Dietary fructose enhances angiotensin II-stimulated Na+ transport via activation of PKC-α in renal proximal tubules.

Angiotensin II (ANG II) stimulates proximal nephron transport via activation of classical protein kinase C (PKC) isoforms. Acute fructose treatment stimulates PKC and dietary fructose enhances ANG II's ability to stimulate Na+ transport, but the mechanisms are unclear. We hypothesized that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation and increases in intracellular Ca2+. We measured total and isoform-specific PKC activity, basal and ANG II-stimulated oxygen consumption, a surrogate of Na+ reabsorption, and intracellular Ca2+ in proximal tubules from rats given either 20% fructose in their drinking water (fructose group) or tap water (control group). Total PKC activity was measured by ELISA. PKC-α, PKC-ß, and PKC-γ activities were assessed by measuring particulate-to-soluble ratios. Intracelluar Ca2+ was measured using fura 2. ANG II stimulated total PKC activity by 53 ± 15% in the fructose group but not in the control group (-15 ± 11%, P < 0.002). ANG II stimulated PKC-α by 0.134 ± 0.026 but not in the control group (-0.002 ± 0.020, P < 0.002). ANG II increased PKC-γ activity by 0.008 ± 0.003 in the fructose group but not in the control group (P < 0.046). ANG II did not stimulate PKC-ß in either group. ANG II increased Na+ transport by 454 ± 87 nmol·min-1·mg protein-1 in fructose group, and the PKC-α/ß inhibitor Gö6976 blocked this increase (-96 ± 205 nmol·min-1·mg protein-1, P < 0.045). ANG II increased intracellular Ca2+ by 148 ± 53 nM in the fructose group but only by 43 ± 10 nM in the control group (P < 0.035). The intracellular Ca2+ chelator BAPTA blocked the ANG II-induced increase in Na+ transport in the fructose group. We concluded that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation via elevated increases in intacellular Ca2+.

Angiotensin II-induced superoxide and decreased glutathione in proximal tubules: effect of dietary fructose.

Angiotensin II exacerbates oxidative stress in part by increasing superoxide (O2-) production by many renal tissues. However, whether it does so in proximal tubules and the source of O2- in this segment are unknown. Dietary fructose enhances the stimulatory effect of angiotensin II on proximal tubule Na+ reabsorption, but whether this is true for oxidative stress is unknown. We hypothesized that angiotensin II causes proximal nephron oxidative stress in part by stimulating NADPH oxidase (NOX)4-dependent O2- production and decreasing the amount of the antioxidant glutathione, and this is exacerbated by dietary fructose. We measured basal and angiotensin II-stimulated O2- production with and without inhibitors, NOX1 and NOX4 expression, and total and reduced glutathione (GSH) in proximal tubules from rats drinking either tap water (control) or 20% fructose. Angiotensin II (10 nM) increased O2- production by 113 ± 42 relative light units·mg protein-1·s-1 in controls and 401 ± 74 relative light units·mg protein-1·s-1 with 20% fructose (n = 11 for each group, P < 0.05 vs. control). Apocynin and the Nox1/4 inhibitor GKT136901 prevented angiotensin II-induced increases in both groups. NOX4 expression was not different between groups. NOX1 expression was undetectable. Angiotensin II decreased GSH by 1.8 ± 0.8 nmol/mg protein in controls and by 4.2 ± 0.9 nmol/mg protein with 20% fructose (n = 18 for each group, P < 0.047 vs. control). We conclude that 1) angiotensin II causes oxidative stress in proximal tubules by increasing O2- production by NOX4 and decreasing GSH and 2) dietary fructose enhances the ability of angiotensin II to stimulate O2- and diminish GSH, thereby exacerbating oxidative stress in this segment.

Deregulation of Cholesterol Homeostasis by a Nuclear Hormone Receptor Crosstalk in Advanced Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) features high intratumoral cholesterol levels, due to aberrant regulation of cholesterol homeostasis. However, the underlying mechanisms are still poorly understood. The retinoid acid receptor-related orphan receptor gamma (RORγ), an attractive therapeutic target for cancer and autoimmune diseases, is strongly implicated in prostate cancer progression. We demonstrate in this study that in mCRPC cells and tumors, RORγ plays a crucial role in deregulation of cholesterol homeostasis. First, we found that RORγ activates the expression of key cholesterol biosynthesis proteins, including HMGCS1, HMGCR, and SQLE. Interestingly, we also found that RORγ inhibition induces cholesterol efflux gene program including ABCA1, ABCG1 and ApoA1. Our further studies revealed that liver X receptors (LXRα and LXRß), the master regulators of cholesterol efflux pathway, mediate the function of RORγ in repression of cholesterol efflux. Finally, we demonstrated that RORγ antagonist in combination with statins has synergistic effect in killing mCRPC cells through blocking statin-induced feedback induction of cholesterol biosynthesis program and that the combination treatment also elicits stronger anti-tumor effects than either alone. Altogether, our work revealed that in mCRPC, RORγ contributes to aberrant cholesterol homeostasis by induction of cholesterol biosynthesis program and suppression of cholesterol efflux genes. Our findings support a therapeutic strategy of targeting RORγ alone or in combination with statin for effective treatment of mCRPC.

RORγ is a context-specific master regulator of cholesterol biosynthesis and an emerging therapeutic target in cancer and autoimmune diseases.

Aberrant cholesterol metabolism and homeostasis in the form of elevated cholesterol biosynthesis and dysregulated efflux and metabolism is well recognized as a major feature of metabolic reprogramming in solid tumors. Recent studies have emphasized on major drivers and regulators such as Myc, mutant p53, SREBP2, LXRs and oncogenic signaling pathways that play crucial roles in tumor cholesterol metabolic reprogramming. Therapeutics such as statins targeting the mevalonate pathway were tried at the clinic without showing consistent benefits to cancer patients. Nuclear receptors are prominent regulators of mammalian metabolism. Their de-regulation often drives tumorigenesis. RORγ and its immune cell-specific isoform RORγt play important functions in control of mammalian metabolism, circadian rhythm and immune responses. Although RORγ, together with its closely related members RORα and RORß were identified initially as orphan receptors, recent studies strongly support the conclusion that specific intermediates and metabolites of cholesterol pathways serve as endogenous ligands of RORγ. More recent studies also reveal a critical role of RORγ in tumorigenesis through major oncogenic pathways including acting a new master-like regulator of tumor cholesterol biosynthesis program. Importantly, an increasing number of RORγ orthosteric and allosteric ligands are being identified that display potent activities in blocking tumor growth and autoimmune disorders in preclinical models. This review summarizes the recent preclinical and clinical progress on RORγ with emphasis on its role in reprogramming tumor cholesterol metabolism and its regulation. It will also discuss RORγ functional mechanisms, context-specificity and its value as a therapeutic target for effective cancer treatment.

Dietary Fructose Increases the Sensitivity of Proximal Tubules to Angiotensin II in Rats Fed High-Salt Diets.

Dietary fructose causes salt-sensitive hypertension. Proximal tubules (PTs) reabsorb 70% of the filtered NaCl. Angiotensin II (Ang II), atrial natriuretic peptide (ANP) and norepinephrine (NE) regulate this process. Although Ang II signaling blockade ameliorates fructose-induced salt-sensitive hypertension, basal PT Na⁺ reabsorption and its sensitivity to the aforementioned factors have not been studied in this model. We hypothesized consuming fructose with a high-salt diet selectively enhances the sensitivity of PT transport to Ang II. We investigated the effects of Ang II, ANP and NE on PT Na reabsorption in rats fed a high-salt diet drinking tap water (HS) or 20% fructose (HS-FRU). Oxygen consumption (QO2) was used as a measure of all ATP-dependent transport processes. Na⁺/K⁺-ATPase and Na⁺/H⁺-exchange (NHE) activities were studied because they represent primary apical and basolateral transporters in this segment. The effect of 10-12 mol/L Ang II in QO2 by PTs from HS-FRU was larger than HS (p < 0.02; n = 7). In PTs from HS-FRU 10-12 mol/L Ang II stimulated NHE activity by 2.6 ± 0.7 arbitrary fluorescence units/s (p < 0.01; n = 5) but not in those from HS. The stimulatory effect of Ang II on PT Na⁺/K⁺-ATPase activity was not affected by HS-FRU. Responses of QO2 and NHE activity to ANP did not differ between groups. The response of QO2 to NE was unaltered by HS-FRU. We concluded that the sensitivity of PT Na⁺ reabsorption specifically to Ang II is enhanced by HS-FRU. This maintains high rates of transport even in the presence of low concentrations of the peptide, and likely contributes to the hypertension.

Dietary Fructose Enhances the Ability of Low Concentrations of Angiotensin II to Stimulate Proximal Tubule Na⁺ Reabsorption.

Fructose-enriched diets cause salt-sensitive hypertension. Proximal tubules (PTs) reabsorb 70% of the water and salt filtered through the glomerulus. Angiotensin II (Ang II) regulates this process. Normally, dietary salt reduces Ang II allowing the kidney to excrete more salt, thereby preventing hypertension. We hypothesized that fructose-enriched diets enhance the ability of low concentrations of Ang II to stimulate PT transport. We measured the effects of a low concentration of Ang II (10-12 mol/L) on transport-related oxygen consumption (QO2), and Na/K-ATPase and Na/H-exchange (NHE) activities and expression in PTs from rats consuming tap water (Control) or 20% fructose (FRUC). In FRUC-treated PTs, Ang II increased QO2 by 14.9 ± 1.3 nmol/mg/min (p < 0.01) but had no effect in Controls. FRUC elevated NHE3 expression by 19 ± 3% (p < 0.004) but not Na/K-ATPase expression. Ang II stimulated NHE activity in FRUC PT (Δ + 0.7 ± 0.1 Arbitrary Fluorescent units (AFU)/s, p < 0.01) but not in Controls. Na/K-ATPase activity was not affected. The PKC inhibitor Gö6976 blocked the ability of FRUC to augment the actions of Ang II. FRUC did not alter the inhibitory effect of dopamine on NHE activity. We conclude that dietary fructose increases the ability of low concentrations of Ang II to stimulate PT Na reabsorption via effects on NHE.