The authors have declared that no competing interests exist.
Conceived and designed the experiments: SRK ABP. Performed the experiments: SJ BTS WW. Analyzed the data: SJ ABP. Contributed reagents/materials/analysis tools: SJ BTS WW ABP. Wrote the paper: SJ ABP SRK. Prepared figures: SJ ABP.
We have previously shown that production of reactive oxygen species (ROS) is an important contributor to renal injury and inflammation following exposure to oxalate (Ox) or calcium-oxalate (CaOx) crystals. The present study was conducted, utilizing global transcriptome analyses, to determine the effect of Apocynin on changes in the NADPH oxidase system activated in kidneys of rats fed a diet leading to hyperoxaluria and CaOx crystal deposition.
Age-, sex- and weight-matched rats were either fed regular rat chow or regular rat chow supplemented with 5% w/w hydroxy-L-proline (HLP). Half of the rats on the HLP diet were also placed on Apocynin-supplemented H2O. After 28 days, each rat was euthanized, their kidneys freshly explanted and dissected to obtain both cortex and medulla tissues. Total RNA was extracted from each tissue and subjected to genomic microarrays to obtain global transcriptome data. KEGG was used to identify gene clusters with differentially expressed genes. Immunohistochemistry was used to confirm protein expressions of selected genes.
Genes encoding both membrane- and cytosolic-NADPH oxidase complex-associated proteins, together with
A strong up-regulation of an oxidative/respiratory burst involving the NADPH oxidase system, activated via the angiotensin-II and most likely the DAG-PKC pathways, occurs in kidneys of hyperoxaluric rats. Apocynin treatment reversed this activation without affecting the levels of hyperoxaluria.
Oxalate (Ox) is a naturally-occurring, highly oxidized organic compound with powerful chelating activity that can cause death at high concentrations in animals and occasionally humans due to its toxic corrosive effects on cells. More commonly, however, higher concentrations of Ox in human fluids can cause a variety of pathological disorders, including hyperoxaluria, cardiomyopathy, cardiac conductance disorders, renal failure and, in particular, calcium oxalate (CaOx) nephrolithiasis
Although Ox is endogenously produced via liver metabolism, dietary Ox is also a major contributor to urinary Ox excretion in most individuals, with recent studies indicating that dietary Ox can contribute as much as 50% of the daily urinary oxalate excretion
In order to study the dynamics of calcium oxalate nephrolithiasis, nephrocalcinosis, metabolic acidosis, hematuria and renal failure, a number of animal models have been developed. The best studied models involve the ingestion of ethylene glycol (EG) or hydroxy-L-proline (HLP). Rats treated with EG or HLP develop hyperoxaluria leading to crystal deposition and production of reactive oxygen species (ROS), lipid peroxidation and cellular injury
Earlier studies from our laboratory
The figure shows various cytosolic and membrane components of NADPH oxidase subunits getting activated via Renin/angiotensin receptor-2 activation of the NADPH-oxidases during the development of hyperoxaluria, resulting in increased production of reactive oxygen species (ROS).
In the present study, we have analyzed changes in the global transcriptome of renal tissues following development of hyperoxaluria in rats fed HLP over a 4 week period, either with or without Apocynin treatment. All rats fed HLP developed hyperoxaluria as well as depositions of CaOx crystals in the kidneys. Results presented herein show an increase in the expression of genes involved in the activation of NADPH oxidase with a concomitant decrease in the expression of genes encoding the ROS scavenger proteins, catalase (Cat) and superoxide dismutase-1 (Sod1), in response to hyperoxaluria and crystal deposition. Oral treatment with Apocynin, a known inhibitor of NADPH oxidase complex assembly and oxidative stress, reversed not only the transcriptome profile of the NADPH oxidase-associated genes, but also multiple molecular pathways involving numerous cell components. These data raise the possibility that apocynin is a broad-spectrum anti-oxidant.
Male Sprague–Dawley rats (n = 18), 8 weeks of age and weighing on average about 150 gm, were purchased from Harlan Laboratories and permitted to acclimate for 2 weeks within the University of Florida's Animal Care Facilities prior to any experimental procedures. Rats were divided into three groups of 6 each: Group 1 rats were fed a normal rat chow and sterile water, Group 2 rats received a diet similar to Group 1 rats, except the food was supplemented with HLP (ICN Biochemicals, Aurora, OH) to 5%, while Group 3 rats received a diet similar to Group 2 rats, except their water was supplemented with Apocynin to 4 mM. To prepare the rat chow, feed pellets were ground, then moistened with water and formed into “cookies”, which were individually weighed. All rats had free access to food and water, and consumption of food and intake of water were recorded daily. Each rat was weighed weekly to check its growth. At day 28 post-treatment, the rats were sedated, then euthanized and their kidneys freshly explanted. All procedures were approved by the University of Florida's IACUC and were in accord with recommendations of the NIH Guide for the Care and Use of Laboratory Animals.
Preparation of total RNA from each of the 36 individual specimens was performed as described in detail elsewhere
Microarray analysis was conducted at the University of Florida's Interdisciplinary Center for Biotechnology Research using the IlluminaTM bead array reader, while all gene expression data were analyzed using The Genome Studio Gene Expression Module V1.0. Before comparative analysis, the individual signal intensity values obtained from the microarray probes were log transformed (using 2 as the base) and normalized between all individual samples within the six sets in this study. After normalizing the signal intensity values for each of the 36 arrays, the Student's t-test was performed considering a probe-by-probe comparison between two groups at a time. In each comparison, p-value and fold changes (FC) were computed for each gene based on the n = 6 replicate samples within each experimental group and volcano plots were drawn for each comparison (
Kidneys from each rat were surgically removed at time of euthanasia, one of which was used for RNA preparation, while the second was placed in 10% phosphate buffered formalin for 24 hours for eventual histological examinations. The formalin-fixed tissues were embedded in paraffin and sectioned to a thickness of 5-μm. Deparaffinization of paraffin-embedded slides was performed by xylene immersion and subsequent dehydration in ethanol. Slides were incubated overnight at 4°C with primary antibodies reactive to either Opn, MCP-1/Ccl2, p47 or Nox-2 (Abcam, Cambridge, MA). Isotype controls were performed using rabbit IgG. Slides were incubated for 30 min in biotinylated goat anti-rabbit IgG followed by incubation with biotinylated horseradish peroxidase using the Vectastain® ABC kit. Staining was developed by addition of diaminobenzidine (DAB) substrate (Vector Labs, Burlingame, CA) and counterstained with hematoxylin. To ensure that positive infiltrate staining for these antibodies was not due to high background staining, an additional run was performed using 10 mM citrate buffer for antigen-retrieval with all other procedures unchanged. Antigen-retrieval was carried out in 25 mMTris/EDTA buffer, pH 9.1 at 60°C for 20 min under 18 psi pressure. Images were taken using the Zeiss Axiovert 200M microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY).
As anticipated, rats fed a HLP-free diet remained normo-oxaluric and devoid of any crystals, while the HLP-supplemented diet led to hyperoxaluria and variable levels of CaOx crystal depositions in all rats
(
Of the 21,000 rat genes represented on the array, 3302 and 2894 were found to be significantly differentially-expressed at a 2-fold change in the cortex and medulla, respectively, in a comparison between control and HLP-fed rats, while 3000 and 2505 genes, respectively, were found to be significantly expressed in the cortex and medulla in the comparison between control and HLP+Apocynin-fed rats. Using KEGG to curate the differentially-expressed genes, several gene clusters were identified that defined specific biological processes for the HLP-fed and HLP+Apocynin-fed rats, as presented in
Category | Signaling Pathway | Count | Percentage | p-value | Benjamini |
|
MAPK signaling pathway | 29 | 3.5 | 3.6E-3 | 1.4E-1 |
|
TGF-beta signaling pathway | 13 | 1.6 | 6.3E-3 | 1.9E-1 |
|
Fc epsilon RI signaling pathway | 12 | 1.5 | 6.6E-3 | 1.7E-1 |
|
Chemokine signaling pathway | 19 | 2.3 | 1.8E-2 | 2.1E-1 |
|
Gap junction | 11 | 1.3 | 2.7E-2 | 2.3E-1 |
|
Complement and coagulation cascades | 10 | 1.2 | 2.8E-2 | 2.2E-1 |
|
Focal adhesion | 19 | 2.3 | 5.6E-2 | 3.5E-1 |
|
Insulin signaling pathway | 14 | 1.7 | 6.4E-2 | 3.5E-1 |
|
ErbB signaling pathway | 10 | 1.2 | 7.8E-2 | 3.9E-1 |
|
Non-small cell lung cancer | 7 | 0.8 | 1.0E-1 | 4.1E-1 |
|
Steroid hormone biosynthesis | 12 | 1.5 | 4.2E-5 | 6.8E-3 |
|
Cytokine-cytokine receptor interaction | 25 | 3.0 | 9.9E-4 | 7.8E-2 |
|
Metabolism of xenobiotics by cytochrome P450 | 11 | 1.3 | 3.4E-3 | 1.7E-1 |
|
Jak-STAT signaling pathway | 17 | 2.1 | 1.1E-2 | 2.4E-1 |
|
Autoimmune thyroid disease | 10 | 1.2 | 1.2E-2 | 2.2E-1 |
|
Graft-versus host disease | 9 | 1.1 | 1.2E-2 | 2.0E-1 |
|
GnRH signaling pathway | 13 | 1.6 | 1.3E-2 | 1.9E-1 |
|
Allograft rejection | 9 | 1.1 | 1.5E-2 | 2.0E-1 |
|
Arginine and proline metabolism | 9 | 1.1 | 1.5E-2 | 2.0E-1 |
|
Calcium signaling pathway | 20 | 2.4 | 1.7E-2 | 2.1E-1 |
|
Neuroactive ligand-receptor interaction | 26 | 3.1 | 1.9E-2 | 2.0E-1 |
|
Taurine and hypotaurine metabolism | 4 | 0.5 | 2.0E-2 | 2.0E-1 |
|
Long-term depression | 10 | 1.2 | 2.1E-2 | 2.0E-1 |
|
Retinol metabolism | 9 | 1.1 | 2.8E-2 | 2.2E-1 |
|
Drug metabolism | 10 | 1.2 | 3.2E-2 | 2.4E-1 |
|
Antigen processing and presentation | 11 | 1.3 | 4.4E-2 | 3.0E-1 |
|
Biosynthesis of unsaturated fatty acids | 5 | 0.6 | 5.7E-2 | 3.4E-1 |
|
ECM-receptor interaction | 10 | 1.2 | 6.1E-2 | 3.5E-1 |
|
Type I diabetes mellitus | 8 | 1.0 | 7.5E-2 | 3.9E-1 |
|
Alpha-Linolenic acid metabolism | 4 | 0.5 | 8.4E-2 | 4.0E-1 |
|
VEGF signaling pathway | 9 | 1.1 | 8.5E-2 | 4.0E-1 |
|
Glutathione metabolism | 7 | 0.8 | 8.6E-2 | 3.9E-1 |
|
Viral myocarditis | 10 | 1.2 | 8.8E-2 | 3.8E-1 |
|
Purine metabolism | 15 | 1.8 | 9.4E-2 | 4.0E-1 |
|
Hedgehog signaling pathway | 7 | 0.8 | 1.0E-1 | 4.1E-1 |
The gene heading indicates number of genes mapped to an ontology category. The first ten pathways are common between the cortex and the medulla. P-values derived from Fisher's exact test and Benjamini multiple test correlation.
Focusing specifically on the proteins involved in the activation of the Agtr-NADPH oxidase pathway
(
In an attempt to define a possible mechanism activating the NADPH oxidase systems, we examined whether the rennin/angiotensin/angiotensin-receptor system might be involved. While the gene encoding angiotensin,
Genes encoding proteins of renin, angiotensin, and angiotensin receptor 2 were up regulated in the cortex and medulla of HLP-treated rats (Red bars) but interestingly down regulated in the HLP+Apocynin rats (Green bars). Angiotensin receptor 1and 1a showed differential expression for both the cortex and medulla in the HLP fed and HLP-Apocynin fed rats.
Genes downstream of angiotensin receptor-2 encoding important immune cell-associated signaling pathways for the activation of NADPH oxidase expressed in the cortex or medulla of HLP-treated rats showing up regulation (Red bars), but exhibiting down-regulation in the cortex and medulla of HLP+Apocynin-treated rats (Green bars).
Multiple published studies have clearly shown that a set of specific proteins are differentially-expressed in the kidneys of rats in response to treatments that induce a state of hyperoxaluria
As expected the HLP-fed rats had osteopontin (
(
Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis of some 3000 genes per run (using a p-value <0.1 cut-off) was carried out for two different sets of genes, one cortex tissue-derived and the other medulla tissue-derived. In this way, we were able to compare the differential expression of renal tissue genes between rats exhibiting primarily hyperoxaluria/CaOx deposition and healthy non diseased rats. Using the pathway analysis of these 3000 gene sets by DAVID (Database for Annotation, Visualization of Integrated Discovery) enrichment analysis tool from National Institute of Allergy and Infectious Diseases (NIAID), we identified several pathways which were highly significant and unique within the experimental groups. Using four common genes whose proteins are well-documented to be differentially-expressed with development of calcium-oxalate urolithiasis (
Two of these gene products, Opn (encoded by
Genes encoding proteins involved in the ROS pathway during development of hyperoxaluria and CaOx crystal deposition in the kidneys suggest two distinct molecular pathways that meld into a similar outcome. First, analyses of differentially-expressed genes from cortical and medullar sections of kidneys explanted from HLP-treated rats revealed similar activations of the multiple genes associated with assembly of NADPH oxidase complexes, as well as genes encoding upstream proteins indicating activation of the rennin/angiotensin/angiotensin receptor-2 signaling pathway(s). Interestingly, however, we also discovered the concomitant up-regulation of genes encoding both gp91
The fact that oxalate and CaOx crystals appear to induce strong ROS activation, especially in the absence of any concomitant up-regulation of ROS scavengers such as SOD and catalase, suggests the renal tissues during prolonged hyperoxaluria are bathed in free radicals and molecules with unpaired electrons, including superoxide anion (O2–), hydroxyl radical (OH), and hydrogen peroxide (H2O2). O2− anions are produced by NADPH oxidases, xanthine oxidase, lipooxigenase, cyclooxygenase, hemeoxygenase, as well as a byproduct of mitochondrial respiration. While lipid radicals can also produce O2−, NO Radicals are produced by the endothelial nitric oxide synthase (eNOS) mediated oxidation of L-arginine. Not surprising, then, that the arginine/proline metabolism pathway is identified as an activated biological process by KEGG. In addition, reactions between superoxide and nitric oxide can produce the highly reactive peroxynitrite molecule, ONOO−. Since oxidative stress ultimately leads to injury and inflammation, it is not surprising that additional biological processes identified in KEGG analyses include cellular recruitment, various inflammatory responses, cellular stress, apoptosis and functional compensations, as listed in
Category | Signaling Pathway | Count | Percentage | p-value | Benjamini |
|
ErbB signaling pathway | 14 | 1.9 | 1.5E-5 | 4.1E-2 |
|
TGF-beta signaling pathway | 14 | 1.9 | 1.7E-3 | 3.5E-2 |
|
MAPK signaling pathway | 28 | 3.7 | 4.7E-3 | 7.5E-2 |
|
Chemokine signaling pathway | 20 | 2.7 | 6.6E-3 | 8.7E-2 |
|
Non-small cell lung cancer | 9 | 1.2 | 1.2E-2 | 1.1E-1 |
|
Insulin signaling pathway | 16 | 2.1 | 1.2E-2 | 1.1E-1 |
|
Focal adhesion | 21 | 2.8 | 1.3E-2 | 1.1E-1 |
|
Complement and coagulation cascades | 10 | 1.3 | 2.3E-2 | 1.7E-1 |
|
Gap junction | 10 | 1.3 | 5.3E-2 | 2.7E-1 |
|
Fc epsilon RI signaling pathway | 9 | 1.2 | 8.4E-2 | 3.7E-1 |
|
Chronic myeloid leukemia | 17 | 2.3 | 6.7E-6 | 1.1E-3 |
|
Pathways in cancer | 36 | 4.8 | 2.8E-4 | 2.3E-2 |
|
Cell cycle | 19 | 2.5 | 4.7E-4 | 2.6E-2 |
|
Pancreatic cancer | 13 | 1.7 | 7.2E-4 | 2.9E-2 |
|
Neutrophin signaling pathway | 18 | 2.4 | 1.3E-3 | 4.2E-2 |
|
Lysosome | 17 | 2.3 | 1.6E-2 | 3.6E-2 |
|
Sphingolipid metabolism | 9 | 1.2 | 3.1E-3 | 5.5E-2 |
|
Acute myeloid leukemia | 10 | 1.3 | 5.0E-3 | 7.3E-2 |
|
Renal cell carcinoma | 11 | 1.5 | 7.6E-3 | 9.3E-2 |
|
Colorectal cancer | 12 | 1.6 | 8.6E-3 | 9.7E-2 |
|
Small cell lung cancer | 12 | 1.6 | 1.0E-2 | 1.1E-1 |
|
Fc gamma R-mediated phagocytosis | 12 | 1.6 | 1.6E-2 | 1.3E-1 |
|
Prion disease | 7 | 0.9 | 1.7E-2 | 1.3E-1 |
|
Notch signaling pathway | 8 | 1.1 | 2.6E-2 | 1.8E-1 |
|
Glioma | 9 | 1.2 | 2.8E-2 | 1.9E-1 |
|
Endometrial cancer | 8 | 1.1 | 3.1E-2 | 2.0E-1 |
|
Endocytosis | 20 | 2.7 | 3.2E-2 | 2.0E-1 |
|
P53 signaling pathway | 9 | 1.2 | 4.3E-2 | 2.4E-1 |
|
Glycerolipid metabolism | 7 | 0.9 | 4.6E-2 | 2.5E-1 |
|
T-cell receptor signaling pathway | 12 | 1.6 | 6.2E-2 | 3.1E-1 |
|
Bladder cancer | 6 | 0.8 | 6.2E-2 | 3.0E-1 |
|
Apoptosis | 10 | 1.3 | 6.8E-2 | 3.1E-1 |
|
Prostate cancer | 10 | 1.3 | 9.0E-2 | 3.8E-1 |
|
Toll-like receptor signaling pathway | 10 | 1.3 | 9.0E-2 | 3.8E-1 |
|
mTOR signaling pathway | 7 | 0.9 | 9.6E-2 | 3.9E-1 |
The gene heading indicates number of genes mapped to an ontology category. The first ten pathways are common in the cortex and the medulla. P-value derived from Fisher's exact test and Benjamini multiple test correlation.
Previous research has suggested that, based on the core protein(s) of Flavocytochrome b558, seven distinct isoforms of NADPH oxidase exist: Nox1 to Nox 5 and the structurally similar Duox1 and Duox2 molecules
Considering the complex structure of the kidney, it is not surprising that there is also a wide distribution in various NADPH oxidase subunit expressions, including the multiple isoforms of Nox. ROS are produced by various cell types of the kidneys, such as tubular cells, endothelial cells, vascular smooth muscle cells, fibroblasts and podocytes
Lastly, one cautionary note must be considered in interpretation of the current data. Although analyses of transcriptome data are a powerful tool, it is also dependent on a number of variables, one of which is the starting tissue. In the current study, it must be pointed out that dissection of the kidney into medulla and cortex is technically difficult, leading to the strong probability that each may have some contaminating cells derived from the other tissue fraction. Similarly, there was no attempt in the current study to isolate renal tissue from infiltrating neutrophils, a fact that no doubt affects the transcriptome data. Thus, assigning precise molecular mechanisms to specific tissues or regions will require more detailed studies. Nevertheless, an in-depth analysis of the genes that identify these specific biological pathways or cellular compartments should provide important up-stream and down-stream steps in renal tissue responses to hyperoxaluria, subsequent development of CaOx crystals and CaOx crystal depositions leading to an eventual onset of the inflammatory responses which clearly involve ROS production. Of clear importance, the current study provides strong evidence that multiple unique and similar changes are occurring in the renal tissues at the molecular and cellular level, whether the rats are treated with HLP or with HLP+Apocynin, despite minimal changes noted at the gross level. These observations raise the question whether apocynin is merely an inhibitor of NADPH oxidase complex assembly or a molecule with strong anti-oxidant inductive activities. Further analyses of the extensive transcriptome data may provide additional clues.
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The authors thank Ms. Ginger Clark, Dr. Jin Yao and Dr. Yijun Sun from the University of Florida's Interdisciplinary Center for Biotechnology Research (ICBR) for running the microarrays and providing expert assistance in data analyses.