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

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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.

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Muthusamy, A., Arivalagan, A., Movsas, T. Z. Demonstrating a Linear Relationship Between Vascular Endothelial Growth Factor and Luteinizing Hormone in Kidney Cortex Extracts. J. Vis. Exp. (155), e60785, doi:10.3791/60785 (2020).

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 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.

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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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Representative 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).

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.

Sample Type Males Females All
Bovine Kidneys n = 4 n = 3 n = 7
LH (mIU/mg total protein) Mean: 27.47 (SD 13.3) Mean: 19.5 (SD 2.1) Mean: 24.06 (SD 10.8)
Median: 25.7 Median: 19.9 Median: 19.9
VEGF (pg/mg total protein) Mean: 126.2 (SD 25.8) Mean: 106.0 (SD 14.5) Mean: 120.6 (SD 25.1)
Median: 131.6 Median: 103.5 Median: 110.8
Porcine Kidneys n = 4 n = 3 n = 7
LH (mIU/mg total protein) Mean: 13.2 (SD 3.6) Mean: 12.3 (SD 5.5) Mean: 12.8 (SD 4.5)
Median: 13.6 Median: 10.3 Median: 11.2
VEGF (pg/mg total protein) Mean: 2987.2 (SD 772.5) Mean: 2354.1 (SD 932.4) Mean: 2715.9 (SD 901.0)
Median: 3324.67 Median: 2377.3 Median: 3226.4

Table 1: Mean and median LH and VEGF levels by animal type and sex.

Figure 1
Figure 1: LH/VEGF linear relationship in adult bovine kidneys (n = 7). Please click here to view a larger version of this figure.

Figure 2
Figure 2: LH/VEGF linear relationship in adult porcine kidneys (n = 7). Please click here to view a larger version of this figure.

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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-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.

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Disclosures

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.

Acknowledgments

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

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

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References

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  2. Majumder, S., Advani, A. VEGF and the diabetic kidney: More than too much of a good thing. Journal of Diabetes and its Complications. 31, (1), 273-279 (2017).
  3. Liu, E., et al. Increased expression of vascular endothelial growth factor in kidney leads to progressive impairment of glomerular functions. Journal of the American Society of Nephrology. 18, (7), 2094-2104 (2007).
  4. Eremina, V., et al. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. Journal of Clinical Investigation. 111, (5), 707-716 (2003).
  5. Ferrara, N. Vascular endothelial growth factor: basic science and clinical progress. Endocrine Reviews. 25, (4), 581-611 (2004).
  6. Movsas, T. Z., Sigler, R., Muthusamy, A. Vitreous Levels of Luteinizing Hormone and VEGF are Strongly Correlated in Healthy Mammalian Eyes. Current Eye Research. 43, (8), 1041-1044 (2018).
  7. Babitha, V., et al. Luteinizing hormone, insulin like growth factor-1, and epidermal growth factor stimulate vascular endothelial growth factor production in cultured bubaline granulosa cells. General and Comparative Endocrinology. 198, 1-12 (2014).
  8. Trau, H. A., Davis, J. S., Duffy, D. M. Angiogenesis in the Primate Ovulatory Follicle Is Stimulated by Luteinizing Hormone via Prostaglandin E2. Biology of Reproduction. 92, (1), 15 (2015).
  9. Movsas, T. Z., et al. Confirmation of Luteinizing Hormone (LH) in Living Human Vitreous and the Effect of LH Receptor Reduction on Murine Electroretinogram. Neuroscience. 385, 1-10 (2018).
  10. Movsas, T. Z., Sigler, R., Muthusamy, A. Elimination of Signaling by the Luteinizing Hormone Receptor Reduces Ocular VEGF and Retinal Vascularization during Mouse Eye Development. Current Eye Research. 43, (10), 1286-1289 (2018).
  11. Hipkin, R. W., Sanchez-Yague, J., Ascoli, M. 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. Molecular Endocrinology. 6, (12), 2210-2218 (1992).
  12. Lei, Z. M., et al. Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene. Molecular Endocrinology. 15, (1), 184-200 (2001).
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  15. Ondruschka, B., et al. Post-mortem in situ stability of serum markers of cerebral damage and acute phase response. International Journal of Legal Medicine. 133, (3), 871-881 (2019).
  16. Swain, R., et al. Estimation of post-mortem interval: A comparison between cerebrospinal fluid and vitreous humour chemistry. Journal of Forensic and Legal Medicine. 36, 144-148 (2015).
  17. Thompson, C. S., Traynor, I. M., Fodey, T. L., Faulkner, D. V., Crooks, S. R. H. Screening method for the detection of residues of amphenicol antibiotics in bovine, ovine and porcine kidney by optical biosensor. Talanta. 172, 120-125 (2017).
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