The metabolic functioning of all tissues depends on the optimal levels of circulating TH that are under the tight regulatory and feedback control of the hypothalamo- pituitary thyroid axis through actions of throtropin(TSH) and Tg. The kidney, a target organ for TH and is also responsible for its metabolism and breakdown along with TSH and thyroglobulin (Tg). Urine, the terminal metabolite of the body produced by the kidney reflects not only kidney function but also the overall metabolism of the body through the presence of urinary vesicles and exosomes [24]. There is a strong interrelationship between the kidney and the thyroid wherein thyroid dysfunction leads to changes in glomerular filtration and tubular absorption as seen in hypothyroidism, decreased TH levels are accompanied by an increased peripheral vascular resistance, reduced renal blood flow, decreased glomerular filtration, altered tubular function, hyponatremia, and an alteration of the ability for water excretion. In the present study, we compared the urinary proteomic profiles in patients with hypothyroidism, before and after treatment with levothyroxine using the 2D-DIGE and MALDI-TOF proteomics approach, to better understand of the differences in the overall metabolism observed between the hypothyroid and euthyroid states. We had previously carried out a similar untargeted plasma proteomic profiling in the same group of patients [20]. Overall, our results showed that a total of 42 proteins, with a > 1.5-fold statistically significant change (P = 0.05) in abundance between the two states were altered, of which 28 proteins were up regulated and 14 proteins were down regulated in the hypothyroid state compared to the euthyroid state. On grouping these proteins based on their biological processes to understand their roles, we found that they were involved in the regulation of processes related to (i) transport (ALB, TF, RBP4, ATP6V1B2, HRAS, KNG1, LMAN2, ARNT2). (i) acute phase response proteins (ALB, TF, AMBP, CTSD, GCNT3, KNG1, KRT10, KRT5, ), (iv) generation of reactive oxygen species (ALB,TF, HRAS, KNG1,) (iii) proliferation of connective tissue cells (TF, CTSD, HRAS, KNG1, KRT10, ) and (ii) endocytosis (ALB, TF, AMBP, ATP6V1B2 up, HRAS ,LMAN2 ,SYNE1). The proteins ALB, RBP, HRAS, CTSD, TF, KNG1 were found to participate in combination with other proteins in more than one metabolic processes and regulate different pathways.
Transport of the TH from its site of synthesis to the target organs mediated via the TH binding proteins is critical for its action. Eight proteins identified in the present study were involved in the transport of TH. The presence of a number of different transport proteins, with different binding capacities ensures a balanced source of bioavailable TH to the different organs and also acts as sensor mechanism which determines the extent of liberation and release.
We found an increase in the levels of human serum albumin (HSA) in the urinary proteome of the hypothyroid group in comparison the euthyroid. HSA is a 66.5 kD protein synthesized by the liver and associates with a wide variety of substances and hormones including circulating TH. HSA, a low-affinity but high-capacity thyroid hormone binding protein binds 10% of the circulating TH and prevents its filtration by the renal tissue [1, 25]. Albumin secretion in the urine, above 150 mg/dl, is considered pathological (proteinuria) and is reflective of different systemic diseases, such as hypertension, diabetes or more locally kidney diseases (for example: glomerulonephritis). Increased albumin in the proteome with no evidence of urinary microalbuminuria, has been documented in number of proteomic studies [5, 26]. The increase in the albumin levels may reflect an increase in the uptake of the thyroid hormones, increased binding to prevent renal loss and in maintaining a balanced supply to the peripheral tissues during lowered thyroxine circulation as is seen in hypothyroidism. We and others have previously reported the presence of increased albumin in post bariatric surgery and in metabolic unhealthy obesity [26]. Hypothyroid patients also present with low grade chronic inflammation [27] and the increased spots relating to albumin along with AMBP levels also point to increase APP response along with and increased oxidative stress in the hypothyroid state. AMBP contains α1-microglobulin and Bikunin an important anti-inflammatory substance that modulates inflammatory events. Urinary α1-microglobulin indicates proximal tubular dysfunction and an increase in AMBP in hypothyroidism may serve to determine early tubular changes and as an increased anti-inflammatory response [28].
The protein spots relating to RBP which circulates as part of the latransthyretin and binds 20% of circulating TH [25] were also found to be significantly increased in the urinary proteome between the hypothyroid and euthyroid states. TTR is known to originate in the liver, but is also found in kidney cells, the choroid plexus, meninges, retina, placenta and pancreatic islet cells and fetal intestine [29]. RBP and TTR are known to have an intermediate binding affinity for TH [30]. They bind 80% of T4 and facilitates its transport to the brain through the blood brain barrier and the CSF wherein it constitutes up to 25% of the total protein. Owing to its low molecular weight, RBP (21-kDa) is freely filtered by the glomerulus with subsequent reabsorption and catabolization by the proximal tubule, making it a marker for tubular function. The binding of RBP to TTR was suggested to prevent extensive loss of the low molecular weight RBP through glomerular filtration. Recently, RBP4 levels have been reported to be elevated in insulin resistant subjects as well as in subjects with obesity and type 2 diabetes (T2DM) [31].
The protein spots relating to LMAN, a vesicular integral membrane type I transmembrane lectin, that plays an important role intracellular protein trafficking was found increased in our study. This protein is involved in the sorting, vesicle-mediated transport, early secretory pathway trafficking, endocytosis and quality control of high mannose type glycoproteins like thyroglobulin (Tg) and erythropoietin. LMAN is a component of the lipid microdomain raft machinery that are responsible for Tg transport to the apical surface and delivery to follicular lumen via raft pathway for secretory endocytosis [32]. Tg is a prohormone that is internalised and highly glycosylated under the influence of TSH in the ER and the golgi where it undergoes significant post translational modifications, conformational maturation and homodimerization before intracellular transport [33]. Mature Tg binds TH in the follicular thyroid cells and is stored as colloid. TSH induces exocytosis and exocytotic vesicles are accumulated in the most apical cell region after being transported on LMAN containing lipid protein microdomains. Increase in LMAN may indicate an attempt to increase Tg synthesis to increase the physiological levels of T4 in the thyrocytes and similarly for erythropoietin synthesis from the kidney.
Another multifunctional protein identified with significantly increased abundance in the urine proteome is transferrin, a metal cofactor involved in the transport of iron and is also required for an effective peripheral deiodination of T3 and for activity of the deiodinase enzyme that are also present in the kidney [20]. We found an increase in the abundance of protein spots relating to urinary transferrin in the hypothyroid state similar to our earlier study where we demonstrated an increased levels in the plasma. Lin et al., in their study in hepatoma cell lines (HepG2-TRa1), showed a direct induction of serotransferrin by T3 [29].
We also found a decrease in the abundance of SYNE 1 a nuclear envelope spectrin-repeat proteins that localise to multiple sub-cellular compartments. It behaves as an interconnecting protein that links the nuclear lamina to the cytoskeleton, and regulates the endothelial shape and migration [34], functions as the intracellular scaffold to maintain the cellular structure and establishes nuclear-cytoskeletal connections. These connections importantly connect the nuclear metabolism to the cytoplasmic actions for regulation of proteins such as Tg. SYNE1 mutations may disrupt tissue specific nesprin scaffolds and explain the tissue specific nature of many nesprin-associated diseases, including laminopathies and related to tumour progression and decreased expression was reported in invasive cancers [35, 36].
Hypothyroidism often leads to an enlargement of the thyroid gland as a compensatory mechanism to overcome the decrease in the circulating TH and in response to an increased TSH in order to increase TH production. The increase in proliferation was supported by our proteins - TF, CTSD, HRAS, KNG1, KRT10, which are known to increase Tg endocytosis and proteolysis, were significantly differentially abundant between the hypothyroid and euthyroid state. These included CTSD, E3 ubiquitin-protein ligase DCST1, putative E3 ubiquitin-protein ligase UBR7, probable E3 ubiquitin-protein ligase TRIML2, protein OS-9. Tg plays a role in the thyroid hormogenesis by its degradation via endosomal lysosomal pathway by cysteine cathepsins (thiol proteases) and the ubiquitin dependant proteolytic pathways. CTSD itself and by activation of cathepsin B breaks down the covalently cross-linked Tg in the acidic enodsomal environment to release soluble Tg for T4 liberation and re-utilization [37, 38]. A similar association was identified in. Cath-D is also a key mediator of induced-apoptosis, essential role in the multiple steps of tumor progression, in stimulating cancer cell proliferation, fibroblast outgrowth and angiogenesis, as well as in inhibiting tumor apoptosis. CTSD expression was increased in human primary breast cancer, thyroid and skin cancers, and was found to be associated with an increased risk of metastasis and shorter survival [39, 40].
We identified an increase in the ubiquitin-proteasome system (UPS) proteins, which includes the E3 ubiquitin ligases involved in the ubiquitin dependant proteosomal degradation of Tg between the hypothyroid and euthyroid state. This included the tripartite motif (TRIM) proteins which are the largest subfamilies of E3 ubiquitin ligases and UBR7 whose dysregulation is associated with chromatin abnormalities, numerous diseases and cancers. UBR7 is also important for the maintenance of the epithelial state and inhibition of the plasticity of a cell.
Aryl hydrocarbon-receptor nuclear translocator (ARNT2) is a transcription factor expressed in nervous system and kidney that also functions as E3 ubiquitin ligase. ARNT2 has been shown to be involved in angiogenesis and nerve development of embryo [41] while mutations in the ARNT2 gene are known to causes hypopituitarism, post-natal microcephaly, visual and renal anomalies [42]. Jia et al showed that ARNT2 inhibits cell proliferation inactivating the AKT signaling pathway in cases of gastric cancer [41]. The increase in all the enzymes involved in the proteolysis, endocytosis aims to liberate TH from the Tg leading to their secretion in the circulation [43].
It is interesting to note that the urinary proteome in our study also identified the enzyme GCNT3 and TGM7 with an increased abundance in the hypothyroid group. These enzymes are responsible for post translational glycosylation of Tg and increasing the covalent cross-links between the Tg molecules to increase its compaction within the thyrocyte [44]. Tg glycosylation is important for its correct folding and trafficking, iodination and TH synthesis [45]. Unglycosylated Tg has been shown to lose its ability to synthesize TH in both in vitro and invivo studies [43]. Protein OS-9 identified in our data set with an increased abundance is an additional participating enzyme of this pathway. It is well known that Tg is a highly glycosylated protein with extended mannose residues that are recognised by protein OS-9 containing mannose 6-phosphate receptor homology domains, were reported to be involved in ER quality control [46].
We found a significant decrease in abundance of Hras in the hypothyroid versus the euthyroid states. Hras is a member of the membrane-bound guanine nucleotide-binding proteins that function as signal transducers from cell membrane to nucleus for TH and other growth factor receptors. Besides the thyroid tissue, Hras is expressed in the kidney and is abundant in the distal collecting duct [47]. Ras interacts with multiple downstream effectors including PI3K and leads to the activation of MAPK kinases resulting in gene induction. The ultimate outcome is regulated induction of cell survival, growth and migration, proliferation, differentiation, adhesion, motility, and morphology [48]. Among their diverse biological functions, Ras poteins are known to regulate activities of ion channels and transporters. Mutations in the other RAS proteins have been identified in benign and malignant thyroid neoplasms,[49, 50] in primary cultures of human thyrocytes and in rat thyroid cell lines except for Hras whose increased oncogenic mutants levels in mice did not develop thyroid lesions [50]. Our results also demonstrated an increase in the cleft lip and palate transmembrane protein 1-like protein (CLPTM1L) that works in conjunction with the RAS proteins through the phosphoinositide 3-kinase (PI3 Kinase) pathway and is an essential molecule its signaling functions. James et al identified an overexpressed protein in human ovarian tumour cell lines that are resistant to cisplatin and in non-small cell lung cancer, where it protects tumour cells from genotoxic apoptosis [51].
The other proteins of interest identified in the study with increased abundance between the hypothyroid and euthyroid state were Zinc-finger proteins (ZNFs) and the keratins that are involved in several cellular processes with key role in development and differentiation, tumorigenesis, cancer progression and metastasis.
The biological significance of the identified proteins in our proteomic analysis was explored by IPA and the network pathway identified the highest scoring interaction network pathway related to amino acid metabolism, molecular transport, small molecule biochemistry. This is true considering the fact that TH are peptide hormones whose synthesis is dependant on the amino acid metabolism. The central nodes with the highest connectivity included the MAP kinase family p38MAPK, ERK1/2,Jnk), PI3 Kinase/Akt, protein kinase C, Nfkb and VEGF, reiterating the involvement of the identified proteins in pathways related to cell proliferation, inflammation, and endothelial function. The proteome analysis also points to the fact that there is a concerted action to increase the TH circulating levels through increase Tg synthesis in the hypothyroid state.