Role of nuclear factor kappa B (NF-kappaB) in oxidative stress-induced defective dopamine D1 receptor signaling in the renal proximal tubules of Sprague-Dawley rats

Abstract

Dopamine promotes sodium excretion, in part, via activation of D1 receptors in renal proximal tubules (PT) and subsequent inhibition of Na, K-ATPase. Recently, we have reported that oxidative stress causes D1 receptor-G-protein uncoupling via mechanisms involving protein kinase C (PKC) and G-protein-coupled receptor kinase 2 (GRK 2) in the primary cultures of renal PT of Sprague-Dawley (SD) rats. There are reports suggesting that redox-sensitive nuclear transcription factor, NF-kappaB, is activated in conditions associated with oxidative stress. This study was designed to identify the role of NF-kappaB in oxidative stress-induced defective renal D1 receptor-G-protein coupling and function. Treatment of the PT with hydrogen peroxide (H(2)O(2), 50 microM/20 min) induced the nuclear translocation of NF-kappaB, increased PKC activity, and triggered the translocation of GRK 2 to the proximal tubular membranes. This was accompanied by hyperphosphorylation of D1 receptors and defective D1 receptor-G-protein coupling. The functional consequence of these changes was decreased D1 receptor activation-mediated inhibition of Na, K-ATPase activity. Interestingly, pretreatment with pyrrolidine dithiocarbamate (PDTC, 25 microM/10 min), an NF-kappaB inhibitor, blocked the H(2)O(2)-induced nuclear translocation of NF-kappaB, increase in PKC activity, and GRK 2 translocation and hyperphosphorylation of D1 receptors in the proximal tubular membranes. Furthermore, PDTC restored D1 receptor G-protein coupling and D1 receptor agonist-mediated inhibition of the Na, K-ATPase activity. Therefore, we suggest that oxidative stress causes nuclear translocation of NF-kappaB in the renal proximal tubules, which contributes to defective D1 receptor-G-protein coupling and function via mechanisms involving PKC, membranous translocation of GRK 2, and subsequent phosphorylation of dopamine D1 receptors.

Figure 1. Effects of hydrogen peroxide (H2O2) and NF-κB inhibitor (PDTC) on translocation of NF-κB into the nucleus

A and B) representative blots and densitometric analysis of NF-κB (p65 subunit) and (p50 subunit) protein respectively, in the nuclear fraction of renal proximal tubules (PT). C and D) representative blots and densitometric analysis of NF-κB (p65 subunit) and (p50 subunit) protein respectively in the cytosolic fraction of PT. Results represent mean ± SEM (N=3 animals). Pretreatment with PDTC prevented H2O2-mediated increase in nuclear translocation of NF-κB. *P < 0.05 compared with vehicle using One-way ANOVA followed by Newman-Keuls Multiple Comparison Test.

Figure 2. H2O2 causes nuclear translocation of NF-κB in intact renal proximal tubular epithelial cells

The nuclear translocation of NF-κB was determined by immunofluorescence (details in methods). H2O2 (20 min/ 50 μM) caused an increase in nuclear fluorescence signal of NF-κB (Fig 2B, upper panel). Ascorbic acid (AA, 60 μg/ ml for 15 min) abolished H2O2-induced increase in nuclear signal of NF-κB (Fig 2C, upper panel). Lower panel represents cells in the corresponding plates showing nuclei stained with DAPI.

Figure 3. H2O2-mediated activation of PKC and subsequent translocation of GRK 2 to the membrane occurs via a mechanism involving NF-κB

A) Upper panel: Protein kinase C activity in control, H2O2, and H2O2 plus PDTC treated proximal tubular membranes. B) Upper panel: representative blot of GRK 2. Lower panel: densitometric analysis of GRK 2 protein in the proximal tubular membranes. Pretreatment with PDTC abolished H2O2-mediated increase in PKC activity and subsequent membranous GRK2 translocation. *P < 0.05 compared with vehicle using One-way ANOVA followed by Newman-Keuls Multiple Comparison Test (mean ± SEM; n=3-4).

Figure 4. NF-κB activation is responsible for H2O2-mediated hyperserine-phosphorylation and defective D1 receptor-G-protein coupling in the renal proximal tubular membranes

A) Proximal tubular cell membranes were used for immunoprecipitation of D1 receptors. Immunoprecipitated samples were then used for immunoblotting of serinephosphorylated D1 receptors and total D1 receptor proteins. Upper panel: representative immunoblots of serine-phosphorylated D1 receptors and total D1 receptor proteins. Lower panel: densitometric analysis of serine-phosphorylated D1 receptor protein, normalized to immunoprecipitated D1 receptor protein density. Pretreatment with PDTC prevented H2O2-induced hyperphosphorylation of D1 receptors. *P < 0.05 compared with Vehicle using One-way ANOVA followed by Newman-Keuls Multiple Comparison Test. B) Proximal tubular membranes from all 3 groups were incubated with [³⁵S] GTPγS, in the presence and absence of SKF-38393 (10^-9-10^-6 mol/l). Pretreatment with PDTC restored D1 receptor G-protein coupling in the proximal tubular membranes of SD rats. *P < 0.05 compared with H2O2 using One-way ANOVA followed by Newman-Keuls Multiple Comparison Test. #P < 0.05 from 10^-8 SKF 38393 using Student’s unpaired t test. (mean ± SEM; n=3).

Figure 5. Role of NF-κB in H2O2 induced impairment in SKF-38393 -mediated inhibition of Na-K-ATPase activity in the renal proximal tubules

Proximal tubular suspensions were incubated with SKF-38393 (10^-9-10^-6 mol/L) at 37°C for 15 minutes. Ouabain sensitive Na, K-ATPase (NKA) activity was measured as described in Methods. Pretreatment with PDTC restored the ability of D1 receptor agonist to inhibit NKA. *P < 0.05 compared with H2O2 using One-way ANOVA followed by Newman-Keuls Multiple Comparison Test (mean ± SEM; n=3). #P < 0.05 from 10^-8 SKF 38393 using Student’s unpaired t test.

Figure 6. A diagrammatic representation of the role of NF-κB in oxidative stress induced renal D1 receptor G-protein uncoupling and loss of functional response to D1 receptor activation

H2O2 induces the translocation of NF-κB from the cytosol to the nucleus. NF-κB increases PKC activity, which in turn, causes translocation of GRK 2 to the membrane. GRK 2 phosphorylates D1 receptors (D1R) preventing D1 receptor-G-protein coupling. The functional consequence is the inability of D1 receptor agonist to inhibit the Na-K-ATPase (NKA) activity (denoted by X). Treatment with PDTC, while inhibiting NF-κB nuclear translocation, also prevents PKC activity and GRK 2 membranous translocation; and therefore, restores D1 receptor-G-protein coupling and D1 receptor agonist-mediated inhibition of the Na-K-ATPase activity.