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 Kidneys
<ol>
	<li>Use sterile scissors, forceps, a knife, and Petri dishes to dissect the kidneys and excise the required tissue portion.</li>
	<li>Prepare RIPA lysis buffer prior to kidney dissection. Dissolve 5 mM NaCl, 0.5 M EDTA, 1 M Tris (pH = 8.0), NP-40 (ID + GEPAL CA-630), 10% sodium deoxycholate, and 10% SDS in double-distilled water, then mix thoroughly. Refrigerate the RIPA lysis buffer when not in use.</li>
	<li>Gently cut the kidney in half (sagittal plane) and cut a piece of tissue (50-70 mm2) from the cortex region in the center of the kidney (weighing 80-100 mg by wet weight).</li>
	<li>Mince the tissue block into small pieces with a knife to assist the homogenization process.</li>
	<li>After mincing the tissues, transfer them into a microfuge tube with 1 mL of ice-cold 1x RIPA lysis extraction buffer. Place the tubes in ice until further extraction.</li>
</ol>
3. Tissue Homogenization
<ol>
	<li>Label the microfuge tubes with specific sample details for tissue supernatant collection.</li>
	<li>Using a handheld homogenizer with a sterile probe, then homogenize the tissues for 1-2 min in cold conditions (samples on ice bucket) until no chunks of tissues are visible.</li>
	<li>Subject the tissue extracts immediately for centrifugation in the refrigerated centrifuge at 9,600 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the tubes from the centrifuge and place them on the ice bucket.</li>
	<li>Collect the supernatant into a new labeled microfuge tube and store on ice. Discard the pellet.</li>
	<li>Prepare separate aliquots of the supernatants for LH and VEGF-A enzyme-linked immunosorbent assays (ELISA) and total protein analysis, respectively, to avoid freeze-thaw cycles.</li>
</ol>
4. Bovine and Porcine LH ELISA Assay
<ol>
	<li>Store all ELISA assay components included in the commercially available luteinizing hormone (LH) ELISA kit (see Table of Materials) at 2-8 &#176;C. This includes the antibody, HRP-conjugate, assay plate (96 well), calibrators, wash buffer (20x concentrate), substrate A, substrate B, and stop solution. Prepare all reagents as recommended by the manufacturer&#39;s instructions.</li>
	<li>Before starting the assay, bring all reagents and assay plate to room temperature (RT). Use the required number of wells for the assay, seal, and keep the unused wells at 4 &#176;C until use.</li>
	<li>Dilute the wash buffer (15 mL of 20x concentrate) to 300 mL with double-distilled water</li>
	<li>Set up the blank wells without any solution.</li>
	<li>Add 50 &#181;L of standard or sample to each well (n = 2), then add another 50 &#181;L of horseradish peroxidase (hrp)-conjugate to each well. Immediately add another 50 &#181;L of antibody solution to each well. Seal the plate, mix well, and incubate for 1 h at 37 &#176;C.</li>
	<li>Wash the wells with 1x wash buffer (200 &#181;L/well) and repeat 4x.</li>
	<li>Add 50 &#181;L of substrate A and 50 &#181;L of substrate B to each well, and mix well by tapping the plate on the side gently. Seal the plate and incubate for 15 min at 37 &#176;C in the dark for 15 min.</li>
	<li>Add 50 &#181;L of stop solution to each well, gently tap the plate, and read the plate using the spectrophotometer set to a 450 nm wavelength.</li>
	<li>Normalize bovine and porcine LH levels to total protein (see section 6).</li>
</ol>
5. Bovine and Porcine VEGF-A ELISA Assay
<ol>
	<li>Store all ELISA assay components included in the commercially available Vascular Endothelial Growth Factor-A ELISA kits (see Table of Materials) at 2-8 &#176;C. This includes the antibody, HRP-conjugate, assay plate (96 well), calibrators, wash buffer (20x concentrate), substrate A, substrate B, and stop solution. Prepare all the reagents as recommended by the manufacturer&#39;s instructions.</li>
	<li>Before starting the assay, bring all reagents and assay plate to RT. Use the required number of wells for the assay, seal, and keep the unused wells at 4 &#176;C until use.</li>
	<li>Dilute the wash buffer (15 mL of 20x concentrate) to 300 mL with double-distilled water</li>
	<li>Add 100 &#181;L of the standard or sample to each well (n = 2). Seal the plate, mix well, and incubate for 2 h at 37 &#176;C.</li>
	<li>Remove the liquid in each well and add 100 &#181;L of detection reagent A to each well, seal the plate, and incubate for 1 h at 37 &#176;C.</li>
	<li>Wash the wells with 1x wash buffer (400 &#181;L/well) and repeat 4x.</li>
	<li>Add 100 &#181;L of detection reagent B to each well and mix well by tapping the plate on the side gently. Seal the plate and incubate the plate for 1 h at 37 &#176;C.</li>
	<li>Wash the wells with 1x wash buffer (400 &#181;L/well) and repeat 4x.</li>
	<li>Add 90 &#181;L of substrate solution to each well, gently tap the plate, and incubate for 1 h at 37 &#176;C.</li>
	<li>Add 50 &#181;L of stop solution to each well, gently tap the plate, and read the plate using the spectrophotometer set to a 450 nm wavelength.</li>
	<li>Normalize bovine and porcine VEGF-A levels to total protein (section 6).</li>
</ol>
6. Total Protein Estimation
<ol>
	<li>Estimate total protein of the bovine and porcine kidney extracts by standard bovine serum albumin (BSA) assay using a commercial kit (see Table of Materials) according to the manufacturer&#39;s recommendations.</li>
</ol>
7. Statistical Analysis
<ol>
	<li>Calculate the mean, median, and standard deviation of each analyte.</li>
	<li>Test the divergence of sample distribution from normal utilizing Kolmogorov-Smirnov Test to decide, upon use, between parametric vs. non-parametric statistical tests. If data is normally distributed, then perform statistical testing via parametric tests.</li>
	<li>Under appropriate circumstances (such as normal data distribution), utilize regression models to examine the linear relationship between LH and VEGF-A.</li>
</ol>

<ol>
	<li>Advani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+VEGF+in+maintaining+renal+structure+and+function+under+normotensive+and+hypertensive+conditions.">Role of VEGF in maintaining renal structure and function under normotensive and hypertensive conditions.</a> Proceedings of the National Academy of Science U. S. A. 104 (36), 14448-14453 (2007).</li><li>Majumder, S., Advani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+and+the+diabetic+kidney:+More+than+too+much+of+a+good+thing.">VEGF and the diabetic kidney: More than too much of a good thing.</a> Journal of Diabetes and its Complications. 31 (1), 273-279 (2017).</li><li>Liu, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+vascular+endothelial+growth+factor+in+kidney+leads+to+progressive+impairment+of+glomerular+functions.">Increased expression of vascular endothelial growth factor in kidney leads to progressive impairment of glomerular functions.</a> Journal of the American Society of Nephrology. 18 (7), 2094-2104 (2007).</li><li>Eremina, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular-specific+alterations+of+VEGF-A+expression+lead+to+distinct+congenital+and+acquired+renal+diseases.">Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases.</a> Journal of Clinical Investigation. 111 (5), 707-716 (2003).</li><li>Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor:+basic+science+and+clinical+progress.">Vascular endothelial growth factor: basic science and clinical progress.</a> Endocrine Reviews. 25 (4), 581-611 (2004).</li><li>Movsas, T. Z., Sigler, R., Muthusamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitreous+Levels+of+Luteinizing+Hormone+and+VEGF+are+Strongly+Correlated+in+Healthy+Mammalian+Eyes.">Vitreous Levels of Luteinizing Hormone and VEGF are Strongly Correlated in Healthy Mammalian Eyes.</a> Current Eye Research. 43 (8), 1041-1044 (2018).</li><li>Babitha, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luteinizing+hormone,+insulin+like+growth+factor-1,+and+epidermal+growth+factor+stimulate+vascular+endothelial+growth+factor+production+in+cultured+bubaline+granulosa+cells.">Luteinizing hormone, insulin like growth factor-1, and epidermal growth factor stimulate vascular endothelial growth factor production in cultured bubaline granulosa cells.</a> General and Comparative Endocrinology. 198, 1-12 (2014).</li><li>Trau, H. A., Davis, J. S., Duffy, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+the+Primate+Ovulatory+Follicle+Is+Stimulated+by+Luteinizing+Hormone+via+Prostaglandin+E2.">Angiogenesis in the Primate Ovulatory Follicle Is Stimulated by Luteinizing Hormone via Prostaglandin E2.</a> Biology of Reproduction. 92 (1), 15 (2015).</li><li>Movsas, T. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confirmation+of+Luteinizing+Hormone+(LH)+in+Living+Human+Vitreous+and+the+Effect+of+LH+Receptor+Reduction+on+Murine+Electroretinogram.">Confirmation of Luteinizing Hormone (LH) in Living Human Vitreous and the Effect of LH Receptor Reduction on Murine Electroretinogram.</a> Neuroscience. 385, 1-10 (2018).</li><li>Movsas, T. Z., Sigler, R., Muthusamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+Signaling+by+the+Luteinizing+Hormone+Receptor+Reduces+Ocular+VEGF+and+Retinal+Vascularization+during+Mouse+Eye+Development.">Elimination of Signaling by the Luteinizing Hormone Receptor Reduces Ocular VEGF and Retinal Vascularization during Mouse Eye Development.</a> Current Eye Research. 43 (10), 1286-1289 (2018).</li><li>Hipkin, R. W., Sanchez-Yague, J., Ascoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+luteinizing+hormone/chorionic+gonadotropin+(LH/CG)+receptor+precursor+in+a+human+kidney+cell+line+stably+transfected+with+the+rat+luteal+LH/CG+receptor+complementary+DNA.">Identification and characterization of a luteinizing hormone/chorionic gonadotropin (LH/CG) receptor precursor in a human kidney cell line stably transfected with the rat luteal LH/CG receptor complementary DNA.</a> Molecular Endocrinology. 6 (12), 2210-2218 (1992).</li><li>Lei, Z. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+luteinizing+hormone/human+chorionic+gonadotropin+receptor+gene.">Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene.</a> Molecular Endocrinology. 15 (1), 184-200 (2001).</li><li>Schrijvers, B. F., Flyvbjerg, A., De Vriese, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+vascular+endothelial+growth+factor+(VEGF)+in+renal+pathophysiology.">The role of vascular endothelial growth factor (VEGF) in renal pathophysiology.</a> Kidney International. 65 (6), 2003-2017 (2004).</li><li>Apaja, P. M., Aatsinki, J. T., Rajaniemim, H. J., Petaja-Repo, U. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+mature+luteinizing+hormone+receptor+in+rodent+urogenital+and+adrenal+tissues+is+developmentally+regulated+at+a+posttranslational+level.">Expression of the mature luteinizing hormone receptor in rodent urogenital and adrenal tissues is developmentally regulated at a posttranslational level.</a> Endocrinology. 146 (8), 3224-3232 (2005).</li><li>Ondruschka, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-mortem+in+situ+stability+of+serum+markers+of+cerebral+damage+and+acute+phase+response.">Post-mortem in situ stability of serum markers of cerebral damage and acute phase response.</a> International Journal of Legal Medicine. 133 (3), 871-881 (2019).</li><li>Swain, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+post-mortem+interval:+A+comparison+between+cerebrospinal+fluid+and+vitreous+humour+chemistry.">Estimation of post-mortem interval: A comparison between cerebrospinal fluid and vitreous humour chemistry.</a> Journal of Forensic and Legal Medicine. 36, 144-148 (2015).</li><li>Thompson, C. S., Traynor, I. M., Fodey, T. L., Faulkner, D. V., Crooks, S. R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+method+for+the+detection+of+residues+of+amphenicol+antibiotics+in+bovine,+ovine+and+porcine+kidney+by+optical+biosensor.">Screening method for the detection of residues of amphenicol antibiotics in bovine, ovine and porcine kidney by optical biosensor.</a> Talanta. 172, 120-125 (2017).</li><li>Konstantinou, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-Linked+Immunosorbent+Assay+(ELISA).">Enzyme-Linked Immunosorbent Assay (ELISA).</a> Methods in Molecular Biology. 1592, 79-94 (2017).</li><li>Levesque, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+urine+vascular+endothelial+growth+factor+levels+are+associated+with+mechanical+ventilation,+bronchopulmonary+dysplasia+and+retinopathy+of+prematurity.">Low urine vascular endothelial growth factor levels are associated with mechanical ventilation, bronchopulmonary dysplasia and retinopathy of prematurity.</a> Neonatology. 104 (1), 56-64 (2013).</li><li>Leviton, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antecedents+and+early+correlates+of+high+and+low+concentrations+of+angiogenic+proteins+in+extremely+preterm+newborns.">Antecedents and early correlates of high and low concentrations of angiogenic proteins in extremely preterm newborns.</a> Clinica Chimica Acta. 471, 1-5 (2017).</li><li>Simo-Servat, O., Hernandez, C., Simo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+the+vitreous+fluid+analysis+in+the+translational+research+of+diabetic+retinopathy.">Usefulness of the vitreous fluid analysis in the translational research of diabetic retinopathy.</a> Mediators of Inflammation. , 872978 (2012).</li><li>Sharma, R. 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Zietchick Research Institute (ZRI) is a private (for-profit) research institute, and Dr. Tammy Movsas (founder and director of ZRI) has a pending patent applications and validated patents for the use of gonadotropin antagonists in the treatment of ocular diseases and diabetes. Other than being an employee (biochemist) at ZRI, Dr. A. Muthusamy has no other financial conflicts to report. A. Arivalagan (summer intern at ZRI, undergraduate student at University of Michigan) has no other financial conflicts to report.

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 ...

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4.7K Views.  Zietchick Research Institute. 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.

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

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...