Uric acid causes kidney injury through inducing fibroblast expansion, Endothelin-1 expression, and inflammation

Chronic Kidney Diseases (CKDs) are one ofthe major global health problems in elderly populations due to their increased incidence and prevalence involving aging and declining health [1]. Recently, it was thought that an elevated uric acid level is common in these conditions and it increases the risk factors associated with heart disease, such as obesity, insulin resistance and gout. Some studies suggested that an elevated uric acid level may not only be a true risk factor but also may be one of the most important risk factors for cardiovascular disease. In renal diseases, the mechanism of hyperuricemia inducing the progression of renal failure is still not well understood. However, uric acid has been reported to play a role in renal inflammation, intra-renal vasoconstriction and renal failure induced by urate crystal formation [2, 3]. In cardiovascular diseases, uric acid may induce endothelial dysfunction with reduction of vasodilatation function [3].

Urate is a result of purine metabolism. In most species, this is an intermediate product which is degraded further by the hepatic enzyme uricase to allantoin, which then is excreted freely in the urine. Humans do not have uricase, due to a genetic mutation resulting in a non-functional form of the gene [4]. As a consequence, humans have higher serum urate levels (>2 mg/dl) compared with most mammals (<2 mg/dl). While hyperuricemia is defined as7 mg/dl in men and 6 mg/dl in women [5], uric acid levels also vary significantly within humans as the result of factors which increase the generation (such as high purine or protein diets, alcohol consumption, conditions with high cell turnover, or enzymatic defects in purine metabolism) or decreased excretion. A reduction in glomerular filtration rate (GFR) increases serum uric acid, although a significant compensatory increase in gastrointestinal excretion occurs. Hyperuricemia also may result from increased net tubular absorption. After filtration, uric acid undergoes both reabsorption and secretion in the proximal tubule, and this process is mediated by a urate/anion exchanger and a voltage-sensitive urate channel [6].

An increasing number of experimental and epidemiological studies suggest that uric acid is an independent risk factor for cardiovascular and renal disease [7, 8]. Uric acid can predict a high mortality in patients with hypertension, heart failure and diabetes [7, 9]. In renal diseases, hyperuricemia has already been known to contribute to the CKDs progression and is known as a marker of renal diseases [10]. In the 1950s-1980s the association between elevated serum uric acid and hypertension was observed and reported, although it still received less attention due to lack of a mechanistic explanation [11, 12]. Twenty-five to 40% of adult patients with untreated hypertension have hyperuricemia (>6.5 mg/dl), and this number increases dramatically when serum uric acid in the high-normal range is included [13]. One study reported that high uric acid levels were present in 25% of untreated hypertensive subjects, in 50% of subjects taking diuretics, and in 75% of subjects with malignant hypertension. The increase in serum uric acid in hypertension may be due to the decrease in renal blood flow which accompanies the hypertensive state, since a low renal blood flow will stimulate urate reabsorption [5].

The mechanisms of organ injury induced by uric acid remain incompletely understood, although growing evidences suggest that uric acid is an agent of inflammation [7, 14]. Raising levels of uric acid also had been reported to occur in rats with administration of uricase inhibitor and led to thickening of afferent arteriole, activation of the renin-angiotensin system, and hypertension [15, 16]. Uric acid also stimulates rat vascular smooth muscle cell (VSMC) proliferation in-vitro with increased expression of platelet-derived growth factor (PDGF), cyclooxygenase-2 (COX-2), and monocyte chemoattractant protein1 (MCP-1) [17, 18]. The autocrine or paracrine ET-1 system has already been known to be involved in the pathogenesis of CKDs for at least two decades. Endothelin-1 (ET-1) is a potent vasoconstrictor for the first time observed by Yanagisawa [19]. Here we demonstrated that hyperuricemia conditions induced renal damage with tubular injury and kidney fibrosis through activation of Endothelin-1 (ET-1) and fibroblast expansion.

In this study we also found that hyperuricemia induced increased inflammation mediators such as MCP-1 and ICAM-1. Those effect might be mediated by reduction of NFκB, a nuclear transcription factor which plays a role in mediating inflammation. Renal epithelial cells have been known as a source of NFκB due to uric acid stimulation [21]. Reduction of uric acid level in the UAL7 and UAL14 groups produced reduction in this effect. This study confirmed Zhou’s et al.’s findings, which showed that induced hyperuricemia increased inflammatory mediators such as MCP-1, TNF-α and RANTES [22].

The process of uric acid in producing renal fibrosis and injury involves many mechanisms especially inflammation and fibroblast expansion. First we showed that 125 mg/kg body weight of daily uric acid injection intraperitoneally could produce hyperuricemia conditions after 7 days and 14 days. Uric acid also induced glomerulosclerosis and tubular injury, while a reduction of serum uric acid level reduced the glomerulosclerosis and tubular injury. Uric acid contributes to the development of end-stage renal diseases, especially before the availability of uric acid lowering agents [23]. Hyperuricemia also is recognized as an independent factor for progression of IgA nephropathy [24, 25]. Based on this study uric acid might become a predictor for the progression of renal diseases [26]. Epidemiological study also revealed that increased levels of uric acid to 8 mg/dL increases renal insufficiency to 2.9 times compared to uric acid with 5 mg/dL [27].

The effect of hyperuricemia in renal diseases might correlate with hypertension, intrarenal vascular disease, and renal injury [2]. Uric acid belongs to the Damage Associated Molucular Pattern (DAMP) and induces inflammation response. Interestingly, the study using transgenic uricase showed reduction of uric acid in interstitial fluid contributing to reduction of inflammation effect due to uric acid. It seems uric acid induces inflammation in the interstitial space [28]. Renal interstitial fluid has been shown to contribute to formation of myofibroblast cells which are responsible for matrix production during fibrosis. Pericytes and fibroblasts induce myofibroblast transition which then leads to kidney fibrosis. We used PDGFR-β to mark the renal interstitial cells. Many studies used the same antibody to examine fibroblasts [29] or pericytes [30].

One study had reported that uric acid induced secretion of fibronectin, a component of the extracellular matrix. This effect is mediated by URAT-1 Transporter [21]. Almost all urate is reabsorbed in the proximal tubules [31]. This process is facilitated by a specific transporter, involving a voltage-sensitive pathway and an urate/anion exchanger. One of these transporters is URAT-1 Transporter. Knockdown of URAT-1 Transporter in epithelial cells culture reduced uric acid induced-fibronectin secretion [21]. In this study we proposed the effect of uric acid in expansion of fibroblasts in-vivo. We found support that uric acid is transported through epithelial cells and acts in inducing fibronectin secretion [21]. Furthermore, we assumed this effect also might continue to interstitial areas consisting of many cells and structures, such as vessels, micro-vessels, fibroblasts and pericytes. Elevation of uric acid due to uricase inhibitor had been reported to induce thickening of afferent arteriole, renin-angiotensin system activation, and hypertension in rats [15, 16]. Uric acid also stimulates rat vascular smooth muscle cell (VSMC) proliferation in-vitro with increased expression of Platelet-Derived Growth Factor (PDGF), Cyclooxygenase-2, and Monocyte Chemoattractant Protein-1 [17, 18]. Interstitial fluid and space may contribute to the pathological effect of hyperuricemia. Uric acid might increase after cell death, then uric acid is secreted to extracellular fluids. High uric acid level may undergo nucleation due to high levels of sodium in extracellular fluids. This process induces inflammation related to DAMP [28]. Those effects might influence the biology of renal interstitial cells, such as fibroblasts. In this study we also revealed that fibroblasts underwent expansion in the UA7 and UA14 groups. We assumed that high levels of uric acid in interstitial spaces may contribute to the expansion of fibroblasts. Our immunoblot and q-RTPCR results showed higher expression of profibrotic factors such as TGF-β1 and ET-1 protein expression which confirm activation of fibrotic pathways after uric acid induction.

Uric acid not only induces endothelial dysfunction and vascular injury [32, 33], but also promotes CKDs through inducing epithelial to mesenchymal transition [34]. Many cells undergo mesenchymal transition thereby inducing the development of renal fibrosis, the final pathway of CKDs [29, 30, 35‐37]. Uric acid might not only promote epithelial cells for mesenchymal transition, but also induce myofibroblast transition from fibroblast. Immunostaining of this study also demonstrated fibroblast expansion (Fig. 2e) and showed αSMA positive staining which represented myofibroblasts in interstitial areas of the UA groups (Fig. 3b-d). We proposed in this study that uric acid might induce renal injury through stimulation of fibroblast expansion to myofibroblast transition. Uric acid might induce fibrotic factors, such as TGF-β1 and ET-1, thereby inducing fibroblast expansion and myofibroblast formation. Further experiments using fibroblast culture with uric acid induction are needed to confirm our results.

We also found that high uric acid is associated with higher expression of ET-1 mRNA (Fig. 3a). We suggested that ET-1 may modulate effect of uric acid in glomerular injury. ET-1 also might be regulated by uric acid, then lead to proliferation of vascular smooth muscle cells of Human Vascular Smooth Muscle cells culture [38]. ET-1 might be secreted by cells in glomerulus and inducing glomerulosclerosis [39]. ET-1 also induces podocyte injury then lead to glomerulosclerosis [40]. Increased ET-1 mRNA had already been shown in congenital pelvic-ureteral junction obstruction in CKD patients [41]. Direct contribution of ET-1 in kidney fibrosis has been shown by studies using ET-1 transgenic mice. ET-1 transgenic mice underwent spontaneous glomerulosclerosis, interstitial fibrosis, and renal cysts but not hypertension [39]. Effects of uric acid in glomerular and tubulointerstitial injury might also be mediated by ET-1. Endothelin receptor-A blocker in the CKDs due to diabetes prevented cytoskeletal and foot processes effacement of podocyte. These effects were equal to Angiotensin Converting Enzyme Inhibitor, especially in reducing proteinuria and podocyte structural maintenance [1]. Although there are only a small number of studies on humans, the recent studies showed the cross-talk between ET-1 and Renin-Angiotensin System (RAS) to be the same as in the study in animals. Uric acid stimulates activation of RAS [32]. Goddard et al. showed that inhibition of endothelial receptor and ACE gives a synergistic effect in ameliorating renal blood flow and sodium excretion [42, 43]. In patients with CKDs due to proliferative glomerulonephritis, selective Endothelin A Receptor (ETAR) blocker could reduce blood pressure due to increasing vasodilation. This finding is associated with the role of ETAR in the efferent arteriole vasoconstriction effect [42]. However, we could not examine and determine significantly higher ETAR protein expression in the uric acid groups. Inflammation in the uric acid group might also be stimulated by higher ET-1 expression. Reduction of ET-1 in kidney ischemic reperfusion injury also showed reduction of inflammation with down-regulation of TLR-2, TLR-4, MCP-1, and ICAM-1 accompanied by reduction of macrophage number [44]. The contribution of the ET-1 pathway in uric acid induction still needs further investigation.

In conclusion, this study highlighted the effect of uric acid in renal deterioration which might be mediated by induction of renal fibroblast expansion and ET-1 stimulation. Further research is needed to specifically elucidate the uric acid effect in renal fibroblast culture with ET-1 or ET receptor blocker addition.