Here we examined the effects of 2% Oxo diet induced hyperuricemia on kidney morphology, inflammation, and markers of oxidative stress in a low-renin model of experimental CRI. Previously, we found that 2% Oxo diet elevated plasma renin and aldosterone, but in parallel improved NO-mediated vasorelaxation in the carotid artery, and reduced oxidative stress in vivo in remnant kidney rats (9, 20). The current results showed that moderately elevated plasma UA level was associated with favorable changes in kidney histology and reduced markers of inflammation in NX rats.
During 2% Oxo feeding a moderate rise in circulating UA levels is achieved due to inhibition of the hepatic enzyme uricase that metabolizes UA to its final end-product allantoin (16). Subsequently, plasma UA levels are elevated to concentrations that are closer to those observed in humans. The present 2.5-3-fold elevations of UA levels induced by the Oxo diet well correspond to previous findings (6, 16, 17, 19). Pharmacological lowering of serum UA levels was not included in the present protocol, as several studies have shown that the xanthine oxidase (XO) inhibitors allopurinol and febuxostat effectively prevent the effects of Oxo diet in rats (5, 16, 17, 19). Of note, the therapeutic effects of XO inhibitors have not been solely related to reduced UA concentrations, but also to the anti-oxidative and anti-inflammatory properties of these compounds (29). The 5/6 NX model has also been characterized by reduced tissue XO activity and a compensatory increase in intestinal UA excretion (30). These mechanisms may explain why plasma UA was not significantly higher in the NX rats on the normal diet than in Sham rats on the normal diet.
The present renal insufficiency 12 weeks after the NX operation was documented by elevated plasma creatinine, reduced creatinine clearance, hyperphosphatemia, and increased urinary protein excretion (19). The histology showed increased indices of arteriosclerosis, glomerulosclerosis, and tubulointerstitial damage in the NX group. The reliability of the histological findings is supported by the good correlation between the glomerulosclerosis score and the amount of 24-hour urinary protein excretion. The hypertrophy of the remnant kidney can be attributed to compensatory tissue growth in an attempt to compensate for the reduced renal function (31, 32). The NX rats presented with low plasma renin activity probably due to the associated volume load, corresponding to previous findings in rats subjected to surgical renal ablation (20). Systolic BP was only modestly elevated 12 weeks after renal ablation, as more marked hypertension is known to develop only later in the course impaired renal function in this surgical low-renin model (33, 34).
Previously, the harmful effects of high UA concentrations in renal tissue have been attributed to the deposition of non-soluble monosodium urate crystals in renal tubules (gouty nephropathy) (35). Intracellularly UA may also mediate biological effects that may play a role in the development of subclinical “non-gouty” types of renal and cardiovascular disease (35). Excess generation of reactive oxygen species (ROS) has been suggested to play a central role in the UA-induced renal disease (5). The interaction between UA and ROS is complex, as the synthesis of UA from its purine and pyrimidine nucleotide precursors is catalyzed by two xanthine oxidoreductase enzymes: xanthine dehydrogenase and XO. In ischemic states such as CRI, the latter is the predominant catalyzer creating ROS, mainly superoxide anion, as a by-product of the UA synthesis. In cell cultures, UA can inhibit renal production of NO synthase, a catalyzing enzyme in NO generation (16). The reaction between ROS and NO may result in renal NO-depletion and afferent artery vasoconstriction, which is an essential step in renal fibrosis (17). On the other hand, the antioxidant properties of UA are widely accepted. By scavenging superoxide anions UA can prevent it from reacting with NO and thus inhibit the formation of the toxic peroxynitrite (10, 36). Also, the reaction of UA with peroxynitrite yields a nitrated UA derivate, which has vasodilatory effects (37). Finally, UA can also counter oxidant-induced renal injury by preventing the inactivation of extracellular superoxide dismutase, an enzyme that provides tissue protection by catalyzing the dismutation of superoxide radical into oxygen and hydrogen peroxide (38, 39).
We found that the number of mast cells was elevated in remnant kidneys, while mast cell quantity was reduced following the Oxo diet. Kidney mast cell density is known to correlate with the severity of renal disease (40). Various etiologies, such as several forms of nephropathies and renovascular ischemia that cause glomerular damage and interstitial fibrosis, are associated with mast cell abundance in the kidney (40). Mast cells can aggravate tissue damage and fibrosis by recruiting leukocytes, profibrogenic cytokines, proteases, and growth factors, and also by directly stimulating collagen synthesis (40). In an experimental rat model, a close association between mast cell density and oxidative stress in the kidney, as indicated by superoxide anion generation, was previously reported (41). We also assessed mast cell activity by measuring the quantity of mast cellderived prostaglandin D2 (PGD2) metabolite, 11-epi-prostaglandin-F2α, in the urine (27, 42). Due to the long half-life and stability, 11-epi-prostaglandin-F2α is a convenient way to evaluate mast cell activity in vivo (27, 42, 43). The present findings of kidney mast cell density and urinary 24-hour 11-epi-prostaglandin-F2α excretion were congruent, and a direct correlation between these variables was observed. Possible explanations to the reduced mast cell infiltration and activity in the renal tissue of hyperuricemic NX rats are decreased amounts of ROS and increased NO bioavailability (9), as both of these factors can reduce tissue inflammation and inhibit mast cell degranulation (41, 44).
Whether the actions of UA are detrimental or beneficial may depend on the distribution of UA between the intra- and extracellular compartments (35). Extracellularly the antioxidant properties predominate, whereas intracellularly UA may be a pro-oxidant (35). For instance, the free radical scavenging capability of plasma UA appears to have favorable effects on kidney tissue in CRI (45). In contrast, the blockade of UA entry into the renal tubular cells by the organic anion transporter inhibitor probenecid prevented epithelial-to-mesenchymal transition, an event contributing to progressive tubular fibrosis (46). We found that kidney tissue HO-1 mRNA content was higher in both NX groups than in the Sham groups but did not differ between the Sham and Sham + Oxo groups, or between the NX and NX + Oxo groups. These findings support the view that the present Oxo diet did not cause oxidative stress even at the cellular level in vivo. Tissue HO-1 content serves as an index of oxidant stress in humans and in animal models of renal disease (47–49). By converting cell toxic heme to biliverdin in a reaction that liberates carbon monoxide (CO) and iron, HO-1 counteracts oxidant burden. Inactivation of heme by HO-1 prevents it from inducing lipid peroxidation, and ROS and hydrogen peroxide generation in tubular epithelial cells, while the reaction by-products biliverdin and CO in low concentrations possess antioxidant and vasodilatory effects (50). In CRI, biliverdin ja CO can even help to preserve normal glomerular filtration rate and sodium handling by suppressing tubule-glomerular feedback and afferent arteriolar vasoconstriction (48).
In the mammalian kidneys, COX-2 has been mainly localized to the macula densa, cortical thick ascending limb, and medullary interstitial cells (28). Increased juxtaglomerular renin and preglomerular arterial COX-2 production have been suggested to contribute to smooth muscle cell proliferation and renal arteriolar obliteration in experimental hyperuricemia (6). However, in the present study the number of glomerular COX-2 positive cells was reduced by 2% Oxo feeding in the Sham rats and was equally further reduced in both NX groups. The explanation for the reduced number of glomerular COX-2 positive cells in both NX groups remains unknown, but may be related to the glomerular hypertrophy and hyperfiltration caused by surgical subtotal nephrectomy (20, 32). We found that experimental hyperuricemia suppressed tubulointerstitial COX2 protein staining. These findings suggest reduced COX-2 derived inflammatory influences in the kidneys after the 2% Oxo diet. Lower kidney tissue COX-2 content is well in line with the beneficial effects of experimental hyperuricemia on renal histology in the NX rats. Of note, in addition to mast cells, 11-epi-prostaglandin-F2α can also originate from prostanoids synthetized via COX-2. Therefore, reduced 11-epi-prostaglandin-F2α excretion may also reflect reduced total COX-2 content in the kidneys of the hyperuricemic rats (51).
Immunohistochemical staining of SMA was done in order to examine the renal preglomerular arterioles, as thickening of the afferent arterioles has been suggested to trigger UA-induced renal fibrosis (17). The renal arterioles were clearly identified adjacent to glomeruli, but we were unable to reliably differentiate the afferent from the efferent renal arterioles. Therefore, the present results are inconclusive with respect to preglomerular small artery structure. Increased interstitial collagen deposition has been suggested to mediate UA-mediated renal fibrosis (16). In the present study, collagen I mRNA expression was elevated in CRI but was not influenced by moderate hyperuricemia.
The causal role of UA in the progression of renal disease has been questioned by Mendelian randomization studies (52, 53). A recent review concluded that the causal association of UA with a range of health outcomes is evident only in gout and nephrolithiasis (54). In hemodialysis patients, lower UA levels were independently associated with higher all-cause and cardiovascular mortality (55), while in patients with end-stage renal disease not receiving dialysis or receiving peritoneal dialysis, higher serum UA associated with higher mortality (53). There is also evidence that subjects genetically predisposed to hypouricemia present with an elevated risk for renal disease (56). The optimal range of circulating UA levels in various health conditions warrants further research.