Takahiko Nakagawa

A Role for Uric Acid in the Progression of Renal Disease

Hyperuricemia is associated with renal disease, but it is usually considered a marker of renal dysfunction rather than a risk factor for progression. Recent studies have reported that mild hyperuricemia in normal rats induced by the uricase inhibitor, oxonic acid (OA), results in hypertension, intrarenal vascular disease, and renal injury. This led to the hypothesis that uric acid may contribute to progressive renal disease. To examine the effect of hyperuricemia on renal disease progression, rats were fed 2% OA for 6 wk after 5/6 remnant kidney (RK) surgery with or without the xanthine oxidase inhibitor, allopurinol, or the uricosuric agent, benziodarone. Renal function and histologic studies were performed at 6 wk. Given observations that uric acid induces vascular disease, the effect of uric acid on vascular smooth muscle cells in culture was also examined. RK rats developed transient hyperuricemia (2.7 mg/dl at week 2), but then levels returned to baseline by week 6 (1.4 mg/dl). In contrast, RK+OA rats developed higher and more persistent hyperuricemia (6 wk, 3.2 mg/dl). Hyperuricemic rats demonstrated higher BP, greater proteinuria, and higher serum creatinine than RK rats. Hyperuricemic RK rats had more renal hypertrophy and greater glomerulosclerosis (24.2 +/- 2.5 versus 17.5 +/- 3.4%; P < 0.05) and interstitial fibrosis (1.89 +/- 0.45 versus 1.52 +/- 0.47; P < 0.05). Hyperuricemic rats developed vascular disease consisting of thickening of the preglomerular arteries with smooth muscle cell proliferation; these changes were significantly more severe than a historical RK group with similar BP. Allopurinol significantly reduced uric acid levels and blocked the renal functional and histologic changes. Benziodarone reduced uric acid levels less effectively and only partially improved BP and renal function, with minimal effect on the vascular changes. To better understand the mechanism for the vascular disease, the expression of COX-2 and renin were examined. Hyperuricemic rats showed increased renal renin and COX-2 expression, the latter especially in preglomerular arterial vessels. In in vitro studies, cultured vascular smooth muscle cells incubated with uric acid also generated COX-2 with time-dependent proliferation, which was prevented by either a COX-2 or TXA-2 receptor inhibitor. Hyperuricemia accelerates renal progression in the RK model via a mechanism linked to high systemic BP and COX-2-mediated, thromboxane-induced vascular disease. These studies provide direct evidence that uric acid may be a true mediator of renal disease and progression.

Uric Acid, Hominoid Evolution, and the Pathogenesis of Salt-Sensitivity

Humans have elevated serum uric acid as a result of a mutation in the urate oxidase (uricase) gene that occurred during the Miocene. We hypothesize that the mutation provided a survival advantage because of the ability of hyperuricemia to maintain blood pressure under low-salt dietary conditions, such as prevailed during that period. Mild hyperuricemia in rats acutely increases blood pressure by a renin-dependent mechanism that is most manifest under low-salt dietary conditions. Chronic hyperuricemia also causes salt sensitivity, in part by inducing preglomerular vascular disease. The vascular disease is mediated in part by uric acid-induced smooth muscle cell proliferation with activation of mitogen-activated protein kinases and stimulation of cyclooxygenase-2 and platelet-derived growth factor. Although it provided a survival advantage to early hominoids, hyperuricemia may have a major role in the current cardiovascular disease epidemic.

Hypothesis: could excessive fructose intake and uric acid cause type 2 diabetes?

We propose that excessive fructose intake (>50 g/d) may be one of the underlying etiologies of metabolic syndrome and type 2 diabetes. The primary sources of fructose are sugar (sucrose) and high fructose corn syrup. First, fructose intake correlates closely with the rate of diabetes worldwide. Second, unlike other sugars, the ingestion of excessive fructose induces features of metabolic syndrome in both laboratory animals and humans. Third, fructose appears to mediate the metabolic syndrome in part by raising uric acid, and there are now extensive experimental and clinical data supporting uric acid in the pathogenesis of metabolic syndrome. Fourth, environmental and genetic considerations provide a potential explanation of why certain groups might be more susceptible to developing diabetes. Finally, we discuss the counterarguments associated with the hypothesis and a potential explanation for these findings. If diabetes might result from excessive intake of fructose, then simple public health measures could have a major impact on improving the overall health of our populace.

Hypothesis: fructose-induced hyperuricemia as a causal mechanism for the epidemic of the metabolic syndrome.

The increasing incidence of obesity and the metabolic syndrome over the past two decades has coincided with a marked increase in total fructose intake. Fructose—unlike other sugars—causes serum uric acid levels to rise rapidly. We recently reported that uric acid reduces levels of endothelial nitric oxide (NO), a key mediator of insulin action. NO increases blood flow to skeletal muscle and enhances glucose uptake. Animals deficient in endothelial NO develop insulin resistance and other features of the metabolic syndrome. As such, we propose that the epidemic of the metabolic syndrome is due in part to fructose-induced hyperuricemia that reduces endothelial NO levels and induces insulin resistance. Consistent with this hypothesis is the observation that changes in mean uric acid levels correlate with the increasing prevalence of metabolic syndrome in the US and developing countries. In addition, we observed that a serum uric acid level above 5.5 mg/dl independently predicted the development of hyperinsulinemia at both 6 and 12 months in nondiabetic patients with first-time myocardial infarction. Fructose-induced hyperuricemia results in endothelial dysfunction and insulin resistance, and might be a novel causal mechanism of the metabolic syndrome. Studies in humans should be performed to address whether lowering uric acid levels will help to prevent this condition.

Excessive fructose intake induces the features of metabolic syndrome in healthy adult men: role of uric acid in the hypertensive response

Background:Excessive fructose intake causes metabolic syndrome in animals and can be partially prevented by lowering the uric acid level. We tested the hypothesis that fructose might induce features of metabolic syndrome in adult men and whether this is protected by allopurinol.Methods:A randomized, controlled trial of 74 adult men who were administered 200 g fructose daily for 2 weeks with or without allopurinol. Primary measures included changes in ambulatory blood pressure (BP), fasting lipids, glucose and insulin, homeostatic model assessment (HOMA) index, body mass index and criteria for metabolic syndrome.Results:The ingestion of fructose resulted in an increase in ambulatory BP (7±2 and 5±2 mm Hg for systolic (SBP) and diastolic BP (DBP), P<0.004 and P<0.007, respectively). Mean fasting triglycerides increased by 0.62±0.23 mmol l−1 (55±20 mg per 100 ml), whereas high-density lipoprotein cholesterol decreased by 0.06±0.02 mmol l−1 (2.5±0.7 mg per 100 ml), P<0.002 and P<0.001, respectively. Fasting insulin and HOMA indices increased significantly, whereas plasma glucose level did not change. All liver function tests showed an increase in values. The metabolic syndrome increased by 25–33% depending on the criteria. Allopurinol lowered the serum uric acid level (P<0.0001) and prevented the increase in 24-h ambulatory DBP and daytime SBP and DBP. Allopurinol treatment did not reduce HOMA or fasting plasma triglyceride levels, but lowered low-density lipoprotein cholesterol relative to control (P<0.02) and also prevented the increase in newly diagnosed metabolic syndrome (0–2%, P=0.009).Conclusions:High doses of fructose raise the BP and cause the features of metabolic syndrome. Lowering the uric acid level prevents the increase in mean arterial blood pressure. Excessive intake of fructose may have a role in the current epidemics of obesity and diabetes.

Diabetic retinopathy is associated with bone marrow neuropathy and a depressed peripheral clock

The present epidemic of diabetes is resulting in a worldwide increase in cardiovascular and microvascular complications including retinopathy. Current thinking has focused on local influences in the retina as being responsible for development of this diabetic complication. However, the contribution of circulating cells in maintenance, repair, and dysfunction of the vasculature is now becoming appreciated. Diabetic individuals have fewer endothelial progenitor cells (EPCs) in their circulation and these cells have diminished migratory potential, which contributes to their decreased reparative capacity. Using a rat model of type 2 diabetes, we show that the decrease in EPC release from diabetic bone marrow is caused by bone marrow neuropathy and that these changes precede the development of diabetic retinopathy. In rats that had diabetes for 4 mo, we observed a dramatic reduction in the number of nerve terminal endings in the bone marrow. Denervation was accompanied by increased numbers of EPCs within the bone marrow but decreased numbers in circulation. Furthermore, denervation was accompanied by a loss of circadian release of EPCs and a marked reduction in clock gene expression in the retina and in EPCs themselves. This reduction in the circadian peak of EPC release led to diminished reparative capacity, resulting in the development of the hallmark feature of diabetic retinopathy, acellular retinal capillaries. Thus, for the first time, diabetic retinopathy is related to neuropathy of the bone marrow. This novel finding shows that bone marrow denervation represents a new therapeutic target for treatment of diabetic vascular complications.

Uric Acid Causes Vascular Smooth Muscle Cell Proliferation by Entering Cells via a Functional Urate Transporter

Background: Soluble uric acid stimulates vascular smooth muscle cell (VSMC) proliferation by activating mitogen-activated protein kinases, and stimulating COX-2 and PDGF synthesis. The mechanism by which uric acid enters the VSMC is not known. We hypothesized that uric acid enters via transporters similar to that observed in the kidney. Methods: We studied the uptake of uric acid into rat VSMC under polarized and depolarized conditions and in the presence of organic anion transport (OAT) inhibitors (probenecid and benzbromarone) or p-aminohippurate (PAH). We also examined the ability of probenecid to inhibit uric acid-induced VSMC proliferation and monocyte chemoattractant protein-1 (MCP-1) synthesis. Results:14C-Urate uptake was shown in VSMC and was enhanced under depolarized conditions. 14C-Uric acid uptake was inhibited by probenecid and benzbromarone, as well as by unlabelled urate and PAH. Probenecid blocked VSMC proliferation and MCP-1 expression in response to uric acid. VSMC did not express rOAT1-3, rOAT-5 or URAT-1 mRNA by PCR, but did express the voltage-sensitive transporter (UAT) by both PCR and RNase protection assay. Conclusions: Urate enters VSMC by both voltage-sensitive and OAT pathways, and the uptake, cell proliferation and MCP-1 expression can be blocked by OAT inhibitors. The specific transporter(s) responsible for the urate uptake remains to be determined.

Nitric Oxide Modulates Vascular Disease in the Remnant Kidney Model

A loss of the microvascular endothelium occurs in the remnant kidney model of renal disease and may play an important role in progression (Kang et al, J Am Soc Nephrol, 12:1434, 2001). Given that nitric oxide (NO) is a potent endothelial cell survival factor, we hypothesized that stimulating (with L-arginine) or blocking (with nitro-L-arginine methyl ester, (L-NAME)) NO synthesis could modulate the integrity of the microvasculature and hence affect progression of renal disease. Rats underwent 5/6 nephrectomy (RK) and then were randomized at 4 weeks to receive vehicle, L-NAME, or L-arginine for 4 weeks. Systolic blood pressure and renal function was measured, and tissues were collected at 8 weeks for histological and molecular analyses. The effect of modulation of NO on vascular endothelial growth factor (VEGF) expression in rat aortic vascular smooth muscle cells (SMC) and mouse medullary thick ascending limb tubular epithelial cells (mTAL) was also studied. Inhibition of NO with L-NAME was associated with more rapid progression compared to RK alone, with worse blood pressure, proteinuria, renal function, glomerulosclerosis, and tubulointerstitial fibrosis. The injury was also associated with more glomerular and peritubular capillary endothelial cell loss in association with an impaired endothelial proliferative response. Interestingly, the preglomerular endothelium remained intact or was occasionally hyperplastic, and this was associated with a pronounced proliferation of the vascular SMCs with de novo expression of VEGF. Cell culture studies confirmed a divergent effect of NO inhibition on VEGF expression, with inhibition of VEGF synthesis in mTAL cells and stimulation of VEGF in vascular SMC. In contrast to the effects of NO inhibition, stimulation of NO with L-arginine had minimal effects in this rat model of progressive renal disease. These studies confirm that blockade of NO synthesis accelerates progression of renal disease in the remnant kidney model, and support the hypothesis that one of the pathogenic mechanisms may involve accelerated capillary loss and impaired angiogenesis of the renal microvasculature. Interestingly, inhibition of NO synthesis did not lead to a loss of the preglomerular endothelium, which may relate to the effect of NO blockade to stimulate VEGF synthesis in the adjacent vascular smooth muscle cell.

Fructokinase activity mediates dehydration-induced renal injury

The epidemic of chronic kidney disease in Nicaragua (Mesoamerican nephropathy) has been linked with recurrent dehydration. Here we tested whether recurrent dehydration may cause renal injury by activation of the polyol pathway, resulting in the generation of endogenous fructose in the kidney that might subsequently induce renal injury via metabolism by fructokinase. Wild-type and fructokinase-deficient mice were subjected to recurrent heat-induced dehydration. One group of each genotype was provided water throughout the day and the other group was hydrated at night, after the dehydration. Both groups received the same total hydration in 24 h. Wild-type mice that received delayed hydration developed renal injury, with elevated serum creatinine, increased urinary NGAL, proximal tubular injury, and renal inflammation and fibrosis. This was associated with activation of the polyol pathway, with increased renal cortical sorbitol and fructose levels. Fructokinase-knockout mice with delayed hydration were protected from renal injury. Thus, recurrent dehydration can induce renal injury via a fructokinase-dependent mechanism, likely from the generation of endogenous fructose via the polyol pathway. Access to sufficient water during the dehydration period can protect mice from developing renal injury. These studies provide a potential mechanism for Mesoamerican nephropathy.

Sucrose induces fatty liver and pancreatic inflammation in male breeder rats independent of excess energy intake

Fructose induces metabolic syndrome in rats; but studies have been criticized for using high concentrations of fructose that are not physiologic, for using only pure fructose, and for not controlling for energy intake. We tested the hypothesis that a 40% sucrose diet (containing 20% fructose) might induce features of metabolic syndrome in male breeder rats independent of excess energy intake. Male Sprague-Dawley breeder rats were pair fed 40% sucrose or isocaloric starch diet for 4 months and evaluated for metabolic syndrome and diabetes. In vitro studies were performed in rat insulinoma cells (RIN-m5F) exposed to uric acid, and markers of inflammation were assessed. Rats fed a 40% sucrose diet developed accelerated features of metabolic syndrome with up-regulation of fructose-dependent transporter Glut5 and fructokinase. Fatty liver and low-grade pancreatic inflammation also occurred. Uric acid was found to stimulate inflammatory mediators and oxidative stress in islet cells in vitro. Sucrose, at concentrations ingested by a subset of Americans, can accelerate metabolic syndrome, fatty liver, and type 2 diabetes mellitus in male breeder rats; and the effects are independent of excess energy intake.