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 homo....

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. Prep....

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).......

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.......

Materials

List of materials used in this article

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