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

Summary

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.

Abstract

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'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.

Introduction

Vascular endothelial growth factor A (VEGF-A), helps to regulate angiogenesis and vascular permeability in the kidney and other organs^1,2 (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 birth^3,4.

On the other hand, overexpression of renal VEGF causes proteinuria and glomerular hypertrophy^3,4. 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 dysregulation^2,5. 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-understood^2,6. 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 testis^7,8. Previous studies have provided evidence that LH also helps regulate VEGF in non-reproductive organs, such as the eyes^6,9,10. LH receptors are found in the medulla and cortex of the kidney^11,12. Of note, kidney tubular epithelial cells, as well as the LH receptor, express VEGF^11,12,13,14. Taking these two observations together, we hypothesized that LH also helps regulate VEGF expression in the kidney^13,14. 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.

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Protocol

No live or experimental animals were used for this study.

1. Tissue Handling

1. Procure bovine and porcine whole kidneys immediately after slaughter from an abattoir. Transport on ice to the laboratory.
2. Upon arrival at laboratory, rinse kidneys with 50 mL of ice-cold phosphate buffered saline (PBS). Repeat this step 2x to remove blood completely.
3. Keep kidneys on ice (or refrigerated) until further extraction.

2. Dissection of Kidneys

1. Use sterile scissors, forceps, a knife, and Petri dishes to dissect the kidneys and excise the required tissue portion.
2. 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.
3. 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).
4. Mince the tissue block into small pieces with a knife to assist the homogenization process.
5. 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.

3. Tissue Homogenization

1. Label the microfuge tubes with specific sample details for tissue supernatant collection.
2. 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.
3. Subject the tissue extracts immediately for centrifugation in the refrigerated centrifuge at 9,600 x g for 5 min at 4 °C.
4. Remove the tubes from the centrifuge and place them on the ice bucket.
5. Collect the supernatant into a new labeled microfuge tube and store on ice. Discard the pellet.
6. 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.

4. Bovine and Porcine LH ELISA Assay

1. Store all ELISA assay components included in the commercially available luteinizing hormone (LH) ELISA kit (see Table of Materials) at 2-8 °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's instructions.
2. 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 °C until use.
3. Dilute the wash buffer (15 mL of 20x concentrate) to 300 mL with double-distilled water
4. Set up the blank wells without any solution.
5. Add 50 µL of standard or sample to each well (n = 2), then add another 50 µL of horseradish peroxidase (hrp)-conjugate to each well. Immediately add another 50 µL of antibody solution to each well. Seal the plate, mix well, and incubate for 1 h at 37 °C.
6. Wash the wells with 1x wash buffer (200 µL/well) and repeat 4x.
7. Add 50 µL of substrate A and 50 µL 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 °C in the dark for 15 min.
8. Add 50 µL of stop solution to each well, gently tap the plate, and read the plate using the spectrophotometer set to a 450 nm wavelength.
9. Normalize bovine and porcine LH levels to total protein (see section 6).

5. Bovine and Porcine VEGF-A ELISA Assay

1. Store all ELISA assay components included in the commercially available Vascular Endothelial Growth Factor-A ELISA kits (see Table of Materials) at 2-8 °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's instructions.
2. 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 °C until use.
3. Dilute the wash buffer (15 mL of 20x concentrate) to 300 mL with double-distilled water
4. Add 100 µL of the standard or sample to each well (n = 2). Seal the plate, mix well, and incubate for 2 h at 37 °C.
5. Remove the liquid in each well and add 100 µL of detection reagent A to each well, seal the plate, and incubate for 1 h at 37 °C.
6. Wash the wells with 1x wash buffer (400 µL/well) and repeat 4x.
7. Add 100 µL 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 °C.
8. Wash the wells with 1x wash buffer (400 µL/well) and repeat 4x.
9. Add 90 µL of substrate solution to each well, gently tap the plate, and incubate for 1 h at 37 °C.
10. Add 50 µL of stop solution to each well, gently tap the plate, and read the plate using the spectrophotometer set to a 450 nm wavelength.
11. Normalize bovine and porcine VEGF-A levels to total protein (section 6).

6. Total Protein Estimation

1. 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's recommendations.

7. Statistical Analysis

1. Calculate the mean, median, and standard deviation of each analyte.
2. 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.
3. Under appropriate circumstances (such as normal data distribution), utilize regression models to examine the linear relationship between LH and VEGF-A.

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Results

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)...

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Discussion

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-mortem^15,16, VEGF-studies in human cadaver kidneys may not reflect rea...

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Materials

Name	Company	Catalog Number	Comments
Bovine LH ELISA Kit	MyBiosource, San Diego, CA.	MBS700951
Bovine VEGF-A ELISA Kit	MyBiosource, San Diego, CA.	MBS2887434
Micro BCA Protein Assay Kit	ThermoFisher Scientific Inc, Columbus, OH.	23235
Porcine LH ELISA Kit	MyBiosource, San Diego, CA.	MBS009739
Porcine VEGF-A ELISA	Ray Biotech, Norcross, GA.	ELP-VEGFA-1
RIPA Lysis and Extraction Buffer	ThermoFisher Scientific Inc, Columbus, OH.	89901