Activation of IKK/NF-κB provokes renal inflammatory responses in guanylyl cyclase/natriuretic peptide receptor-A gene-knockout mice

Abstract

The present study was aimed at determining the consequences of the disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) on proinflammatory responses of nuclear factor kappa B, inhibitory kappa B kinase, and inhibitory kappa B alpha (NF-κB, IKK, IκBα) in the kidneys of mutant mice. The results showed that the disruption of Npr1 enhanced the renal NF-κB binding activity by 3.8-fold in 0-copy (−/−) mice compared with 2-copy (+/+) mice. In parallel, IKK activity and IκBα protein phosphorylation were increased by 8- and 11-fold, respectively, in the kidneys of 0-copy mice compared with wild-type mice. Interestingly, IκBα was reduced by 80% and the expression of proinflammatory cytokines and renal fibrosis were significantly enhanced in 0-copy mice than 2-copy mice. Treatment of 0-copy mice with NF-κB inhibitors andrographolide, pyrrolidine dithiocarbamate, and etanercept showed a substantial reduction in renal fibrosis, attenuation of proinflammatory cytokines gene expression, and significantly reduced IKK activity and IkBα phosphorylation. These findings indicate that the systemic disruption of Npr1 activates the renal NF-κB pathways in 0-copy mice, which transactivates the expression of various proinflammatory cytokines to initiate renal remodeling; however, inhibition of NF-κB pathway repairs the abnormal renal pathology in mutant mice.

hypertension is tightly regulated by two cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP) (12). ANP and BNP are released from the heart into the circulation and elicit natriuretic, diuretic, vasorelaxant, and antiproliferative responses, all directed to reduce blood pressure and blood volume (11, 18, 38, 65). Distinct natriuretic peptide receptors have been identified and characterized by molecular cloning (51, 56), which includes natriuretic peptide receptor -A, -B, and -C, also designated, respectively, as NPRA, NPRB, and NPRC (18, 37). Both ANP and BNP bind to guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA), which is considered to be the principal natriuretic peptide receptor that synthesizes intracellular second messenger cGMP (15, 50, 65). Mice carrying targeted disruption of the Npr1 gene (encoding for GC-A/NPRA) exhibit hypertension, congestive heart failure, reduced kidney function, and altered plasma renin and angiotensin II (ANG II) levels (47, 58, 59, 65).

Nuclear factor kappa B (NF-κB) is a ubiquitous inducible transcription factor that activates various genes, including inflammatory cytokines, and has a critical role in the pathogenesis of many diseases (5, 6, 25, 42). NF-κB transcription factors, including p105/50, p100/52, p65, cRel, and RelB, contain a Rel homology domain that mediates their dimerization and DNA binding properties (6). These proteins are sequestered in the cytoplasm of most cells, where they are bound to a family of inhibitory proteins known as inhibitory kappa B (IκB) (5, 21). Treatment of cells with cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), stimulates the activity of IκB kinases (IKKs), which phosphorylates IκBα, leading to its ubiquitination and degradation by the proteosome (20, 21, 33, 34). This process results in the nuclear translocation of NF-κB proteins, where they bind to the κB element of the promoter of a variety of genes involved in the control of immune and inflammatory responses (5, 6, 20, 21, 33, 34). Earlier studies have shown that different forms of IκB proteins (IκBα, β, γ, ϵ) preferentially interact with specific subunits of NF-κB (23). Upon stimulation by various activators, these proteins undergo degradation to activate NF-κB (16). In the in vitro system, the degradation of IκBα has been reported to be transient, indicating a potential mechanism for limiting NF-κB activation (61). It is important to note, however, that the role of in vivo IκBα activation has largely been remained unclear. In the present study, we sought to examine whether ablation of the Npr1 gene has a critical function in NF-κB activation through the regulation of IκBα, resulting in altered renal function and abnormal renal morphology in mutant mice. We further examined the compensatory effects of different drugs in suppressing IKK/NF-κB signaling to determine their beneficial effects on renal protection in Npr1 mutant mice.

Systemic deletion of Npr1 gene leads to high blood pressure, cardiac hypertrophy, and congestive heart failure after 6 mo of age (47, 64). In the present studies, we utilized 6 mo old age-matched wild-type and Npr1 gene-disrupted mice to examine the consequences of NPRA deletion on proinflammatory responses of NF-κB, IKK, and IκBα in the kidneys of Npr1 null mutant mice.

MATERIALS AND METHODS

Materials.

Primary antibodies, TNF-α, interleukin-1alpha (IL-1α), interleukin-1beta (IL-1β), IL-6, interleukin-10 (IL-10), transforming growth factor (TGF)-β1, p65, phosphorylated p65 at Ser276 (p-p65, Ser276) and p-p65 (Ser536), IκBα, and phosphorylated IκBα (p-IκBα, Ser32/36) were obtained from Santa Cruz Biotechnology (San Diego, CA). Goat anti-rabbit secondary antibody conjugated with Alexa Fluor 647 and ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Molecular Probes (Invitrogen, Eugene, OR). The electrophoretic mobility shift assay (EMSA) kit was purchased from Promega (Madison, WI). Radioisotope for [γ-³²P] ATP (3,000 Ci/mmol) was purchased from Perkin Elmer (Waltham, MA). Andrographolide (Andro) and pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma Chemicals (St. Louis, MO). The TNF-α inhibitor, etanercept, was kindly provided by Amgen (Thousand Oaks, CA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA). The creatinine kit was purchased from BioAssay System (Hayward, CA). Microalbumin assay kit was purchased from Bethyl Laboratories (Montgomery, TX). Quantifast SYBR Green RT-PCR kit and RNase-free DNase were obtained from Qiagen (Valencia, CA). A multiplex kit for mouse cytokine assay was purchased from Millipore (Billerica, MA). All other chemicals were of reagent grade.

Generation of Npr1 gene-knockout mice.

Npr1 gene-disrupted mice were produced by homologous recombination in embryonic stem cells as previously described (47). These mice were bred and maintained at the animal facility of the Tulane University Health Sciences Center. Animals were handled under protocols approved by the Institutional Animal Care and Use Committee. The mouse colonies were housed under 12 h light/dark cycles at 25°C and fed regular chow (Purina Laboratory) and tap water ad libitum. All experimental animals were littermate progenies of the C57/BL6 genetic background and were designated as Npr1 gene-disrupted homozygous null mutant (Npr1^−/−, 0-copy), heterozygous (Npr1^+/−, 1-copy), and wild-type (Npr1^+/+, 2-copy) mice. The animals were genotyped by polymerase chain reaction (PCR) analyses of DNA isolated from tail biopsies using primer A (5′ GCTCTCTTGTCGCCGAATCT-3′), corresponding to 5′ sequence of the mouse Npr1 gene common to both alleles (Npr1^+/+): primer B (5′-TGTCACCATGGTCTGATCGC-3′), corresponding to the exon 1 sequence present only in the intact allele (Npr1^+/−), and primer C (5′-GCTTCCTCGTGCTTTACGGT-3′), corresponding to a sequence in the neomycin-resistance cassette present only in the null mutant allele (Npr1^−/−). PCR was carried out in 25 μl of reaction mixture containing 50 mM Tris·HCl (pH 8.3), 20 mM ammonium sulfate, 1.5 mM MgCl₂, 10% DMSO, 100 μM dNTPs, 2 units of Taq DNA polymerase, and 40 nM primers with a 60 s denaturation step at 94°C, a 60 s annealing step at 60°C, and a 60 s extension step at 72°C for 35 cycles using the GeneAmp 9700 (Applied Biosystems). PCR product was resolved on 2% agarose gel with the endogenous band of 500 bp and the targeted band of 200 bp.

The initial Npr1 gene-disrupted male mice were littermate progenies of a mixed 129 × C57/BL6 genetic background and showed high blood pressure and sudden death after 6 mo of age. However, after 12 generations of backcross breeding into C57/BL6 background, the progenies showed less mortality rate, while cardiac remodeling and fibrosis still persist. In the present studies, we utilized 24 wk old age-matched 0-copy and 2-copy male mice.

NF-κB inhibitor treatment.

We compared the efficacy of three types of NF-κB inhibitors. PDTC, a well-known inhibitor of NF-κB, showed potential responses. Andrographolide (Andro) is the major active principle of the medicinal plant Andrographis paniculata, which has been extensively used in traditional herbal medicine for the treatment of inflammatory diseases. Etanercept is used to treat autoimmune disease, generally, by interfering with TNF-α and acting as its inhibitor. Twenty-four week adult 0-copy (n = 32) and age-matched 2-copy (n = 32) mice were infused with the drugs in the following treatment groups: group I, 2-copy + saline; group II, 2-copy + Andro (4 mg/kg/day); group III, 2-copy + PDTC (50 mg/kg/day); group IV, 2-copy + etanercept (1 μg/kg/day); group V, 0-copy + saline; group VI, 0-copy + Andro (4 mg/kg/day); group VII, 0-copy + PDTC (50 mg/kg/day); and group VIII, 0-copy + etanercept (1 μg/kg/day). Thereafter, saline-treated mice are referred to as vehicle-treated groups. All drugs were subcutaneously infused for 30 days using Mini-Osmotic pump.

Blood pressure analysis.

Systolic blood pressure (SBP) was measured by a noninvasive computerized tail-cuff method using Visitech 2000 and was calculated as the average of three to five sessions per day for 7 consecutive days as previously described (58). In brief, all the mice were first undergone training procedure (for acclimatization) for 7 days. Afterward, actual SBP was measured on and from 24th day till 30th day of the treatment and data were collected. The results are expressed as the pooled average of 40 measurements for each group.

Blood and tissue collection.

Blood was collected by cardiac puncture under CO₂ anesthesia in prechilled tubes containing 10 μl of heparin (1,000 USP units/ml). Plasma was separated by centrifugation at 3,000 rpm for 20 min at 4°C and stored at −80°C until use. Animals were euthanized by administration of a high concentration of CO₂. Kidney tissues were collected, frozen in liquid nitrogen, and stored at −80°C.

Morphological studies.

Kidney tissues from each group were fixed in 10% buffered paraformaldehyde solution. Paraffin-embedded tissue sections (5-μm) were stained with hematoxylin and eosin (H&E) and with Masson's Trichrome for the presence of interstitial collagen fiber accumulation as a marker of renal fibrosis. The percentage of matrix mesangial expansion (MME) relative to the total kidney area was determined by calculations made in 20 randomly selected microscopic fields in five sections per kidney using ImagePro Plus image analysis software (Media Cybernetics, Silver Spring, MD). The percentage of fibrosis to the total kidney area was also determined.

Immunofluorescence of p-p65.

Immunofluorescence staining was done on 5 μm sections of paraffin-embedded kidney tissues. After dehydration and antigen retrieval, the sections were sequentially incubated at room temperature with blocking reagent, primary antibodies (rabbit polyclonal p-p65 antibody), and goat anti-rabbit IgG conjugated with Alexa Fluor for 30 min. After that the sections were washed with PBST and an appropriate amount of ProLong-Gold Antifade reagent with DAPI was added. The nonspecific binding of secondary antibodies was excluded by omitting the primary antibody and served as a control. Immunofluorescence was observed and photographed under a fluorescence microscope (Olympus BX51) with an integrated Magnafire Digital Firewire Camera Software. The percentage of p-p65-positive area to the total kidney was calculated using ImagePro Plus image analysis software (Media Cybernetics, Silver Spring, MD).

Assay of albumin and creatinine in urine and plasma.

Albumin levels were measured in 24 h urine samples collected from mice in a metabolic cage, using the ELISA kit (Bethyl Laboratories, Montgomery, TX). Plasma and urine creatinine concentrations were measured using the creatinine assay kit (BioAssay Systems, Hayward, CA) as previously described (68). Creatinine levels were calculated according to the manufacturer's protocol. Creatinine clearance (CCr) was calculated from the creatinine concentration in the collected urine samples (UCr), plasma concentration (PCr), and urine volume (V), and expressed as ml/24 h.

Determination of collagen in kidney tissues.

Total collagen concentrations in kidney tissue samples were quantified from the hydroxyproline content as previously described (29). In brief, the tissue samples were homogenized and hydrolyzed in 6 N HCl at 110°C for 18 h in a sealed reaction vial. The samples were then dried under vacuum, and the residue was resuspended in 50% isopropanol and then treated with chloramine T. After 10 min of incubation, the samples were mixed with Ehrlich's reagent and incubated at 50°C for 90 min. The absorbance was read at 558 nm using water as a reference; readings were corrected with a reagent blank. To obtain the total collagen content, a conversion factor of 8.2 was used.

Preparation of cytosolic and nuclear extracts.

Cytosolic and nuclear proteins were extracted from frozen kidney tissues as previously described (14). Briefly, the kidney tissues were homogenized in an ice-cold 10 mM Tris·HCl buffer (pH 8.0) containing 0.32 M sucrose, 3 mM calcium chloride (CaCl₂), 2 mM magnesium acetate (MgOAc), 0.1 mM ethylene diaminetetraacetic acid (EDTA), 0.5% Nonidet P-40 (NP-40), 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 4.0 μg/ml each of leupeptin, aprotinin, and pepstatin. The homogenate was centrifuged at 800 g, and the supernatant was separated and saved as a cytosolic fraction. The precipitate was resuspended in a low-salt buffer (20 mM HEPES, pH 7.9; 1.5 mM MgCl₂; 20 mM KCl; 0.2 mM EDTA; 25% glycerol; 0.5 mM DTT; and 0.5 mM PMSF), incubated on ice for 5 min, and mixed with an equal volume of high-salt buffer containing 20 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 800 mM KCl, 0.2 mM EDTA, 1% NP-40, 25% glycerol, 0.5 mM DTT, 0.5 mM PMSF, and 4.0 μg/ml each of leupeptin, aprotinin, and pepstatin. The mixture was incubated on ice for 30 min and centrifuged at 14,000 g for 20 min. The supernatant was separated and stored as a nuclear fraction at −80°C.

Assay of plasma and renal levels of proinflammatory cytokines.

The concentration of proinflammatory cytokines, including TNF-α, IL-1α, IL-1β, IL-6, and TGF-β1, were measured in both plasma and kidney tissue homogenates by multiplex bead array format (Milliplex and Lincoplex from Millipore), using a Bio-Plex Instrument (Bio-Rad) according to the manufacturer's guidelines. Spectrally addressed polystyrene beads coated with cytokine-specific monoclonal antibodies were used to capture the cytokine of interest. The instrument sorted out and measured the fluorescent signal from each bead by dual excitation sources.

Western blot analysis.

For Western blots, a cytoplasmic fraction was mixed with an equal volume of 2× sodium dodecyl sulfate (SDS) sample loading buffer containing 125 mM Tris·HCl, 4% SDS, 20% glycerol, 100 mM DTT, and 0.2% bromphenol blue, then separated in a 10% SDS polyacrylamide gel. Proteins were eletrotransferred onto a polyvinyl difluoride (PVDF) membrane. The membrane was blocked with 1× Tris-buffered saline-Tween 20 (TBST; 25 mM Tris, 500 mM NaCl, and 0.05% Tween 20, pH 7.5) containing 5% fat-free milk and incubated overnight in TBST containing 3% fat-free milk at 4°C with primary antibodies (1:200 dilution). The membrane was then treated with corresponding secondary horseradish peroxidase-conjugated antibodies (1:5,000 dilution). Protein bands were visualized by enhanced chemiluminescence (ECL) plus detection system with Alpha Innotech phosphoimager.

EMSA.

EMSA was done as previously described (13). Double-stranded oligonucleotides containing the consensus-binding site for NF-κB were used. Oligonucleotides were end-labeled using [γ-³²P]ATP and T4 polynucleotide kinase. For binding reaction, 5 μg of nuclear proteins was incubated for 30 min at room temperature in binding buffer [50 mM Tris·HCl, pH 8.0; 750 mM KCl; 2.5 mM EDTA; 0.5% Triton X-100; 20% glycerol (vol/vol) and 1 mM DTT] containing 2 μg poly(dI-dC) and 50,000 cpm radiolabeled oligonucleotides. Cold competitor assays were performed by adding 100-fold excess molar concentrations of unlabeled NF-κB. The supershift assay was carried out by incubating nuclear extract with p65 antibody and subsequently with radiolabeled oligonucleotides. The DNA-protein complex was resolved from the free-labeled DNA using 4% native polyacrylamide gel electrophoresis and autoradiography.

IKK activity assay.

The IKK activity assay was done by the method previously described (39). Cytoplasmic proteins (200 μg) from the kidney tissues of all experimental groups were immunoprecipitated with 2 μg IKKβ antibody at 4°C for 2 h. Protein G plus-agarose beads (20 μl) were added, and the mixture was incubated for another 1 h at 4°C. The content was centrifuged at 3,000 rpm for 5 min at 4°C. The pellet was then washed twice with lysis buffer (50 mM HEPES, pH 7.4; 250 mM NaCl; 1% NP-40; 1 mM PMSF; and 5.0 μg/ml each of leupeptin and aprotinin) and once in kinase buffer (10 mM HEPES, pH 7.4; 1 mM MnCl₂; 5 mM MgCl₂, 12.5 mM β-glycero-2-phosphate; 50 μM Na₃VO₄; 2 mM NaF; 50 μM DTT; and 10 μM ATP). The pellet was then resuspended in kinase buffer. The IKK reaction was carried out in the presence of 1 μg glutathione-S-transferase-IkBα substrate and 5 μCi [γ-³²P]ATP (3,000 Ci/mmol) at 37°C for 30 min. The reaction was stopped by the addition of 3× Laemmli sample buffer; the phosphorylated protein was resolved by 15% SDS-PAGE and autoradiography.

NF-κB targeted and nontargeted gene quantitation by qRT-PCR.

Total RNA was isolated from each kidney sample, using the TRIZOL protocol. The purified RNA from each sample was used for qRT-PCR. The genetic expressions of IKK enzyme isoforms (IKK α, β, γ, and ε), NF-κB proteins (IκB-α, p65, p50, p52, RelB, cRel), and proinflammatory cytokines such as TNF-α, IL-1α, IL-1β, and IL-6 were determined by using the mouse RT² profiler PCR array kit according to manufacturer's guidelines. Data were analyzed by RT² profiler PCR array data analysis software. For TGF-β1 and Col1α, extracted RNA samples were amplified with specific primers by real-time PCR using the Qiagen Quantifast Sybergreen RT-PCR kit. Amplification data were collected by the Stratagene Mx3005P system. For each assay, a standard curve was constructed using a different concentration of RNA. The quantitative fold changes in mRNA expression were determined relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels in each corresponding group and calculated using the REST2009 software from Qiagen.

Statistical analysis.

Statistical analysis was done using GraphPad software (San Diego, CA). The results are presented as means ± SE. Differences between groups were determined by one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons post hoc test. We also used Student's t-test for comparison between groups. A P value of 0.05 was considered significant.

RESULTS

NF-κB activity and IκBα phosphorylation.

The protein expression of NF-κB (p65) and its phosphorylated form was markedly enhanced in the cytosolic fractions of 0-copy null mutant mice kidneys (Fig. 1A, lanes i–iii). Densitometric analysis demonstrated that the phosphorylation of p65 protein (p-p65) was increased by eightfold at Ser276 and by fivefold at Ser536 in the kidneys of 0-copy mice compared with wild-type mice (Fig. 1, B and C). The expression of p65 protein was also increased 2.3-fold in 0-copy mice compared with 2-copy wild-type mice (Fig. 1D). Treatment with Andro reduced the phosphorylation of p65 by 80% at both phosphorylation sites in 0-copy mice compared with vehicle-treated control mice. PDTC and etanercept treatment reduced phosphorylation at Ser536 by 80%, but at Ser 276 by only 54%. To investigate the functional role of IκBα in Npr1 gene-disrupted mice kidney, we also phosphorylated IκBα (Fig. 1A, lane iv–vi). Densitometric analysis showed that the phosphorylation of IκBα was increased by 11-fold in 0-copy mouse kidneys compared with 2-copy mice (Fig. 1E). However, the IκBα level was reduced by 80% in 0-copy mice compared with 2-copy control mice. After treatment with Andro, PDTC, and etanercept, the levels of IκBα were restored in 0-copy mice kidneys compared with vehicle-treated controls (Fig. 1F).

Fig. 1.

Comparative analysis of p65, IκBα phosphorylation, and IκBα degradation in Npr1 gene-disrupted mouse kidneys with or without inhibitor treatment. A: renal expression levels of p-p65 (Ser276), p-p65 (Ser536), p65, p-IκBα (Ser32/36), and IκBα proteins as determined by Western blot analysis. β-Actin was used as a loading control. Densitometric analyses of the respective bands were done by Alpha Innotech phosphoimager and are shown in B–F for each protein. 2-Copy and 0-copy mice were treated with different inhibitors, andrographolide (Andro), pyrrolidine dithiocarbamate (PDTC), and etanercept (Eta), at different doses. Veh, saline-treated group. Bar represents mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.001; ♣P < 0.01; n = 7 mice per group.

To examine whether the increase in NF-κB activity in Npr1 0-copy mice kidney was caused by IKK activation, an in vitro kinase assay was done with or without inhibitor treatment. IKKβ antibody was used to immunoprecipitate the IKK complex from each sample. The IKK activity was assessed by analyzing the phosphorylation of GST-tagged substrate (IκBα) by the immunoprecipitated IKK complex. IKK activity was increased by eightfold in Npr1 0-copy kidney compared with 2-copy control kidney; it was reduced by 60% after treatment with Andro, PDTC, and etanercept (Fig. 2, A and B). The IKK activities were comparable in 2-copy vehicle-treated control mice with or without inhibitor treatment. We also used EMSA to determine the NF-κB binding activity in 0-copy and 2-copy mouse kidneys (Fig. 2C). NF-κB binding activity was increased by 3.8-fold in 0-copy mice compared with 2-copy mice kidney. Treatment with Andro, PDTC, and etanercept reduced NF-κB binding activity in 0-copy mice by almost 60%. The NF-κB binding activity was comparable in 2-copy control mice with or without inhibitor treatment. The specific DNA binding of NF-κB was confirmed by the addition of a 100-fold molar excess of unlabeled NF-κB DNA competitor into the EMSA reaction. The presence of NF-κB in the protein complex was also demonstrated by antibody supershift assays (Fig. 2C).

Fig. 2.

Comparative analysis of IKK activity by in vitro phosphorylation assay and measurements of NF-κB DNA binding activity by EMSA in Npr1 gene-disrupted mice. A: autoradiogram of phosphorylation ability of IKK enzyme complex to phosphorylate IκBα in kidneys of 2-copy and 0-copy mice with or without inhibitor treatment. B: the respective densitometric analyses. C: autoradiogram of the binding activity of NF-κB DNA binding by EMSA. Veh, saline-treated group. Bar represents mean ± SE. **P < 0.01; ♣P < 0.01; n = 6 mice per group.

Systemic and renal proinflammatory cytokine levels.

As shown in Fig. 3A, plasma TNF-α levels were increased by 6.3-fold (13.32 ± 1.13 pg/ml, P < 0.01) in 0-copy mice compared with 2-copy mice (2.10 ± 0.35 pg/ml). Plasma TNF-α levels were significantly reduced in 0-copy mice after treatment with Andro (3.54 ± 0.43 pg/ml), PDTC (4.57 ± 1.06 pg/ml), and etanercept (2.49 ± 0.81 pg/ml). Similarly, TNF-α concentrations in the kidney were significantly increased (22.02 ± 1.15 pg/mg protein, P < 0.01) in 0-copy mice compared with 2-copy vehicle-treated control mice (9.89 ± 0.85 pg/mg protein) (Fig. 3B). Similar to plasma, renal TNF-α levels were also significantly reduced in 0-copy mice after treatment with Andro (9.24 ± 1.03 pg/mg protein), PDTC (11.51 ± 0.99 pg/mg protein), and etanercept (6.39 ± 0.61 pg/mg protein) compared with vehicle-treated 0-copy mice. Compared with plasma and kidney IL-6 levels in 2-copy mice, those in 0-copy mice were significantly (P < 0.01) increased (52.95 ± 3.59 pg/ml and 79.85 ± 4.62 pg/mg protein, respectively) (17.34 ± 0.70 pg/ml and 42.70 ± 3.43 pg/mg protein, respectively) (Fig. 3, C and D). IL-6 levels were reduced by 50–60% in both the plasma and kidneys of 0-copy mice after treatment with Andro, PDTC, and etanercept. In 0-copy mice, IL-1α levels were significantly increased by 3.7-fold in plasma (15.96 ± 1.56 vs. 4.29 ± 1.01 pg/ml) and 2.3-fold in kidney tissues (798.2 ± 39.38 vs. 342.4 ± 14.96 pg/mg protein) compared with vehicle-treated wild-type controls (Fig. 3, E and F). In addition, IL-1β levels were also markedly higher in 0-copy kidneys (56.31 ± 5.76 vs. 16.40 ± 1.25 pg/mg protein, 3.4-fold compared with 2-copy controls) (Fig. 3G). However, there was no change in plasma IL-1β levels in 2-copy and 0-copy mice (data not shown). Levels of active TGF-β1 were significantly increased by 3.2-fold in plasma (47.22 ± 4.50 vs. 14.63 ± 1.45 pg/ml, P < 0.01) and 3.7-fold in kidney tissues (159.1 ± 21.83 vs. 42.05 ± 9.32 pg/mg protein) of 0-copy mice compared with 2-copy control mice (Fig. 3, H and I). Treatment with inhibitors reversed the increase in IL-1α, IL-1β, and TGF-β1 levels in both plasma and kidney tissues. Figure 4A shows the protein expression of proinflammatory and anti-inflammatory cytokines in the kidneys of mice in all experimental groups. A 4.7-fold increase in TNF-α protein expression was observed in 0-copy mouse kidney compared with 2-copy controls (Fig. 4B). Similarly, significant increases in IL-6 (10.2-fold, Fig. 4C), TGF-β1 (4.2-fold, Fig. 4D), IL-1α (9-fold, Fig. 4E), and IL-1β (4-fold, Fig. 4F) protein expression levels were observed in the kidneys of Npr1 0-copy mice compared with 2-copy mice. However, IL-10 protein expression was significantly reduced in the kidneys of 0-copy mice compared with 2-copy control animals (Fig. 4A). While treatment with inhibitors led to a reduction in proinflammatory cytokine expression in the kidneys of 0-copy mice (Fig. 4, B–F), IL-10 expression was increased in 0-copy mouse kidneys treated with Andro, PDTC, and etanercept (Fig. 4G).

Fig. 3.

Quantitative analysis of plasma and renal proinflammatory cytokines and growth factor in Npr1 gene-disrupted mice. A, C, E, and H: plasma levels of TNF-α, IL-6, IL-1α, and active TGF-β1, respectively. B, D, F, G, and I: concentrations of TNF-α, IL-6, IL-1α, IL-1β, and active TGF-β1, respectively, in kidney tissues. All analytes were determined with multiplex kits from Millipore according to the manufacturer's guidelines. Veh, saline-treated group. Values are expressed as means ± SE. **P < 0.01; ♣P < 0.01; n = 8 mice per group.

Fig. 4.

Renal expression of proinflammatory cytokines, anti-inflammatory cytokine, and growth factor in Npr1 gene-disrupted mice. A: renal protein expression of proinflammatory cytokines, TNF-α, IL-6, IL-1α, and IL-1β and anti-inflammatory cytokine, IL-10, with growth factor, TGF-β1 by Western blot. B–G: the densitometric analyses for TNF-α, IL-6, TGF-β1, IL-1α, IL-1β, and IL-10, respectively. Veh, saline-treated group. **P < 0.01; ♣P < 0.01; n = 6 mice per group.

Blood pressure and renal functional measurements.

Blood pressure measurements showed a significant increase in SBP in 0-copy mice (138.2 ± 4.0 mmHg) vs. 2-copy mice (104.0 ± 1.9 mmHg). Treatments with Andro, PDTC, and etanercept markedly reduced the SBP of 0-copy mice (Table 1). A significant (P < 0.01) increase occurred in the urinary ratio of albumin and creatinine in 0-copy mice (4.6-fold, 5.60 ± 1.0) compared with wild-type controls (1.20 ± 0.2). In 0-copy mice, a significant reduction occurred in the ratio of urinary albumin to creatine after treatment with Andro (2.1 ± 0.3), PDTC (2.3 ± 0.2), and etanercept (2.5 ± 0.3). On the other hand, CCr was reduced by 59% in 0-copy mice (108.1 ± 7.7 ml/24 h, P < 0.001) compared with wild-type mice (263.5 ± 15.8 ml/24 h) and was attenuated following treatment with Andro (205.9 ± 21.8 ml/24 h), PDTC (171.3 ± 13.8 ml/24 h), and etanercept (178.5 ± 11.8 ml/24 h) (Table 1). Similarly, renal collagen content was significantly increased by twofold (P < 0.01) in 0-copy mice (3.1 ± 0.2 μg/g kidney wt) compared with 2-copy mice (1.6 ± 0.1 μg/g kidney wt). The collagen content reverted to normal in 0-copy mice treated with Andro (1.8 ± 0.1 μg/g kidney wt), PDTC (1.7 ± 0.1 μg/g kidney wt), and etanercept (1.5 ± 0.1 μg/g kidney wt).

Table 1.

Measurement of SBP, renal function, and kidney collagen in Npr1 gene-disrupted and wild-type mice with or without Andro, PDTC, and etanercept treatment

Parameters	SBP, mmHg	Alb/Cr (Urine)	CCr, ml/24 h	KC, μg/g
Vehicle
2-Copy	104.0 ± 1.9	1.2 ± 0.2	263.5 ± 15.8	1.6 ± 0.1
0-Copy	138.2 ± 4.0b	5.6 ± 1.0a	108.1 ± 7.7b	3.1 ± 0.2a
Andro
2-Copy	100.6 ± 2.2	1.0 ± 0.2	273.5 ± 11.9	1.5 ± 0.1
0-Copy	108.8 ± 2.7	2.1 ± 0.3†	205.9 ± 21.8†	1.8 ± 0.1
PDTC
2-Copy	104.4 ± 2.1	0.8 ± 0.1	264.1 ± 18.2	1.4 ± 0.1
0-Copy	110.4 ± 3.0	2.3 ± 0.2†	171.3 ± 13.8♣	1.7 ± 0.1
Etanercept
2-Copy	98.6 ± 1.2	1.1 ± 0.2	266.5 ± 22.8	1.4 ± 0.1
0-Copy	117.0 ± 2.7	2.5 ± 0.3†	178.5 ± 11.8♣	1.5 ± 0.1

Values are expressed as means ± SE. Blood pressure was measured by computerized tail-cuff method. The urine albumin, creatinine, kidney collagen levels were determined as described in materials and methods.

SBP, systolic blood pressure; CCr, creatinine clearance; Alb/Cr, urine albumin-creatinine; KC, kidney collagen. Andro, andrographolide; PDTC, pyrrolidine dithiocarbamate.

^aP < 0.01,

^bP < 0.001, (vehicle-treated 2-copy vs. vehicle-treated 0-copy),

^♣P < 0.05,

^†P < 0.01 (vehicle-treated 0-copy vs. drug-treated 0-copy) n = 6 in each group.

Immunofluorescence detection of p-p65 and renal morphological changes.

To confirm the enhanced expression of p-p65 in glomerular mesangial cells and renal tubular cells, localization of active p65 (p-p65) protein in paraffin-embedded kidney tissue sections was done. There was a ninefold increase in p-p65 expression (NF-κB, red fluorescence) in 0-copy mouse kidney compared with 2-copy mice (Fig. 5A, top). The robust expression of p-p65 protein in glomerular mesangial cells and renal tubular epithelial cells (arrow, Fig. 5A) represents marked activation of NF-κB in 0-copy mice kidney. On the other hand, p-p65 protein was not discernible in the glomerular mesangial cells and renal tubular epithelial cells (marked by arrowheads) in 2-copy wild-type mice (Fig. 5A, bottom). Treatment with Andro, PDTC, and etanercept showed marked attenuation of NF-κB expression in the glomerular mesangial cells and tubular epithelial cells in the kidneys of 0-copy mice (Fig. 5D). H&E staining of tissues showed progressive expansion of the interstitial spaces, generally known as MME, in kidney sections of 0-copy mice, but not in wild-type mice (Fig. 5B). The percentage of MME was eightfold higher in 0-copy mice than in 2-copy mice (Fig. 5E). The percentages of such changes were minimum and are comparable in 2-copy animals with or without inhibitor treatment (Fig. 5E). However, attenuation of this pathology was observed in 0-copy mouse kidney treated with Andro (63%), PDTC (60%), and etanercept (58%). Masson trichrome stain of the kidney tissues showed that compared with wild-type mice, Npr1 gene-disrupted mice had increased matrix production and increased collagen accumulation (Fig. 5C). The percentage of blue-stained area (renal fibrosis) was significantly elevated (eightfold) in 0-copy mice compared with wild-type control mice (Fig. 5F). The blue stained area was much less in 0-copy mice treated with Andro (62%), PDTC (52%), and etanercept (58%) than that in vehicle-treated controls.

Fig. 5.

Comparative analysis of p-p65 immunoexpression and renal histology for mesangial matrix expansion (MME) and renal fibrosis in Npr1 gene-disrupted mice. A: immunofluorescence of p-p65 protein in the kidney sections is shown by arrows. Top: images of p-p65 immunoexpression in glomerular mesangial cells and tubular epithelial cells. Middle: images with DAPI for nucleus. Bottom: respective merged images in 2-copy and 0-copy mice kidney sections. The p-p65 immunofluorescence was reduced in glomeruli, and in tubular epithelial cells (shown by arrowhead) in 0-copy mice kidney after treatment with Andro, PDTC, and Eta compared with saline-treated Npr1 0-copy mice. B: renal kidney sections stained with hematoxylin and eosin (H&E), indicating the MME. Increased MME were observed in 0-copy kidney compared with 2-copy control. C: accumulation of collagen (renal fibrosis) in the kidney sections of 0-copy mice, after staining with Masson's trichrome. D–F: their quantitative analysis, respectively. Veh, saline-treated group; ***P < 0.001; ♣P < 0.01; n = 5 mice per group.

Expression of NF-κB targeted and nontargeted genes in the kidneys.

We did real-time RT-PCR from all experimental groups to determine the specific expression profile of renal NF-κB targeted and nontargeted genes, including TNF-α, IL-1α, IL-1β, IL-6, TGF-β1, and Col1α, in Npr1 gene-disrupted mice with or without inhibitor treatments. We also determined the mRNA expression levels of different isoforms of IKK (α, β, γ, and ε) and NF-κB family proteins involving p65 (RelA), p50 (NFKB1), p52 (NFKB2), RelB, cRel, and IκBα to examine the levels in 0-copy mouse kidneys during the course of each inhibitor treatment. Significant increases in TNF-α (10-fold), IL-6 (9-fold), IL-1α (6.5-fold), and IL-1β (4.7-fold) mRNA expression levels were observed in the kidneys of 0-copy mice compared with their respective 2-copy controls (Fig. 6A). GAPDH was used as an internal control to compare the relative expression of each gene. Treatment with inhibitors attenuated the TNF-α mRNA expression levels in 0-copy mice treated with Andro, PDTC, and etanercept by 67, 54, and 77%, respectively. Simultaneously, treatment with Andro, PDTC, and etanercept also showed a substantial reversal of IL-6 and IL-1α mRNA expression in the kidneys of 0-copy mice compared with vehicle-treated 0-copy control mice. A significant attenuation of IL-1β mRNA was observed in 0-copy mouse kidneys after treatment with each inhibitor. TGF-β1 and Col1α mRNAs were also increased by 8.5-fold and 7.4-fold, respectively, in 0-copy mice kidney compared with controls (Fig. 6B). TGF-β1 mRNA expression was attenuated by 60, 40, and 67%, respectively, in Andro, PDTC, and etanercept-treated 0-copy mice kidney. Similarly, the inhibitor treatment normalized Col1α mRNA expression in the kidneys of 0-copy animals.

Fig. 6.

Effect of inhibitors on NF-κB targeted and nontargeted genes in the kidneys of Npr1 gene-disrupted mice. A: relative mRNA expression of NF-κB targeted genes (TNF-α, IL-6, IL-1α and IL-1β) normalized to GAPDH mRNA in the kidney tissues with or without inhibitor treatment. B: mRNA expression of NF-κB nontargeted genes (TGF-β1 and Col1α) relative to GAPDH mRNA in the kidney tissues. Veh, saline-treated group. Bar represents mean ± SE. **P < 0.01; ***P < 0.001; ♣P < 0.01; n = 7 mice per group.

The mRNA expression of different isoforms of IKK enzyme-complex (α, β, γ, and ε) were significantly activated in 0-copy mice kidneys by sevenfold (IKKα), sixfold (IKKβ), fourfold (IKKγ), and 4.8-fold (IKKε), respectively, compared with 2-copy mice (Fig. 7A). Although treatment with inhibitors led to substantial inhibition in the mRNA expression of IKKα, IKKβ, and IKKγ isoforms, a complete reversal was observed with IKKε mRNA expression in 0-copy mice kidneys treated with Andro, PDTC, and etanercept. On the other hand, the mRNA expression levels of NF-κB proteins showed differential regulation in the kidneys of 0-copy mice compared with wild-type controls. The mRNA expression levels were significantly increased for p65 (6.5-fold), p50 (3.1-fold), p52 (2.2-fold), relB (3.2-fold), and cRel (4.9-fold) in 0-copy mice kidney compared with 2-copy controls (Fig. 7, B and C). Inhibitor treatment of 0-copy mice led to a partial and/or complete reversal of such increases in all NF-κB proteins.

Fig. 7.

Effect of inhibitors on the IKK and NF-κB family member genes by qRT-PCR in the kidneys of Npr1 gene-disrupted mice. A: relative mRNA expression of IKK enzyme subtypes (IKK-α, β, γ, ε) normalized to GAPDH mRNA. B and C: mRNA expression of NF-κB family members, IκBα, p65 (RelA), p50 (NFKB1), p52 (NFKB2), relB, and cRel normalized with GAPDH mRNA in the kidney tissues. Veh, saline-treated group. Values are expressed as mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.001; ♣P < 0.01; n = 7 mice per group.

DISCUSSION

The present results demonstrate that systemic disruption of the Npr1 gene activates NF-κB in 0-copy mice, transactivating the expressions of various inflammatory cytokines to initiate renal derangement and remodeling in mutant mice. Npr1 gene disruption significantly enhanced the phosphorylation of p65 protein at Ser276 and Ser536 residues in 0-copy mice kidney compared with 2-copy wild-type mice. Andro treatment abolished the phosphorylation of p65 at both sites, whereas PDTC and etanercept showed partial attenuation in phosphorylation at Ser276. However, both PDTC and etanercept also reversed the phosphorylation of p65 at Ser536. Previous studies have suggested that the major inducible form of NF-κB corresponds to a heterodimeric complex composed of p65 and p50 subunits (4). Several studies have reported that Ser276 and Ser536 are required for p65 activation (30, 66). It has been suggested that insufficient phosphorylation of p65 contributes to a lack of NF-κB activation (27). Interestingly, phosphorylation of p65 seems to occur at the multiple sites; for example, in mouse embryonic fibroblasts, TNF-α has been shown to stimulate phosphorylation of p65 at Ser 529 by casein kinase II (67), whereas Ser 536 was phosphorylated by coexpressed IKKs (55). However, a number of studies have indicated that p65 is also phosphorylated at Ser 276 position (10, 35, 52). It has been shown that IκB-associated protein kinase A catalytic subunit phosphorylates p65 at Ser 276 positions (72). Furthermore, Ser 276 was also found to be constitutively phosphorylated in endothelial cells (3). Intriguing was the finding that mitogen and stress-activated protein kinase-1 targets p65 for direct phosphorylation at Ser 276 as an essential element for gene expression (66). In the present study, 0-copy mice kidney showed significantly upregulated p-p65 immunoexpression, as confirmed by comparison of the immunofluorescence images of the glomeruli and tubular epithelial cells in 0-copy and 2-copy mouse kidneys. The increased p65 may serve as reservoir or ready store in order for the constitutive phosphorylation process to maintain induction of NF-κB in the kidneys of 0-copy mice. The constitutive activation of NF-κB observed in many types of cancer, including different types of leukemia and lymphoma, has been associated with increased cytoplasmic degradation of IκBα, resulting in increased nuclear translocation of NF-κB proteins and high levels of NF-κB DNA binding activity (32, 48, 54).

A parallel increase was observed in IκBα phosphorylation (11-fold, at Ser32/36) in the cytosol of 0-copy mouse kidneys. IκBα phosphorylation is a critical regulatory step and is considered to be a marker of NF-κB activation. The phosphorylated IκBα undergoes ubiquitin-dependent degradation by 26S proteosome complex (22). Nevertheless, IκBα reappears in <2 h in the cells as it simultaneously stimulates its own transcriptional activity, suggesting a feedback mechanism for the inhibition of the general transcription process (61). We observed an 80% reduction in IκBα levels in the cytosol of 0-copy mice compared with control 2-copy animals. The IκBα levels in 2-copy mice were significantly higher than those in 0-copy mice and were comparable in all 2-copy animals with or without treatment with inhibitors. The reduction of IκBα in 0-copy mice kidneys that we found suggests total exhaustion of IκBα by its increased degradation, as well as its simultaneously reduced synthesis.

Importantly, we also found a slight decrease in IκBα mRNA instead of an increase, which was expected in the kidneys of 0-copy mice. Our results suggest that the abolishment of IκBα is critical for sustained and increased NF-κB activity, which in turn activates the transcription of various inflammatory genes involved in the progression of renal diseases. Treatment with Andro, PDTC, and etanercept restored IκBα levels in the cytosol of 0-copy mice, which was associated with attenuated renal morphology and function. We observed that the treatment with etanercept markedly attenuated proinflammatory cytokines gene expression and their relative quantity in plasma and renal tissues. Several other studies have also shown that treatment with etanercept reduces proinflammatory cytokines gene expression in myocardial tissues (24) and spinal cord (19). The present results suggest that etanercept inhibits NF-κB activity as a mechanism by which it blocks IKK activity and thus exerts immunomodulatory effects. TNF-α has been shown to act as a upstream regulator of NF-κB inducing kinase, which may regulate IKK (36, 46). Similarly, a number of studies have revealed that Andro inhibits oral squamous cell carcinogenesis (69) and platelet activation (40), induces apoptosis (53), and reduces proinflammatory cytokine secretion from LPS/IFNγ-stimulated cells (9) through NF-κB inactivation. Previously, it has also been indicated that Andro did not alter IkBα after 30 min of treatment (70). However, in our present studies, Npr1 mice were treated with Andro for 30 days, which showed an inhibitory effect on IKK, IκBα degradation, and reduced p65 nuclear accumulation. Recent studies are in agreement with our findings that Andro inhibits IKK, IκBα activation, p65 nuclear translocation, and NF-κB DNA-binding activity (8, 26).

The present data indicate that the availability of IκBα is important for masking p65 protein so that it remains as an inactive complex in the cytosol. Based on this concept, we hypothesized that an increased level of IκBα may be beneficial, inhibiting NF-κB from DNA binding and preventing its further activation (Fig. 8). Earlier reports showed that in cells treated with either phorbol myristate acetate or TNF-α, cytoplasmic IκBα completely disappeared within 40 min of stimulation but reappeared at a normal level after 4 h of treatment (61). A recent study with mouse embryonic fibroblast cells also showed a biphasic expression of IκBα (1). The present results demonstrate a constitutive loss of IκBα, which indicates overriding of NF-κB activation in the kidneys of 0-copy mice.

Fig. 8.

Diagrammatic representation of the impact of NPRA deficiency in renal remodeling and impairment. Disruption of Npr1 gene leads to impaired renal functions, which triggers specific structural and molecular changes in 0-copy mice kidney. Activated IKK enzymes induce the phosphorylation of IκBα, which then undergoes enzymatic degradation. The reduction of cytosolic IκBα favors the constitutive activation of NF-κB, which would translocate into the nucleus to activate the transcription of proinflammatory cytokines, growth factors, and extracellular matrix protein genes. In the contrary, the increased amount of cytosolic IκBα favors the formation of trimeric complex between p65, p50 and IκBα and thereby withdraws the free p65 molecule to inhibit the transcription process. ANP, atrial natriuretic peptide; KHD, kinase homology domain; GCD, guanylyl cyclase domain; IKK, IκB kinase; IκBα, inhibitory κB α.

Activation of NF-κB is mediated through the induction of IKK activity. An eightfold increase in IKK activity was observed in 0-copy mice kidneys compared with vehicle-treated 2-copy controls. The present study showed that the disruption of Npr1 activates NF-κB by inducing IKK activity in the kidneys of 0-copy mice. At the same time, we also observed that IKK activity is attenuated in 0-copy mouse kidneys after treatment with Andro, PDTC, and etanercept. Studies using fibroblasts isolated from IKKα and IKKβ knockout mice have confirmed that IKKβ is the dominant kinase in regulating NF-κB activity, although IKKα could potentiate the effect (28, 62, 63). It was recently observed that IKKα and IKKβ can also function separately to regulate NF-κB activation (1). Several other studies with aspirin and sodium salicylate have also suggested that reduction of IKK activity could be used as a target for therapeutic strategy (2, 71).

Our results indicate that increased DNA binding activity of NF-κB stimulates the transcription of proinflammatory cytokines, including TNF-α, IL-6, IL-1α, IL-β, and TGF-β1, in the kidney tissues of 0-copy mice. Similar activation in the DNA binding activity of NF-κB was also reported in kidneys with ureteral obstruction (44) and in ANG II-induced renal damage (45), as well as salt-sensitive hypertension (24). The increased DNA binding ability of NF-κB is believed to contribute to its ability to enhance the transcription of several specific genes, including cytokines and growth factors, adhesion molecules, immunoreceptors, and acute-phase proteins, which are thought to be important in the pathogenesis of numerous diseases (7). Treatment with Andro, PDTC, and etanercept showed the reversal of both protein and mRNA expression in the kidneys of 0-copy mice. In parallel, we observed a twofold increase in kidney collagen content and a sevenfold increase in Col1α mRNA expression in 0-copy mice. TNF-α and IL-1β have been reported to regulate TGF-β1 signaling to stimulate collagen synthesis and accumulation to produce interstitial fibrosis (31, 41, 60).

The results of our study showed an upregulation of the relative mRNA expression of all isoforms of the IKK enzyme complex, along with increased expression of p65 (relA), p50 (NFKB1), p52 (NFKB2), relB, and cRel mRNA in 0-copy mice kidneys compared with 2-copy control animals. The genetic upregulation of IKK and NF-κB family members demonstrated here suggests that disruption of Npr1 activates the proinflammatory cytokine genes, which in turn activate the expression of IKK and NF-κB by a positive feedback mechanism. Treatment with Andro, PDTC, and etanercept downregulated the mRNA expression of IKK and NF-κB family members, indicating that these inhibitors exert beneficial effects in the kidneys of 0-copy mice. On the other hand, we found a tendency toward the reduction of IκBα mRNA and a simultaneous increase in the degradation of IκBα protein in the cytosol, which correlates with the reduced amount of IκBα protein in 0-copy mice kidneys. Our results indicate that the absence of IκBα favors sustained transcriptional activity of NF-κB in 0-copy mice kidneys. The IκBα mRNA level was reduced only in the PDTC-treated group at a lower level (P < 0.05) but remained unaltered in Andro- and etanercept-treated 0-copy mice compared with 2-copy mice, suggesting that a constitutive signal for IκBα prevails within the cell for its self-renewal, which exerts an inhibitory effect on NF-κB activity. We hypothesize that at a certain point of time in an injured tissue a relative amount of IκBα protein and its message are the prime regulators of NF-κB activity. Our results could be partially explained by the previously reported observation that transcriptional activity of NF-κB is regulated by IκBα (17).

Our results suggest that systemic disruption of Npr1 activates NF-κB in the kidney with progressive loss of renal function and abnormal renal pathology. The ratio of urinary albumin and creatinine was significantly increased in 0-copy mice compared with 2-copy mice. The CCr, which is a marker of glomerular filtration rate, was also markedly decreased in 0-copy mice kidneys. Treatment with NF-κB inhibitors showed a considerable restoration of renal function in 0-copy mice. The reduction of blood pressure in Andro-, PDTC-, and etanercept-treated 0-copy mice may be attributed to the secondary effects of these agents in improving renal function and pathology. Our previous studies have indicated that NPRA negatively regulates mitogen-activated protein kinase (MAPKs) and proliferation of mesangial cells (49, 57). Agonist hormones such as platelet-derived growth factor, ANG II, and endothelin-1, all stimulated the immunoreactive MAPK/ERK2 activity in rat mesangial cells and human vascular smooth muscle cells; however, ANP blocked the agonist-stimulated activity of MAPK/ERK2 by 65–75% in cells overexpressing NPRA (49). NPRA antagonist A71915 and cGMP-dependent protein kinase inhibitor KT5823 completely reversed the inhibitory effect of ANP on MAPK/ERK2 activity. Thus, MAPKs pathway might also be considered another stimulatory pathway for renal mesangial cell growth and hypertrophy in Npr1 0-copy mice. Our findings indicate that renal remodeling, as represented by percentage of renal fibrosis and MME in 0-copy animals, could be corrected by prolonged treatment with Andro, PDTC, and etanercept. Although earlier studies have shown that suppression of NF-κB with drugs like aspirin (45), enalapril (44), and captopril (43) provide a beneficial effect, the exact molecular mechanism has yet to be deciphered. In contrast, our study demonstrates that the beneficial effects of Andro, PDTC, and etanercept in attenuating altered renal function and abnormal renal pathology in Npr1 gene-disrupted mice can be attributed to their primary effects in blunting IKK activity in vivo.

In conclusion, several key findings have emerged from this study. First, it provides direct evidence that, in the absence of functional Npr1, renal NF-κB activity is increased, which in turn stimulates the synthesis of proinflammatory cytokines to produce renal abnormalities. Second, we found that although IKK complex is an important player for NF-κB activation, IκBα has the central role in regulating the NF-κB activity. Third, this study shows that the IKK/NF-κB signaling pathway is a dominant target for therapeutic strategy. Our results demonstrate that the Npr1 gene contributes to counterregulatory effects on NF-κB signaling and provides reno-protection and prevention of hypertension.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-62147.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: S.D. and R.P. performed experiments; S.D. and R.P. analyzed data; S.D. interpreted results of experiments; S.D. prepared figures; S.D. drafted manuscript; K.N.P. conception and design of research; K.N.P. edited and revised manuscript; K.N.P. approved final version of manuscript.