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. 2011 Apr;22(4):635-48.
doi: 10.1681/ASN.2009111130. Epub 2011 Mar 3.

Luminal alkalinization attenuates proteinuria-induced oxidative damage in proximal tubular cells

Tomokazu Souma  1 , Michiaki Abe, Takashi Moriguchi, Jun Takai, Noriko Yanagisawa-Miyazawa, Eisuke Shibata, Yasutoshi Akiyama, Takafumi Toyohara, Takehiro Suzuki, Masayuki Tanemoto, Takaaki Abe, Hiroshi Sato, Masayuki Yamamoto, Sadayoshi Ito
Affiliations

Luminal alkalinization attenuates proteinuria-induced oxidative damage in proximal tubular cells

Tomokazu Souma et al. J Am Soc Nephrol. 2011 Apr.

Abstract

A highly acidic environment surrounds proximal tubular cells as a result of their reabsorption of HCO(3)(-). It is unclear whether this luminal acidity affects proteinuria-induced progression of tubular cell damage. Here, we investigated the contribution of luminal acidity to superoxide (O(2)(·-)) production induced by oleic acid-bound albumin (OA-Alb) in proximal tubular cells. Acidic media significantly enhanced OA-Alb-induced O(2)(·-) production in the HK-2 proximal tubular cell line. Simultaneous treatment with both OA-Alb and acidic media led to phosphorylation of the intracellular pH sensor Pyk2. Highly phosphorylated Pyk2 associated with activation of Rac1, an essential subcomponent of NAD(P)H oxidase. Furthermore, knockdown of Pyk2 with siRNA attenuated the O(2)(·-) production induced by cotreatment with OA-Alb and acid. To assess whether luminal alkalinization abrogates proteinuria-induced tubular damage, we studied a mouse model of protein-overload nephropathy. NaHCO(3) feeding selectively alkalinized the urine and dramatically attenuated the accumulation of O(2)(·-)-induced DNA damage and proximal tubular injury. Overall, these observations suggest that luminal acidity aggravates proteinuria-induced tubular damage and that modulation of this acidic environment may hold potential as a therapeutic target for proteinuric kidney disease.

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Figures

Figure 1.
Figure 1.
An acidic environment increases OA-Alb–induced superoxide (O2·−) production. HK-2 cells were incubated in HBSSH/l-arginine at a physiologically relevant pH (pH 7.0 to pH 6.0) with or without OA-Alb stimulation (15 g/L). O2·− production was assayed using the O2·−-specific fluorescence probe, DHE. (A) OA-Alb, administered at 200 seconds after DHE (10 μM) loading (arrow) led to increased O2·− production at pH 6.0 (red) and pH 6.4 (blue). (B) Acidification without OA-Alb stimulation did not induce O2·− production in HK-2 cells (P = 0.074 among all pH conditions). (C) The O2·− production rate was significantly increased up to 7-fold at pH 6.4, and up to 13-fold at pH 6.0, compared with the OA-Alb− control (***P < 0.001). (D) Representative photomicrographs of HK-2 cells, with or without OA-Alb stimulation at the indicated pH, 1000 seconds after DHE loading, are shown. The white signals indicate O2·− production. All the experiments were repeated at least six times, and data are expressed as the mean ± SEM.
Figure 2.
Figure 2.
OA-Alb administration induces superoxide (O2·−) production mediated by NAD(P)H oxidase in HK-2 cells. (A) OA-Alb administration (5 to 30 g/L) dose dependently induced O2·− production (orange symbols) in HK-2 cells. FFA-Alb–induced O2·− production in HK-2 cells was only slightly higher than that induced by the vehicle (yellow symbols). OA- or FFA-Alb was administered at 200 seconds after DHE (10 μM) loading (arrow) at pH 6.4. (B) The O2·− production rate was significantly increased by OA-Alb administration in a dose-dependent manner. (C) OA-Alb–induced O2·− production was attenuated by pretreatment with either the NAD(P)H oxidase inhibitor, apocynin (100 μM), or by the ROS scavenger, tiron (10 mM). Simultaneous preincubation with apocynin and tiron did not show additive attenuation (P = 0.66 versus apocynin treatment). Pretreatment with either apocynin or tiron alone did not increase O2·− production (P = 0.27 versus no treatment). All the experiments were repeated at least four times, and data are expressed as the mean ± SEM. The statistical significance of the differences are indicated (**P < 0.01 and ***P < 0.001).
Figure 3.
Figure 3.
Pyk2 is strongly phosphorylated after OA-Alb administration under acidic conditions. HK-2 cells were incubated at pH 6.4 (A) or pH 7.0 (B), with or without OA-Alb stimulation (15 g/L) for the indicated times. Total cell lysates (diluted to 1 mg/ml) were immunoprecipitated with an anti-Pyk2 antibody and were immunoblotted with anti-Pyk2 and anti-phosphotyrosine (p-Tyr) antibodies. (A) At pH 6.4, Pyk2 was marginally phosphorylated. After OA-Alb stimulation marked Pyk2 phosphorylation was observed at pH 6.4 from 90 seconds onward. The lower panel indicates quantification of the phosphorylated Pyk2 level, which was normalized to the level of total Pyk2. The data are expressed as the mean ± SEM of at least four independent experiments. The statistical significance of the differences are indicated (*P < 0.05). (B) At pH 7.0 Pyk2 was hardly phosphorylated. OA-Alb administration did not induce phosphorylation. All gel data are representative of at least four independent experiments.
Figure 4.
Figure 4.
c-Src phosphorylation is involved in the signaling pathway of OA-Alb–induced superoxide production. HK-2 cells were incubated at pH 6.4 (A) or at pH 7.0 (B), with or without OA-Alb stimulation (15 g/L) for the indicated times. Total cell lysates (diluted to 1 mg/ml) were immunoprecipitated with an anti-Pyk2 antibody and were then immunoblotted with anti-Pyk2, anti–c-Src, or p-Tyr antibodies. (A) Phosphorylated c-Src was coimmunoprecipitated with Pyk2 upon OA-Alb stimulation at pH 6.4. The lower panel indicates quantification of phosphorylated c-Src, which was normalized to the level of total Pyk2 protein. The data are expressed as the mean ± SEM of four independent experiments. The statistical significance of the differences are indicated (*P < 0.05). (B) At pH 7.0, c-Src was not coimmunoprecipitated with Pyk2. All gel data are representative of four independent experiments. p-Tyr, anti-phosphotyrosine.
Figure 5.
Figure 5.
Rac1, a subcomponent of NOX, is activated upon OA-Alb exposure at pH 6.4. (A) HK-2 cells were stimulated with OA-Alb (15 g/L) at pH 6.4 for the indicated times. The amount of GTP-bound active Rac1 was assessed using a glutathione-S-transferase (GST) pull-down assay with p21 activated kinase 1(PAK)-p21 binding domain (PBD) GST-fusion beads followed by immunoblotting with an anti-Rac1 antibody. GTP-bound active Rac1 increased in a time-dependent manner. (B) Quantification of GTP-bound active Rac1 normalized to total Rac1 levels. The total level of Rac1 did not change over time. The data are expressed as the mean ± SEM of three independent experiments. The statistical significance of the difference is indicated (*P < 0.05).
Figure 6.
Figure 6.
Pyk2 knockdown attenuates OA-Alb–induced O2·− production. (A) Pyk2-specific siRNA (siPyk2) reduced the protein level of Pyk2 by 58% as assessed by Western blotting. α-Tubulin was assayed as a loading control. (B) Pyk2 knockdown attenuated OA-Alb–induced O2·− production at pH 6.4. OA-Alb was administered 200 seconds after DHE (10 μM) loading (arrow). (C) OA-Alb–induced O2·− production was significantly attenuated by 70% by Pyk2 knockdown. (D) Representative photomicrographs of HK-2 cells with and without OA-Alb stimulation at 1000 seconds after DHE loading are shown. All the experiments were repeated at least four times, and the data in (A) and (C) are expressed as the mean ± SEM. The statistical significance of the differences in (A) and (C) are indicated (**P < 0.01 and ***P < 0.001).
Figure 7.
Figure 7.
Protein overload with acidic exposure reduces cellular viability through Pyk2-dependent pathways. (A and B) Cellular viability was assessed using flow cytometry and Annexin V/PI costaining. Early apoptotic cells resided in the Annexin-V–single positive fraction (lower right), viable cells were negative for both Annexin-V and PI (lower left), and late apoptotic and necrotic cells stained positively for both stains (upper right). The percentage of cells in each quadrant is indicated. (C) The number of Annexin-V+/PI− early apoptotic cells (red rectangles in A) was increased upon exposure to OA-Alb (15 g/L) at pH 6.4. siRNA knockdown of Pyk2 suppressed the OA-Alb–induced apoptotic cell death. All the experiments were repeated at least four times. The data in (C) are expressed as the mean ± SD. The statistical significance of the differences are indicated (**P < 0.01 and ***P < 0.001).
Figure 8.
Figure 8.
Luminal alkalinization attenuates oxidative DNA damage in a mouse model of protein-overload nephropathy. (A) C57B6/J mice were treated with daily intraperitoneal (ip) FFA- or OA-Alb injections for 5 consecutive days. The mice were analyzed on day 6. PBS was injected into control animals (ip). Luminal alkalinization was induced by feeding 200 mM NaHCO3 in the drinking water. (D) Staining of kidney sections with H&E (D, left column) revealed that protein overload induced tubular dilation, flattening of tubular cells, cast formation (*), and interstitial edema (D-b and -d). Note OA-Alb injection induced vacuolization of tubular cells (inlet in D-d). Luminal alkalinization by feeding NaHCO3 attenuated the proteinuria-induced tubular damage (D-c and -e). Upon FFA- or OA-Alb injections, the cytoplasmic, that is, mitochondrial, and nuclear 8-OHdG-ir were detected in renal tubular cells located mainly in the outer stripe of outer medulla, extending to the cortex (D-g, -l, -i, and -n). Luminal alkalinization markedly attenuated the accumulation of 8-OHdG-ir (D-h, -m, -j, and -o). There were no abnormalities in the control sections (D-a, -f, and -k). Scale bars: 100 μm. (B) The number of casts formed in the randomly selected cortical area was markedly increased with protein overload, and luminal alkalinization reduced this histologic change (n > 8 in each group). (C) The mean 8-OHdG-ir density was significantly increased with protein overload and dramatically reduced by luminal alkalinization (n ≥ 4 in each group). The statistical significance of the differences are indicated (*P < 0.05, **P < 0.01, and ***P < 0.001).
Figure 9.
Figure 9.
Oxidative DNA damage accumulates in the proximal tubular cells. Kidney sections from FFA-Alb–injected (B, G) or OA-Alb–injected (D, I) or control PBS-injected (A, F) mice were analyzed by immunhistochemistry for colocalization of 8-OHdG-ir (black arrow heads) with LTL (red arrow heads), a marker of proximal tubule apical membranes [(F) through (J) are higher magnifications of the dotted square in (A) thorugh (E), respectively]. 8-OHdG-ir colocalized with LTL after FFA- or OA-Alb injections. Note that 8-OHdG-ir was observed in both dilated and nondilated tubules (B). Tubular vacuolization was more predominantly observed in the OA-Alb–injected group (asterisk, I). 8-OHdG-ir was not observed in the PBS-injected control section. Scale bars: 100 μm (A through E) and 50 μm (F through J). LTL, Lotus tetragonobulus lectin.
Figure 10.
Figure 10.
Luminal alkalinization attenuates ROS production in a mouse model of protein-overload nephropathy. ROS level of CD13+ renal PT cells from C57BL6/J mice treated with FFA-Alb or OA-Alb were analyzed by FACS using DCFDA. (A) A representative histogram of unstained kidney cells (blue line) and CD13+ PT cells (red line). The CD13+ PT cell fraction (indicated by the horizontal bar) was subjected to the DCFDA analysis. CD45+ hematopoietic cells and PI+ dead cells were gated out throughout the FACS experiments. (B) A diagram showing the tissue distribution of CD13+ PT cells in the nephron. (C) and (D) DCFDA FACS analysis. The ROS level in the CD13+ PT cell fraction (red line) was strongly increased upon FFA- or OA-Alb administration compared with the CD13− cell fraction (blue line). (E) The median intensity of the DCFDA fluorescence in (C) and (D). OA-Alb treatment induces more marked ROS accumulation in CD13+ PT cells than FFA-Alb treatment does. (F) and (G) FFA- or OA-Alb–induced ROS accumulation (red line) was attenuated by the luminal alkalinization induced by NaHCO3 feeding (blue line). The green line indicates the ROS level in the PBS-injected mouse. (H) The median intensity of the DCFDA fluorescence in (F) and (G). All data are expressed as the mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.001; n = 6 in each group). LOH, loop of Henle; mTAL, medullary thick ascending limb; DT, distal tubule; CD, collecting duct; OS, outer stripe; IS, inner stripe.
Figure 11.
Figure 11.
Model of the intracellular signaling mechanisms underlying OA-Alb induced superoxide production in renal proximal tubular cells. Proteinuria associated with physiologic luminal acidification cooperatively induces robust Pyk2 activation in proximal tubular cells. The activated Pyk2 induces extensive ROS accumulation through activation of c-Src and NAD(P)H oxidase.
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References

    1. Risdon RA, Sloper JC, De Wardener HE: Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis. Lancet 2: 363–366, 1968 - PubMed
    1. Benigni A, Zoja C, Remuzzi G: The renal toxicity of sustained glomerular protein traffic. Lab Invest 73: 461–468, 1995 - PubMed
    1. D'Amico G: Tubulointerstitium as predictor of progression of glomerular diseases. Nephron 83: 289–295, 1999 - PubMed
    1. Tang S, Leung JC, Abe K, Chan KW, Chan LY, Chan TM, Lai KN: Albumin stimulates interleukin-8 expression in proximal tubular epithelial cells in vitro and in vivo. J Clin Invest 111: 515–527, 2003 - PMC - PubMed
    1. Morigi M, Macconi D, Zoja C, Donadelli R, Buelli S, Zanchi C, Ghilardi M, Remuzzi G: Protein overload-induced NF-kappaB activation in proximal tubular cells requires H(2)O(2) through a PKC-dependent pathway. J Am Soc Nephrol 13: 1179–1189, 2002 - PubMed

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