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 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.
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.
No live or experimental animals were used for this study.
1. Tissue Handling
2. Dissection of Kidneys
3. Tissue Homogenization
4. Bovine and Porcine LH ELISA Assay
5. Bovine and Porcine VEGF-A ELISA Assay
6. Total Protein Estimation
7. Statistical Analysis
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: LH/VEGF linear relationship in adult bovine kidneys (n = 7). Please click here to view a larger version of this figure.
Figure 2: LH/VEGF linear relationship in adult porcine kidneys (n = 7). Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
The authors thank Scholl's Slaughterhouse (Blissfield, MI) for providing the bovine and porcine kidneys. No grant funding was utilized for this study.
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 |