Name: demonstrating a linear relationship between vascular endothelial growth factor and luteinizing hormone in kidney cortex extracts
Uploaded: 2020-01-22T14:00:00
Description: Watch this Scientific Journal Video about Demonstrating a Linear Relationship Between Vascular Endothelial Growth Factor and Luteinizing Hormone in Kidney Cortex Extracts at JoVE.com

The authors thank Scholl&#39;s Slaughterhouse (Blissfield, MI) for providing the bovine and porcine kidneys. No grant funding was utilized for this study.

No live or experimental animals were used for this study.
1. Tissue Handling
<ol>
	<li>Procure bovine and porcine whole kidneys immediately after slaughter from an abattoir. Transport on ice to the laboratory.</li>
	<li>Upon arrival at laboratory, rinse kidneys with 50 mL of ice-cold phosphate buffered saline (PBS). Repeat this step 2x to remove blood completely.</li>
	<li>Keep kidneys on ice (or refrigerated) until further extraction.</li>
</ol>
2. Dissection of Kidney...

<ol>
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Procuring kidneys from the abattoir immediately after animal death is the key to success in this methodology. This is the main advantage of utilizing organs from cows and pigs instead of human cadavers. There is usually at least a 12-24 h delay from the time of death until human cadaver organs are procured. Because the chemical composition of bodily organs significantly changes within 2 h post-mortem15,16, VEGF-studies in human cadaver kidneys may not reflect rea...

Vascular endothelial growth factor A (VEGF-A), helps to regulate angiogenesis and vascular permeability in the kidney and other organs1,2 (hereafter, VEGF-A will be referred to as VEGF). VEGF levels in the kidney are under tight homeostatic control. When renal VEGF levels are elevated or depressed, the kidney can malfunction. For example, within 3 weeks after birth, mice with podocyte-specific heterozygosity for VEGF develop endotheliosis and bloodless glomeruli (i.e, renal lesions seen in human preeclampsia), and end-stage kidney failure occurs in these heterozygotes by 3 months of age. Podocyte-specific homozygotic knockouts die from hydrops and kidney failure within 1 day of birth3,4.
On the other hand, overexpression of renal VEGF causes proteinuria and glomerular hypertrophy3,4. For example, transgenic rabbits that overexpress VEGF exhibit progressive proteinuria with increased glomerular filtration rates in early stages of nephropathy, followed by decreased glomerular filtration rates in later stages3. Diabetic nephropathy, a major cause of end-stage renal disease in diabetic adults, is strongly associated with VEGF dysregulation2,5. A great deal of attention has been paid to the role of hypoxia in inducing VEGF expression under pathologic conditions5. However, the factors governing VEGF under physiologic conditions (both in the kidney and other organs) are not well-understood2,6. Identifying these factors (except for oxygen) that are involved in physiologic and pathologic VEGF regulation is an important undertaking.
Luteinizing hormone (LH), a pro-angiogenic hormone, helps regulate physiologic VEGF expression in reproductive organs such as the ovary and testis7,8. Previous studies have provided evidence that LH also helps regulate VEGF in non-reproductive organs, such as the eyes6,9,10. LH receptors are found in the medulla and cortex of the kidney11,12. Of note, kidney tubular epithelial cells, as well as the LH receptor, express VEGF11,12,13,14. Taking these two observations together, we hypothesized that LH also helps regulate VEGF expression in the kidney13,14. To provide evidence of this LH/VEGF relationship, the presented protocol aims to show that LH levels are able to predict VEGF levels in the kidney. Many previous VEGF-related investigations involving the kidney have used lower order mammal models (i.e., rodents and rabbits)2. To translate this work to the human body, the study examines the relationship between VEGF and LH in higher order mammals (here, bovine and porcine models). To carry out this objective, total protein lysate was prepared from the cortex region of bovine and porcine kidneys.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine LH ELISA Kit</td>
			<td>MyBiosource, San Diego, CA.</td>
			<td>MBS700951</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine VEGF-A ELISA Kit</td>
			<td>MyBiosource, San Diego, CA.</td>
			<td>MBS2887434</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific Inc, Columbus, OH.</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine LH ELISA Kit</td>
			<td>MyBiosource, San Diego, CA.</td>
			<td>MBS009739</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine VEGF-A ELISA</td>
			<td>Ray Biotech, Norcross, GA.</td>
			<td>ELP-VEGFA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>ThermoFisher Scientific Inc, Columbus, OH.</td>
			<td>89901</td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstrating a linear relationship between vascular endothelial growth factor and luteinizing hormone in kidney cortex extracts

Vascular endothelial growth factor (VEGF) helps to control angiogenesis and vascular permeability in the kidney. Renal disorders, such as diabetic nephropathy, are associated with VEGF dysregulation in the kidney. The factors that govern VEGF under physiologic conditions in the kidney are not well-understood. Luteinizing hormone (LH), a pro-angiogenic hormone, helps regulate physiologic VEGF expression in reproductive organs. Given that LH receptors are found in the kidney, we, at Zietchick Research Institute, hypothesized here that LH also helps regulate VEGF expression in the kidney as well. To provide evidence, we aimed to show that LH levels are able to predict VEGF levels in the mammalian kidney. Most VEGF-related investigations involving the kidney have used lower order mammals as models (i.e., rodents and rabbits). To translate this work to the human body, it was decided to examine the relationship between VEGF and LH in higher order mammals (i.e., bovine and porcine models). This protocol uses the total protein lysate from the kidney cortex. Keys to this method&#39;s success include procurement of kidneys from slaughterhouse animals immediately after death as well as normalization of analyte levels (in the kidney extract) by total protein. This study successfully demonstrates a significant linear relationship between LH and VEGF in both bovine and porcine kidneys. The results are reproducible in two different species. The study provides supporting evidence that the use of kidney extracts from cows and pigs are an excellent, economical, and abundant resource for the study of renal physiology, particularly for examining the correlation between VEGF and other analytes.

The mean and median levels of LH and VEGF by animal type and by sex are shown in Table 1. After verifying normality of data by Kolmogorov-Smirnov Testing of normality, linear regression models were utilized to examine the relationship between LH and VEGF. LH was found to be a strong and significant predictor of VEGF in both bovine and porcine kidneys (bovine kidney model: n = 7, R2 = 0.86, p = 0.002; porcine kidney model: n = 7; R2 = 0.66, p = 0.025)...

Watch this Scientific Journal Video about Demonstrating a Linear Relationship Between Vascular Endothelial Growth Factor and Luteinizing Hormone in Kidney Cortex Extracts at JoVE.com

Demonstrating a Linear Relationship Between Vascular Endothelial Growth Factor and Luteinizing Hormone in Kidney Cortex Extracts

Presented here is a protocol for utilizing a cortical kidney extract preparation and total protein normalization to demonstrate the correlation between vascular endothelial growth factor and luteinizing hormone in the mammalian kidney.

Vascular endothelial growth factor (VEGF) helps to control angiogenesis and vascular permeability in the kidney. Renal disorders, such as diabetic ...

Our protocol observes that the use of kidney extracts from cows and pigs are an excellent and economical resource for the study of renal physiology, particularly examining the correlation between vascular endothelial growth factor and other analytes. It has always been a challenge to correlate VEGF, an essential growth factor in organ angiogenesis, with other proteins. This technique allows us to examine the association between VEGF and analytes such as hormones.This will certainly advance our base of knowledge regarding this important growth factor. This method may be useful for correlating other analytes in kidney cortical extracts besides the ones mentioned in this video. Visual demonstration will help others to obtain reproducible results without any sampling errors.The use of fresh tissue is essential to the success of this technique. It is important to obtain fresh whole kidneys immediately after slaughter from an animal. Always transfer the tissue to the laboratory on ice.Use sterile scissors, forceps, a knife, and Petri dishes to dissect kidneys and excise the required tissue portion. Gently cut the kidney in half along the sagittal plane and cut a piece of tissue from the cortex region in the center of the kidney. Mince the tissue into small pieces with the knife and transfer it into a microfuge tube with one milliliter of ice cold 1X RIPA lysis extraction buffer.Label the tube with specific sample details and place it on ice. Use a handheld homogenizer with a sterile probe to homogenize the tissue for one to two minutes until no chunks are visible, then immediately centrifuge the tube at 9, 600 times g for five minutes at four degrees Celsius. After centrifugation, prepare separate aliquots of the sample for ELISA and total protein analysis to avoid multiple freeze/thaw cycles.Prior to starting the assay, bring all reagents and the assay plate to room temperature removing the excess wells and storing them at four degrees Celsius until further use. Dilute the 20X wash buffer to 300 milliliters with double distilled water and set up the blank wells without any solution. Add 50 microliters of standard or sample and another 50 microliters of horseradish peroxidase conjugate to each well, then immediately add 50 microliters of antibody solution to each well and seal the plate.Mix the plate and incubate it at 37 degrees Celsius for one hour. After the incubation, wash each well with 200 microliters of wash buffer. Add 50 microliters of substrate A and substrate B to each well, mix the plate gently by tapping on the side, then seal and incubate it in the dark for 15 minutes at 37 degrees Celsius.Add 50 microliters of stop solution to each well. Gently tap the plate and read the absorbance at 450 nanometers with a spectrophotometer. Prepare the reagents and wash buffer as previously described, then add 100 microliters of standard or sample to each well, seal the plate, mix well, and incubate it for two hours at 37 degrees Celsius.Remove the liquid in each well and add 100 microliters of detection reagent A.Seal the plate and incubate it for another hour. Next, wash each well with 400 microliters of wash buffer, then add 90 microliters of substrate solution to each well, gently tap the plate, and incubate it for another hour at 37 degrees Celsius. After the last incubation, add 50 microliters of stop solution to each well, gently tap the plate, and read the absorbance at 450 nanometers with a spectrophotometer.Estimate the total protein of the bovine and porcine kidney extracts with a standard BSA assay and use this estimate to normalize the LH and VEGF-A-ELISA results. The mean and median levels of LH and VEGF were calculated for both animals and sexes. After verifying normality of data, linear regression models were utilized to examine the relationship between LH and VEGF.LH was found to be a strong and significant predictor of VEGF in both bovine and porcine kidneys. When attempting this procedure, it is important to remember to sample identical areas in each tissue. A similar procedure can be used to extract any type of tissue.Remember to normalize analytes by total protein extract of whatever tissue you use.

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