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. K., Rowe-Rendleman, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+molecular+and+genomic+biomarkers+of+retinal+drug+efficacy:+use+of+ocular+fluid+sampling+to+evaluate+VEGF.">Validation of molecular and genomic biomarkers of retinal drug efficacy: use of ocular fluid sampling to evaluate VEGF.</a> Neurochemical Research. 36 (4), 655-667 (2011).</li></ol>

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 real-life circumstances. Although the protocol greatly emphasizes the importance of immediate procurement and placement of animal organs on ice after extraction, it is not known if other researchers also prioritize this step. For example, the methodology section of a recent study (utilizing bovine and porcine kidneys for the detection of antibiotic residues) did not specify the time delay between animal death and procurement/refrigeration of the organs17.
This study measures the analytes of interest (VEGF and LH) with commercially available, species-specific ELISAs. ELISAs are highly sensitive, simple to perform assays with, and yield robust results18. A critical step in the protocol is the normalization of (ELISA-measured) analyte levels by total protein. The cortical kidney extract is a highly heterogeneous biological substance. In the light of this, a correction factor is essential so that analyte levels can be compared between animals. Thus, normalized by total protein was performed, since we and others have successfully normalized other heterogeneous biological substances (i.e., urine, dried blood spots, and vitreous fluid) in the same manner9,19,20.
A prior study showed that the correlation between LH and VEGF in vitreous fluid (from bovine and porcine eyes) only manifests after normalization by total protein6. Importantly, this normalization step is frequently omitted in published VEGF studies, particularly in those involving ELISA assays. Instead, VEGF levels are often expressed in units such as picogram per milliliter (and not as picogram per milligram of total protein). For example, none of the vitreous VEGF measurements in nine different ELISA studies (that were included in a vitreous VEGF review article) were normalized by any correction method21,22. This lack of VEGF normalization in ELISA studies may partially explain why VEGF has not yet been verified as a valid biomarker21,22.
Despite the limited sample size of the representative data (bovine, n = 7; porcine, n = 7), this protocol demonstrates a strong and significant linear relationship between LH and VEGF in both bovine and porcine kidneys. That said, there was not a large enough sample size to perform multivariate analyses adjusted for gender. We plan to repeat this study with larger sample sizes so that such analyses can be performed. Nevertheless, the presented results support the potential association between LH and VEGF in the mammalian kidney.
It is expected that this work will help further the understanding of homeostatic regulation of VEGF in the kidney. Both the quality of this methodology and robustness of the findings are illustrated by the reproducibility of the results in two different species. Because animals destined for meat production are healthy, the use of kidney extracts from slaughterhouse animals is primarily for studying physiology; however, their organs are less helpful for studying pathology, which is the main limitation of their use. All in all, the use of renal extracts from cows and pigs are an excellent, economical, and abundant resource for the study of normal adult kidneys. Finally, the protocol demonstrates the effectiveness of utilizing total protein for normalization, particularly when examining correlations between VEGF and other analytes.

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><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).
The LH/VEGF linear relationship is illustrated in Figure 1 (bovine regression model) and Figure 2 (porcine regression model). The bovine linear equation is as follows: VEGF level = 2.156 x LH level + 68.75. The porcine linear equation is as follows: VEGF level = 196.7 x LH levels + 47.94.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample Type</td>
			<td>Males</td>
			<td>Females</td>
			<td>All</td>
		</tr>
		<tr>
			<td>Bovine Kidneys</td>
			<td>n = 4</td>
			<td>n = 3</td>
			<td>n = 7</td>
		</tr>
		<tr>
			<td rowspan="2">LH (mIU/mg total protein)</td>
			<td>Mean: 27.47 (SD 13.3)</td>
			<td>Mean: 19.5 (SD 2.1)</td>
			<td>Mean: 24.06 (SD 10.8)</td>
		</tr>
		<tr>
			<td>Median: 25.7</td>
			<td>Median: 19.9</td>
			<td>Median: 19.9</td>
		</tr>
		<tr>
			<td rowspan="2">VEGF (pg/mg total protein)</td>
			<td>Mean: 126.2 (SD 25.8)</td>
			<td>Mean: 106.0 (SD 14.5)</td>
			<td>Mean: 120.6 (SD 25.1)</td>
		</tr>
		<tr>
			<td>Median: 131.6</td>
			<td>Median: 103.5</td>
			<td>Median: 110.8</td>
		</tr>
		<tr>
			<td>Porcine Kidneys</td>
			<td>n = 4</td>
			<td>n = 3</td>
			<td>n = 7</td>
		</tr>
		<tr>
			<td rowspan="2">LH (mIU/mg total protein)</td>
			<td>Mean: 13.2 (SD 3.6)</td>
			<td>Mean: 12.3 (SD 5.5)</td>
			<td>Mean: 12.8 (SD 4.5)</td>
		</tr>
		<tr>
			<td>Median: 13.6</td>
			<td>Median: 10.3</td>
			<td>Median: 11.2</td>
		</tr>
		<tr>
			<td rowspan="2">VEGF (pg/mg total protein)</td>
			<td>Mean: 2987.2 (SD 772.5)</td>
			<td>Mean: 2354.1 (SD 932.4)</td>
			<td>Mean: 2715.9 (SD 901.0)</td>
		</tr>
		<tr>
			<td>Median: 3324.67</td>
			<td>Median: 2377.3</td>
			<td>Median: 3226.4</td>
		</tr>
	</tbody>
</table>
Table 1: Mean and median LH and VEGF levels by animal type and sex.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60785/60785fig1.jpg" /> 
Figure 1: LH/VEGF linear relationship in adult bovine kidneys (n = 7). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60785/60785fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60785/60785fig2.jpg" /> 
Figure 2: LH/VEGF linear relationship in adult porcine kidneys (n = 7). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60785/60785fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

demonstrating-linear-relationship-between-vascular-endothelial-growth

Nanyang Technological University

Research

JoVE Journal

Biology

4.7K Views.  Zietchick Research Institute. Ons protocol merkt op dat het gebruik van nierextracten van koeien en varkens een uitstekende en economische hulpbron is voor de studie van nierfysiologie, met name het onderzoeken van de correlatie tussen vasculaire endotheelgroeifactor en andere analyten. Het is altijd een uitdaging geweest om VEGF, een essentiële groeifactor in orgaan angiogenese, te correleren met andere eiwitten. Deze techniek stelt ons in staat om de associatie tussen VEGF en analyten zoals hormonen te onderzoeken....

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

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

Developmental Biology

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Neuroscience

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Medicine

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Comparative Proteomic Analysis of Whole Kidney, Medulla, and Cortical Tubules in Diabetic Pathogenesis of Kidney Injury in Mice

Defining the sequence of events in renal disease is the cornerstone of clinical practice in the nephrologist toolkit. Tissue proteomic analyses are a significant approach to understanding the fundamental physiological and molecular processes of renal pathophysiology. The methods and protocols we present here will allow for the molecular dissection of the kidney in each specific region of interest related to disease sequelae. To determine the effects of disease on specific kidney regions and structures with unique functions, the goals of this protocol are to demonstrate simplified mouse kidney compartmentalization and renal cortical tubule isolation techniques in tandem with streamlined label-free quantitative proteomic workflows. Combining these methods will assist in the identification of perturbed molecular patterns in the whole kidney, medullary compartments, and cortical tubule structures of kidneys, with the ultimate and eventual goal of single-cell proteomics in pathological contexts. Applying these methods in virtually any disease model will be helpful in delineating mechanisms of pathology related to kidney dysfunction.

Here, we present a detailed protocol for proteomic analysis of the whole kidney, isolated cortical tubule, and medullary proteomes. The study also compares regional proteomes in a diabetic mouse model and non-diabetic mice.

Defining the sequence of events in renal disease is the cornerstone of clinical practice in the nephrologist toolkit. Tissue proteomic analyses are a ...

Biochemistry

Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing factor. Single-point sampling measures are inadequate to fully understand the biological significance of the secretory pattern of pulsatile hormones either under normal physiologic conditions or during conditions of dysregulation. Luteinizing hormone (LH) is synthesized by the anterior pituitary gonadotrope&#160;cells and secreted in a pulsatile pattern which requires frequent collection of blood samples for pulse assessment. This has not been possible in mice until recently, due to the development of a high-sensitivity LH assay and advancement in a technique for frequent low-volume sample collection, initially described by Steyn and colleagues.1 Here we describe a protocol for the frequent peripheral blood sample collection from mice with sufficient handling acclimatization to detect pulsatile secretion of LH. The current protocol details an expanded acclimatization period that allows assessment of robust and continuous pulses of LH over multiple hours. In this protocol, the tip of the tail is clipped&#160;and blood is collected from the tail using a hand-held pipette. For assessment of pulsatile LH in gonadectomized mice, serial samples are collected every 5-6 min for 90-180 min. Importantly, the collection of blood and measurement of robust pulses of LH can be accomplished in awake, freely behaving mice, given adequate handling acclimatization and effort to minimize environmental stressors. Sufficient acclimatization can be achieved within 4-5 weeks prior to blood collection. This protocol highlights advances in the methodology to ensure collection of whole blood samples for assessment of pulsatile LH secretion patterns over multiple hours in the mouse, a powerful animal model for neuroendocrine research.

Here we present a tail-tip blood sampling protocol for frequent sample collection in unrestrained mice. This method is useful for assessing patterns of pulsatile luteinizing hormone secretion and could be adapted for analysis of other circulating factors.

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

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 ...

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes patients to CKD later in life. Kidney epithelial cells have been identified as key signaling nodes in both AKI and CKD, whereby the cells can determine the course of the disease through the secretion of cytokines and other proteins. In CKD especially, several lines of evidence have demonstrated that maladaptively repaired tubular cells drive disease progression through the secretion of transforming growth factor-beta (TGF-&#946;), connective tissue growth factor (CTGF), and other profibrotic cytokines. However, identifying the source and the relative number of secreted proteins from different cell types in vivo remains challenging.
This paper describes a technique using brefeldin A (BFA) to prevent the secretion of cytokines, enabling the staining of cytokines in kidney tissue using standard immunofluorescent techniques. BFA inhibits endoplasmic reticulum (ER)-to-Golgi apparatus transport, which is necessary for the secretion of cytokines and other proteins. Injection of BFA 6 h before sacrifice leads to a build-up of TGF-&#946;, PDGF, and CTGF inside the proximal tubule cells (PTCs) in a mouse cisplatin model of AKI and TGF-&#946; in&#160;a mouse aristolochic acid (AA) model of CKD. Analysis revealed that BFA + cisplatin or BFA + AA increased TGF-&#946;-positive signal significantly compared to BFA + saline, cisplatin, or AA alone. These data suggest that BFA can be used to identify the cell type producing specific cytokines and quantify the relative amounts and/or different types of cytokines produced.

Identifying the cell type responsible for secreting cytokines is necessary to understand the pathobiology of kidney disease. Here, we describe a method to quantitatively stain kidney tissue for cytokines produced by kidney epithelial or interstitial cells using brefeldin A, a secretion inhibitor, and cell-type-specific markers.

Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes ...

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

Samenvatting

Meer video's verkennen