In this study, we determined the body weight and relative organ weight to body weight of rats exposed to TAA to evaluate TAA general toxicity. The body weight decreased, however, the relative organ weights including those of the liver and kidneys were increased. A previous report suggested that a reduction in body weight of TAA-treated rats might be due, in part, to gastrointestinal toxicity and the concomitant loss of the animal’s appetite with subsequent reduction in food intake or the excessive loss of water, salts, and proteins as a result of renal injury, resulting in dehydration and weight loss21. In addition, the enlarged livers in the TAA-treated rats indicated hepatic lesions and liver injury associated with the toxicological effects of TAA21. In our study, the relative liver and kidney weight to body weight showed similar results of significant increases in rats exposed to the high concentration of TAA.
In a previous study, TAA-treated rats showed leukocytosis, granulocytosis, and thrombocytopenia with decreasing red blood cell (RBC) and MCV and increasing WBCs and PLTs22. In our study of the leukocytic parameters, the total WBC counts in rats exposed to TAA were not changed by TAA exposure. However, the numbers of MOs and PLTs were significantly increased in exposed rats compared to the controls. MOs are the largest type of leukocytes and can differentiate into macrophages and conventional dendritic cells. In a previous study, ionized calcium-binding adaptor molecule 1 and galectin-3, which are associated with increasing numbers of CD68 + and CD163 + macrophages, were highly expressed in TAA-induced acute rat liver lesions23. Furthermore, the platelet-activating factor levels were significantly increased in rat blood for one month after the administration of TAA in the drinking water (300 mg/L)24. In erythrocytic parameters, the MCV and HCT values showed similar results, with significant decreases in rats exposed to the high concentration of TAA.
To evaluate the pathological toxic effects in the kidneys of rats exposed to TAA, we performed histopathological observations of basophilia, casts, cysts, inflammatory cell foci, and interstitial fibrosis in kidney tissue. TAA has shown pathological toxic effects in kidneys, resulting in the deposition of collagen in the renal medulla and fibrin in the tubules8, cell death in the terminal portion of the proximal renal tubules7, and the renal tissue infiltration of inflammatory cells, degeneration, sclerosis and necrosis of the glomeruli, interstitial fibrosis, and epithelial shedding11. However, no significant histopathologic abnormalities were found in the kidneys of rats exposed to TAA in the present study (Fig. 1 and Supplementary Table S1). Based on previous pathological studies, a low dose of 25 mg/kg BW was the highest dose that might lead to mild or no kidney injury, but a high dose of 100 mg/kg BW was the minimum dose of TAA that could result in organ injury7,8,11,25.
Therefore, further biochemical studies were performed to evaluate the toxic effects of TAA in the kidneys of rats exposed to TAA. A panel of novel urinary kidney biomarkers was recently approved for the improved detection of acute nephrotoxicity by the U.S. Food and Drug Administration, the European Medicines Agency, and the Pharmaceuticals and Medical Devices Agency (Japan)26. Among them, Fuchs et al. proposed that the most promising biomarkers were NGAL, Kim-1, osteopontin, clusterin, RPA-1, and GSTYb1, detected by multiplexing technologies27. In this study, four protein biomarkers including Kim-1, NGAL, osteopontin, and clusterin, which are biomarkers of injury to the proximal and distal tubules26, were applied to evaluate the nephrotoxicity of TAA using Western blot assays. The expression levels of Kim-1 and NGAL were significantly increased in the kidneys and urine of rats exposed to the high concentration of TAA, resulting in significant nephrotoxicity.
In proteomic analysis, 5221 proteins in the kidneys of rats exposed to TAA were analyzed to determine the protein changes. Twenty-four proteins were significantly up- and downregulated. Of these, the expression of seven proteins including ASAP2, regulator of G protein signaling 14 (RGS14), MAP7 domain containing 1 (MAP7D1), interleukin 3 receptor, alpha (IL-3Rα), Tropomodulin 1 (Tmod1), N-ribosyldihydronicotinamide dehydrogenase quinone (NQO2), and MUP were validated by Western blot assays.
ASAP2 encodes for a 1006-amino acid multi-domain protein and is involved in the regulation of vesicular transport, cellular migration, and autophagy28,29. ASAP2 was also reported to promote tumor growth by facilitating cell cycle progression by phosphorylating the epidermal growth factor receptor30. In this study, the expression levels of ASAP2 showed a threefold increase in the kidney tissue of rats exposed to the high concentration of TAA compared to the control group. TAA has been known as a carcinogen, which might be involved in the activation of cancer cell signaling.
Regulators of G protein signaling (RGS) are intracellular signaling regulators31,32,33. Among the RGS proteins, RGS14 is a highly unusual RGS protein with a multidomain structure that allows it to interact with binding partners from multiple signaling pathways34. It has been reported that RGS14, an upstream regulator of the AC-PKA and Raf⁄MEK⁄ERK signaling pathways, functions globally in stress resistance and the longevity of several species, beyond its specific cellular functions. When RGS14 expression was reduced in rat fibroblast cells, the resistance to oxidative stress increased with higher MnSOD expression35,36. In this study, the expression levels of RGS 14 were significantly decreased in the kidney tissue of rats exposed to the high concentration of TAA compared to the control group. A decrease in RGS14 expression by TAA may reduce the resistance to oxidative stress and renal cellular longevity.
Microtubule-associated proteins (MAPs) are a family of proteins that bind to and stabilize microtubules37,38. Four MAP7 paralogs, MAP7, MAP7D1, MAP7D2, and MAP7D3, are encoded in the mammalian genome, and phylogenetic analysis suggested that MAP7D1 was the most conserved MAP739,40. MAP7 may enhance the membrane expression of transient receptor potential vanilloid 4 (TRPV4) and possibly, link cytoskeletal microfilaments. In renal epithelial cells, TRPV4 channel activation results mainly from hypotonic cell swelling, which suggests that TRPV4 acts as an osmosensor and could play a critical role in renal ischemic/reperfusion injury41,42 and in autosomal recessive polycystic kidney disease43. TRPV4 dysfunction promotes renal cystogenesis in autosomal recessive polycystic kidney disease43. In this study, the expression levels of MAP7D1 were significantly decreased in the kidney tissue of rats exposed to the high concentration of TAA compared to the control group. TA might be involved in kidney damage, causing TRPV4 dysfunction by decreasing the expression of MAP7D1.
IL-3Rα, which is also called CD123, is a cell surface protein that is widely expressed across the various subtypes of acute leukemia44,45,46. Many studies have been published in the immunology, signaling pathway, hematological malignancy, and immunotherapy fields44,45,46,47,48,49. However, few studies have evaluated the renal toxicology of IL-3Rα. In the present study, IL-3Rα was significantly downregulated in the kidney tissue of rats exposed to the high concentration of TAA. The lower expression of IL-3Rα by TAA may reduce the heterodimer formation of IL-3 with other receptors, which might inhibit physiological functions including cell proliferation and survival.
Tmods are unique actin-binding proteins that cap the slow-growing ends of actin filaments, a major component of the cytoskeleton50,51,52. Recently, Wang et al. found that Tmod1 was specifically expressed in distal tubules and the collecting ducts of the kidney that regulate water homeostasis. Furthermore, Tmod1 was found to be closely related to metabolic processes, protein phosphorylation, and multiple signaling pathways by proteomic and bioinformatic analyses53. These results indicate the critical role of Tmod1 in renal function and provide new molecular mechanisms for the regulation of water balance53. In the present study, Tmod1 was significantly downregulated in the kidneys of rats exposed to the high concentration of TAA. The higher expression of the two kidney injury biomarkers Kim-1 and NGAL showed that TAA caused injury to the kidney proximal and distal tubules. Therefore, the significant downregulation of Tmod1 showed that TAA may play a critical role in kidney function.
NQO2 is a cytosolic enzyme. The initial Northern blot findings in human specimens indicated that the highest expression was in skeletal muscle followed by kidneys, liver, lungs, and heart54,55. In previous studies using an NQO2 knockdown cell line and NQO2 siRNA, the inhibition of NQO2 activity induced the upregulation of antioxidant enzymes, which resulted in increased cellular resistance to oxidants and protected cellular components from oxidation-related damage56,57,58. Furthermore, high concentrations of resveratrol, which is a natural antioxidant agent, induced the downregulation of NQO2 expression, which inhibited the angiotensin II-induced generation of reactive species oxygens in rat vascular smooth muscle cells57,58. An immunohistochemical toxicity study showed that TAA significantly reduced the expression level of NQO1 in rat renal tissues59. In the present study, NQO2 was significantly downregulated in the kidneys of rats exposed to the high concentration of TAA. The downregulation of NQO2 by TAA exposure might protect renal cellular components against oxidative damage.
MUPs are low-molecular-weight (approximately 19 kDa) members of the large lipocalin family60,61,62,63. They are synthesized in the liver as precursors and, after excision of the signal peptide and formation of disulfide bonds, the small proteins are secreted into the bloodstream to be finally excreted into the urine60,61,62,63. MUPs are also expressed in other tissues, including the salivary, lachrymal, meibomian, mammary preputial, and perianal glands, nasal tissue, and respiratory epithelia60,61,62,63.
In the present study, MUPs were significantly downregulated in the kidneys of rats exposed to the high concentration of TAA. The downregulation of MUPs in kidney tissue could be caused by TAA hepatotoxicity because MUP is mostly synthesized in the liver and TAA induced a significant downregulation of MUPs in the liver of rats exposed to TAA (Supplementary Fig. S4). On the other hand, MUP down-regulation might be attenuated by the renal injury of kidney proximal and distal tubules, resulting in the inhibition of MUP resorption. It has been reported that MUPs were reabsorbed by kidney renal tubule cells by the general mechanism for low-molecular-weight proteins64. In this study, we found three MUP isoforms, which were located in the 5.0 pI to 5.75 pI range, and they were all significantly downregulated. However, in a previous proteomic study, which showed protein expression level changes in rat livers exposed to TAA, three major urinary proteins were present as isoforms with pI ranges of 5.19, 5.29, and 5.43. Among them, two MUPs were significantly downregulated, but one major urinary protein remained unchanged65. Therefore, we evaluated the changes in MUP isoforms in the kidney tissue of rats exposed to TAA using the 2-DE immunoblot assay with a large polyacrylamide gel (size 35 × 45 cm) with a pI range from 5.2 to 6.5 and a molecular weight range from 16 kDa to 17.5 kDa. Sixteen MUP isoforms were found across the pI ranges and they were all significantly downregulated by exposure to increasing concentrations of TAA (Fig. 7).
In previous studies, IEF of male mouse urine resolved up to 15 MUP isoforms in a pI range from 4.6 to 5.360,61. To identify all MUP isoforms in kidney tissues, the 2-DE immunoblot assay was applied with extended pI ranges from 4.5 to 6.5 and molecular weights from 10 to 20 kDa (Fig. 8B). Furthermore, MUP isoform patterns in the liver, kidneys, and urine were compared. In this study, different amounts of protein samples from the liver, kidneys, and urine obtained from untreated male rats were used for the fine separation of protein bands and spots in 1-DE Western blots and 2-DE immunoblot assays (Fig. 8). In the 1-DE Western blot assay, liver, kidney, and urine showed different protein bands (Fig. 8A). One protein band in the liver was barely visible in the kidneys and the other two protein bands showed more intensity in the kidneys than in the liver. However, the urine showed all bands, although the low-molecular-weight protein bands were only slightly seen. It has been reported that 60% of MUPs, which are synthesized in the liver, were reabsorbed in the proximal tubule and degraded in the lysosomal compartment in the kidney, resulting in the generation of proteins truncated at ∼15.5 kDa, which accumulated in the cytosol66. In contrast, a different pattern of protein bands between the liver and kidneys might be derived from MUPs that were synthesized from other tissues including the salivary, lachrymal, meibomian, mammary preputial and perianal glands, nasal tissue, and respiratory epithelia60,61,62,63. The 2-DE immunoblot assay showed variable patterns of MUP isoforms in the liver, kidneys, and urine, although the major MUP isoform spots were found in all samples (Fig. 8B). To obtain the fine separation of each MUP isoform spot, different amounts of protein samples of the liver, kidneys, and urine were analyzed. This methodological approach could reveal a limitation in comparing the MUP isoform patterns in the liver, kidneys, and urine using the 2-DE immunoblot assay because the small-sized MUP isoforms were not detected in the gel. A total of 43 different MUP isoforms were found in the liver, kidneys, and urine. Previous studies have been performed to evaluate MUP profiles in different kinds of rodents and investigate MUP profile dynamics62,63,67,68,69,70,71. The present study also provides additional information for MUP profiles in the synthesis, circulatory, and secretory biological systems of laboratory male rats.
In conclusion, TAA is a carcinogen and a hepatotoxicant and is known as a nephrotoxicant, which induced structural kidney damage including severe tubular epithelial cell death associated with inflammatory cell infiltration, sclerosis and necrosis of the glomeruli, and fibrosis9,11,13,14,15. In this study, we determined the hematological, pathological, and biochemical toxicity of TAA and evaluated the proteomic changes in kidney tissue under different conditions after exposure to TAA. In the hematological study, total WBC counts were not changed by TAA exposure. However, the numbers of MOs and PLTs were significantly increased, and the MCV and HCT values were decreased significantly in rats exposed to 30 mg/kg BW TAA. No significant histopathologic abnormalities were found in the kidneys of rats exposed to TAA. Nonetheless, the expression levels of Kim-1 and NGAL, which are kidney injury biomarkers, showed significant increases in the kidney tissue of rats exposed to TAA, indicating nephrotoxicity. In proteomic analysis, seven proteins including ASAP2, RGS14, MAP7Dl, IL-3Rα, Tmod1, NQO2, and MUP were found to be up- and down-regulated and a total of 43 MUP isoforms were found in the liver, kidneys, and urine.
