To our knowledge, strategies to target Cu regulation for pharmacotherapy of renal fibrosis in diabetes have not previously been systematically explored. This study is the first to demonstrate marked suppression of renal fibrosis in vivo by a highly selective CuII chelator , as demonstrated both histologically (Figs 2 and 5) and by decreased renal content of hydroxyproline, collagen and fibronectin-specific biochemical markers for tissue fibrosis (Figs 4 and 5). Perhaps more significantly, TETA also suppressed diabetes-evoked increases in UACR, a key measure of renal function, in diabetic rats.
We had previously reported that TETA improved hallmarks of heart disease in diabetic rats and patients with type 2 diabetes [6, 7]. However, until the current study, it was not known whether TETA could exert any renoprotective effect in diabetes. To address this question, we evaluated the effect of TETA on urinary albumin excretion in diabetic rats. Urinary albumin has been suggested as a predictive indicator for prognosis of diabetic nephropathy and its elevated excretion is also said to impair renal function . Furthermore, reductions in urinary albumin in diabetic nephropathy were reportedly associated with renal protection . Here we show that diabetes caused significant increases in the UACR, whereas TETA suppressed this effect (Fig. 3a), demonstrating a renoprotective effect of TETA in a rat model of diabetic nephropathy. In another report, renal Cu levels were reportedly increased in 3 week streptozotocin diabetic rats, but elevated renal Cu in those animals did not affect renal function in the early stages of diabetes , possibly consistent with the shorter diabetic period in that study. Previous studies have also shown TETA dihydrochloride to act as a Cu-selective chelator [25, 26]. We are interested in comparing the effects of TETA with those of other Cu chelators and agents shown to attenuate or prevent indices of diabetic nephropathy. Here, the increased blood glucose in diabetic rats was not altered by TETA, indicating that its renoprotective effect was independent of any possible hypoglycaemic action. It remains unknown whether TETA prevents renal Cu accumulation by sequestering the metal in the serum and thus preventing Cu entry into the renal cells or by stimulating its excretion from renal cells or by both mechanisms.
Although the pathogenesis of diabetic kidney disease is multi-factorial, tissue fibrosis is a key pathological hallmark of this disease, in which build-up of ECM proteins is a key pathogenic process. Hyperglycaemia in diabetes is known to increase the synthesis of ECM components in the kidneys. In this study, the increase in total collagen and content of the major ECM components, fibronectin-1, collagen III and collagen IV, occurred in experimental diabetes (Figs 4 and 5). Our recent transcriptomic analysis has also identified increased content of mRNAs corresponding to fibronectin-1 and collagen IV, respectively, in the heart and aorta of diabetic rats . Here, we found that TETA normalised diabetes-induced elevations in total collagen, fibronectin-1, collagen III and collagen IV proteins (Figs 4 and 5). Our recent proteomic analysis showed that diabetes-induced upregulation of the major receptor, β
1 integrin, of another ECM protein, laminin, was attenuated by TETA in the diabetic aorta (X. Chen, D. Gong, A. R. J. Phillips and G. J. S. Cooper, unpublished observations), consistent with our previous findings in the diabetic heart . It remains unknown whether TETA modifies content of laminin and its receptors in the diabetic kidney.
TGF-β1 reportedly induces mesangial expansion and glomerular basement membrane thickening through ECM protein build-up in diabetic kidneys . Here, we have shown that increased TGF-β1 levels in diabetic kidneys were significantly suppressed by TETA. TGF-β1 is synthesised as a 391-amino acid precursor with little activity, which requires cleavage of its NH2-terminal latency-associated peptide to produce the active form . Its activity may also be modified by the proteoglycan decorin  and by the scavenging protein α2-macroglobulin  such that changes in corresponding Tgfb2 mRNA expression or TGF-β1 protein content may not accurately reflect its biological activity. In this study, therefore, we investigated the biological effects of TGF-β1 by analysing pSMAD2, a marker of TGF-β signalling activation . Diabetes-evoked increases in renal pSMAD2 protein were attenuated by TETA (Fig. 6e). This suppression of pSMAD2 formation was well correlated with the concomitant decreases in other fibrogenic markers observed here.
TGF-β1 is a key cytokine in the pathway underlying tissue fibrosis and can be activated by multiple upstream effectors. Elevation of its content in mesangial cells was shown to be activated by the Cu-dependent matricellular protein, secreted protein acidic and rich in cysteine (SPARC) , while collagen accumulation was reportedly decreased in SPARC-null mice with pulmonary fibrosis . Moreover, inhibition of TGF-β content was associated with inhibition of lung and liver fibrosis by tetrathiomolybdate-mediated Cu-lowering . However, it remains unknown whether increased renal Cu (Fig. 1) upregulates SPARC and whether TETA inhibits diabetes-induced renal fibrosis through inhibition of SPARC.
Diabetic nephropathy is characterised by relentless accumulation of ECM components. Decreased ECM degradation also plays a role in the progression of renal lesions to fibrosis . Here, TETA normalised Serpine1/Pai1 mRNA and its corresponding SERPINE1/PAI1 protein in diabetic renal cortex, accompanied by decreased levels of TGF-β1 and reversal of numerous pathophysiological changes. PAI-1 has been reported to control TGF-β production and thereby regulate overall ECM production in diabetes . Conversely, TGF-β, acting via SMAD activation, can induce expression of Serpine1/Pai1, whose promoter contains SMAD-binding elements. TETA could thus interfere with the vicious cycle of reciprocal stimulation between PAI-1 and TGF-β1, thus ameliorating excessive ECM deposition and subsequent renal fibrosis in diabetes.
The Cu-dependent enzyme SSAO reportedly plays an important role in ECM deposition and maintenance in vascular smooth muscle . Here, SSAO protein was increased in the diabetic kidney and this increase was downregulated by TETA (ESM Fig. 1). Interestingly, our recent proteomic analysis also showed that content of SSAO in the aortas of diabetic rats was significantly increased and that TETA normalised this overproduction (D. Gong, X. Chen, A. R. J. Phillips and G. J. S. Cooper, unpublished observations). By contrast, TETA had no effect on levels of two other Cu-containing enzymes, SOD-1 and SOD-3 (ESM Fig. 1). In the current study, total Cu content was increased in the diabetic kidney (Fig. 1), a finding consistent with our prior report of elevated chelatable Cu in the hearts of diabetic rats ; both of these abnormalities were ameliorated by TETA treatment. Thus, TETA could ameliorate diabetic nephropathy by inhibiting the elevated SSAO levels associated with increased Cu concentrations in diabetic kidneys.
One answer to the question of how renal Cu accumulation occurs in diabetes is that it could be due to elevated renal AGE . Multiple pathways lead to AGE accumulation in tissues in diabetes and diverse AGE products are formed. AGE deposition has been implicated in animal models of diabetic nephropathy , and diabetes exacerbated AGE accumulation in the veins of end-stage renal failure patients . Glycation reportedly increased in vitro Cu binding to collagen . A second possibility is endothelin-1, which was reported to mediate alteration of trace metals including Cu in the liver and kidneys of chronically diabetic rats . Both Menkes’ disease and Wilson’s disease are also known to cause renal Cu accumulation associated with renal pathology. Accumulation of Cu in the proximal tubules causes mildly disturbed tubular function in Menkes’ disease [42, 43], a disorder caused by mutations in the Cu-transporting ATPase gene, ATP7A. Patients with Wilson’s disease suffer from haematuria, renal stones and tubular dysfunction . Increased renal Cu levels [45, 46] and tissue damage  were also observed in Long–Evans Cinnamon rats, an animal model of Wilson’s disease. This increased Cu accumulation is due to a defect in a second Cu-transporting ATPase, ATP7B. Thus, these Cu-induced renal pathologies appear similar to that in diabetic nephropathy, but the molecular defects that cause tissue localisation and, of relevance to the current findings, renal Cu accumulation in these diseases differ from that of diabetic nephropathy.
Diabetes-evoked renal Cu accumulation may induce expression of the metal-binding protein, metallothionein. Although metallothionein mainly acts as a regulator of metal homeostasis, it may also be an adaptive protein that protects cells from oxidative stress. The protein is highly expressed in proximal convoluted tubular cells, which are known to be the primary site of the nephrotoxicity caused by heavy metals . Elevated levels of renal metallothionein-I and metallothionein-II were identified in insulin-deficient diabetic rats. The altered metabolism of renal metallothionein was largely due to accumulation of excessive dietary Cu in the kidney . These studies suggest that metallothionein could play a renoprotective role in diabetic nephropathy.
In summary, we have shown here that TETA, a synthetic tetramine that acts as a highly selective tetradentate CuII chelator [7, 50], attenuates diabetic kidney disease. It does this, at least in part, by suppressing activation of the TGF-β signalling pathway, which otherwise mediates increased content of ECM proteins and consequent renal fibrosis. The TETA-mediated decrease in the CuII-dependent enzyme SSAO (ESM Fig. 1) could contribute to this effect. Our findings implicate diabetes-evoked renal CuII build-up, probably mainly in the ECM , as a key early defect in the mechanism by which diabetes leads to or causes diabetic nephropathy. They also provide a molecular explanation, at least in part, for the mechanism by which TETA acts to improve renal function in diabetes.