the responses to the elevation of RPP and acute sodium loading. elevation of RPP and acute sodium loading. OverexpressionOverexpression of PHD2 of PHD2 transgenetransgene in the renal medulla in the renal medulla increased salt sensitivity of blood pressure.increased salt sensitivity of blood pressure.
RESULTSRESULTS
SUMMARYSUMMARY
CONCLUSIONCONCLUSIONRenal Renal medullarymedullary PHD senses the high salt intake PHD senses the high salt intake and mediates renal salt adaptation, thereby, and mediates renal salt adaptation, thereby, regulates salt sensitivity of blood pressure. regulates salt sensitivity of blood pressure.
Figure 3. Effects of renal medullary PHD2 transfection on urinary sodium excretion in response to acute sodium loading. To further evaluate the impact of renal medullary PHD2 transfection on renal salt handling, we examined the sodium excretion after acute sodium loading. Acute sodium loading dramatically increased urine volume (U·V) and urinary sodium excretion (UNa·V). These increases in U·V and UNa·V were considerably attenuated in PHD2-treated rats. These results demonstrated that overexpression of PHD2 transgene in the renal medulla remarkably impaired the capability of the kidneys to remove extra sodium load. These data additionally suggest that renal medullary PHD2 is a crucial determinant in the regulation of sodium excretion. * indicates P < 0.05 vs. PHD2 rats. (n = 5)
Figure 2. Effect of renal medullary PHD2 tranfection on pressure natriuresis. We compared diuretic and natriuretic responses to the elevation of renal perfusion pressure (RPP) between control animals and PHD2 transfected animals. Both the urine flow and urinary sodium excretion rates were remarkably increased in response to the elevation of RPP. However, these pressure diuretic and natriuretic responses were significantly blunted in PHD2 plasmids transfected group compared with the control group. These data indicate that overexpression of PHD2 transgene impaired renal medullary function.* P<0.05 vs. PHD2 rats. (n=5)
Figure 1. Overexpression of PHD transgene increased PHD2 mRNA levels and decreased HIF-1α protein levels in the renal medulla. A: In the animals transfected with PHD2 plasmids, the PHD2 levels were increased by two folds compared with that in control animals. Although high salt (HS) intake significantly suppresses the expression of PHD2, the levels of PHD2 in PHD2-transfected animals were preserved after high slat diet to the same levels as that in control animals on a low salt diet. These results demonstrated a successful PHD2 gene transfection that prevented the decrease in PHD2 levels in the renal medulla after high salt intake. * P < 0.05 vs. low salt (LS) from control rats or PHD2 rats, respectively; # P< 0.05 vs. control on the same salt diet rats. n = 6. B: PHD transfection reduced HIF-1α levels in the renal medulla (low salt diet, n = 3).
Overexpression of a HIF-4-prolyl hydroxylase Transgene in the Renal Medulla Increases the Salt Sensitivity of Arterial Blood Pressure
Min Xia, Qing Zhu, Zhengchao Wang, Fan Zhang, Pin-Lan Li, Ningjun LiDepartment of Pharmacology and Toxicology, Medical College of Virginia,Virginia Commonwealth University, Richmond, VA, 23298
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80 120 150RPP (mmHg)
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wt)
Ctrl PHD2
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We have shown that Hypoxia-inducible factor-1α (HIF-1α), a transcription factor, is abundantly expressed in the renal medulla and plays an important role in the control of renal mdeullary functions and arterial pressure, through regulating many oxygen-sensitive genes such as nitric oxide synthase, cyclooxygenase-2 and heme oxygenase-1.
High salt intake up-regulates HIF-1α and its target genes in the renal medulla. Inhibition of HIF-1α within the renal medulla reduces sodium excretion and increases salt sensitivity of arterial blood pressure.
HIF prolyl-4-hydroxylases are enzymes that promote the degradation of HIF-1α by catalyzing the proline hydroxylation of HIF-1α, which is one of the most important pathways for the regulation of HIF-1α. Prolyl-4-hydroxylase domain-containing proteins (PHDs) are highly expressed in the renal medulla with PHD2 as the predominant isoform of PHDs.
We have also shown that renal medullary PHDs importantly contribute to the control of renal sodium excretion through regulation of HIF-1α and its targeted genes, and high salt intake up-regulates HIF-1α through inhibition of PHD2 in the renal medulla.
The present study was designed to test the hypothesis that overexpressionof a PHD transgene in the renal medulla increases the salt sensitivity of blood pressure. We first transfected PHD2 plasmids into the renal medulla and then detect the renal sodium excretion and arterial pressure after high salt challenge. Our data shows renal medullary PHD2 is an important determinant in salt sensitivity of blood pressure.
Animals. Experiments were performed on the male Sprague-Dawley rats, 300-350 g (Harlan, Madison, WI). All animal procedures were approved by the Institutional Animal Care and Use Committee of the Virginia Commonwealth University.
In vivo transfection of PHD2 expression plasmids into the renal medulla. Animals were uninephroctemized one week before the experiment. A medullary interstitial catheter (tapped tip, 4-5mm) was implanted into the remaining left kidney and anchored into place on the kidney surface with Vetbond Tissue Adhesive. A mixture of PHD2 expression plasmid (50µg, a generous gift from Dr. Frank Lee, University of Pennsylvania) in 600 µL in vivo-jetPEI™ Transfection Reagent was infused into the renal medulla at a speed of 20 µl/min. After infusion, the catheter was cut and blocked by a piece of fat tissue with Vetbond Tissue Adhesive. Luciferase plasmids were used as control. The position of the catheters was confirmed at the end of experiments.
Chronic monitoring of arterial blood pressure in conscious rats. Mean arterial pressure (MAP) was measured using a telemetry blood pressure recording system (Data Sciences International). Telemetry transmitter was implanted after the injection of PHD2 plasmids in the same surgery. The blood pressure signals was recorded through a remote receiver, which allowed rats to be housed unrestrainedly. After baseline MAP was recorded on 3 control days while the rats remained on the normal salt diet, animals were switched to high salt diet containing 4% NaCl (Dyets, Inc) and MAP was recorded for additional three weeks. At the end of chronic MAP recording, the kidneys were collected for the detection of PHD2 and HIF-1α levels.
Measurement of pressure natriuresis in response to the elevations of RPP. One week after PHD2 transfection, rats were surgically prepared for measurement of renal perfusion pressure (RPP) and urine flow. After 1-h equilibration followed by two 20-min control collection periods, RPP was acutely increased to 160 mmHg by tying off the celiac and mesenteric arteries. The RPP was set to 160, 120 and 80 mmHg, respectively, by an adjustable aorta clamp above the renal arteries. At each RPP level, after a 10-min equilibration period, urine were collected for 10-min clearance period. The urine flow rate was determined gravimetrically, and sodium (Na+) concentrations of urine samples were measured using a flame photometer (Buck Scientific). The urine flow and urinary Na+ excretion were factored per gram kidney weight (kwt).
Measurement of urinary sodium excretion in response to acute sodium loading. Animals, the same as above, were surgically prepared for measurement of arterial pressure and urine flow. After 1-hour equilibration and two 10-minute control period sample collections, a 5% body weight isotonic saline load was administered intravenously and three 10-minute samples were collected over 30 minutes, and then 3 more 10-minute postcontrol samples were taken.
RNA extraction and quantitative RT-PCR analysis of PHD2 mRNA levels. Total RNA from renal tissues was extracted and then reverse-transcribed (RT). The RT products were amplified using a TaqMan® Gene Expression Assays kit (Applied Biosystems), with 18S ribosomal RNA as an endogenous control. The relative mRNA levels were calculated in accordance with the ΔΔCt method. The ΔCt value in PHD2 from the control animals was used as a reference to calculate ΔΔCt values for other samples. Relative mRNA levels were expressed by the values of 2-ΔΔCt.
Western blot analysis of HIF-1α levels. Nuclear extraction from renal medullarytissue was subjected to Western blot using anti-HIF-1α antibody (monoclonal, Novus)
Figure 4. Effect of renal medullary PHD2 transfection on salt sensitivity of blood pressure. After the rats were challenged with a high salt diet for 3 weeks, the MAP were significantly increased in PHD2-treated rats compared with control rats. When PHD2-treated rats were maintained on normal salt diet, there was no increase in MAP. These data suggest that PHD2-mediated pathway the renal medulla importantly participates in high salt adaptation of the kidneys and consequently mediates salt sensitivity of arterial blood pressure. * P< 0.05 vs. other groups. (n = 5)
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