Hemoglobin is expressed by mesangial cells and reduces oxidant stress

Abstract

Hemoglobin (Hb) serves as the main oxygen transporter in erythrocytes, but it is also expressed in nonhematopoietic organs, where it serves an unknown function. In this study, microarray and proteomic analyses demonstrated Hb expression in the kidney. Rat kidneys were perfused extensively with saline, and glomeruli were isolated by several techniques (sieving, manual dissection, and laser capture-microdissection). Reverse transcriptase-PCR revealed glomerular alpha- and beta-globin expression, and immunoblotting demonstrated expression of the protein. In situ hybridization studies showed expression of the globin subunits in the mesangium, and immunostaining confirmed this localization of Hb. Furthermore, globin mRNA expression was detected in primary cultures of rat mesangial cells but not in cultured glomerular endothelial or epithelial cells. For investigation of Hb function in mesangial cells, the SV40-MES13 murine mesangial cell line was transfected with a vector expressing alpha- and beta-globins; this overexpression reduced production of hydrogen peroxide-induced intracellular radical oxygen species and enhanced cell viability against oxidative stress. In summary, Hb is expressed by rat mesangial cells, and its potential functions may include antioxidative defense.

Figure 1.

Expression of globin in the rat kidney glomeruli. (A) Two-dimensional differential in gel electrophoresis of proteins sampled from chronic hypoxia kidney was analyzed. The arrows represent one of the spots that were increased by hypoxia with respect to the control rat group with sham operation (top) and the rat group with renal artery stenosis for 7 d (bottom). Subsequent proteomic studies identified a significant increase in β-globin in the kidney rendered to chronic hypoxia. (B) Expression of globins in the normal rat glomeruli obtained by sieving was confirmed by RT-PCR. Arrowheads indicate the predicted size of the PCR product by each primer pair for cDNA (not genomic DNA). Sequence-specific primers: Hb-α, rat α-globin (429 bp); Hb-β, rat β-globin (444 bp); AE1, mouse/rat erythrocyte anion exchanger (467 bp); β-actin (306 bp). (C and D) Furthermore, expression of globins in the manually dissected (C) and the laser capture–microdissected (D) rat nephron segments was confirmed by RT-PCR. Arrowheads indicate the predicted size of the PCR product by each primer pair for cDNA (not genomic DNA). Sequence-specific primers: Hb-α, rat α-globin (335 bp); Hb-β, rat β-globin (416 bp). (E) Immunoblot analyses for the detection of Hb in isolated normal rat glomeruli (80 μg of total protein) confirmed Hb protein expression. Rat blood lysate served as a positive control. M, marker, ϕX174/HaeIII; G, glomeruli; B, blood; no, no template; PCT, proximal convoluted tubule; PST, proximal straight tubule; MTAL, medullary thick ascending limb; CTAL, cortical thick ascending limb; DCT, distal convoluted tubule; CCD, cortical collecting duct; OMCD, outer medullary collecting duct; IMCD, inner medullary collecting duct; T, tubules.

Figure 2.

In situ hybridization studies showed globin subunit mRNA expression in the mesangial region. (A) Hybridization using antisense probe evidenced expression of α- and β-globin genes in rat glomeruli. Of note, the signals were localized in the mesangial region. Specificity of hybridization was confirmed using sense probes, which resulted in complete lack of signal of both globins. (B) Higher magnification images showed localization of these antisense signals of each globin to the cytosol of mesangial cells. Magnifications: ×400 in A; ×600 in B.

Figure 3.

Double-immunofluorescence studies of rat kidney showed localization of Hb protein in mesangial region. (A) Immunofluorescence of the normal rat kidney with antibody to Hb was observed in the glomerular mesangial region in green. (B) Higher magnification image showed immunofluorescence in mesangial cells. (C) Immunofluorescence without primary antibody revealed negligible signals in the glomerulus. (D) Immunofluorescence of the normal rat kidney with antibody to α-globin subunit also showed some glomerular cells in green. Double immunostaining with OX-7, a marker of rat mesangial cells, demonstrated that Hb was expressed by mesangial cells. Magnifications: ×400 in A, C, and D; ×600 in B.

Figure 4.

Rat cultured mesangial cells express globin subunit mRNA. Expression of globins in several cultured rat glomerular cells, such as primary cultured rat mesangial cells, podocytes, and endothelial cells, was confirmed by RT-PCR. MES, mesangial cells; POD, podocytes; GEN, endothelial cells; B, rat blood. Sequence-specific primers: Hb-α, rat α-globin (335 bp); Hb-β, rat β-globin (416 bp).

Figure 5.

Transfection of murine mesangial cells with the globin-expressing vector suppressed intracellular ROS generation. (A) The construct pIRES/α-globin/β-globin was designed to express effectively both α- and β-globin subunits in transfected cells. Immunoblotting with rabbit polyclonal anti-Hb antibody identified overexpressed globin protein in lysate of murine mesangial cells, SV40-MES13, transfected with this vector. Control and Hb-overexpressing cells were stained with fluorogenic probes to detect intracellular H₂O₂ and superoxide anion and exposed to extracellular H₂O₂ of 1000 μM for 2 min. (B and C) Immunocytochemistry confirmed that the intracellular generation of H₂O₂ (B) and superoxide anion (C) was suppressed in Hb-overexpressing cells. (D and E) Intracellular levels of H₂O₂ (D) and superoxide anion (E) in these cells were estimated by flow cytometry analysis. Although those levels in control (pink histogram) and transfected cells (blue) exposed to extracellular stimuli were higher as total than control (black) and transfected cells (green) with no exposures, respectively, Hb-overexpressing cells included a lower level of cellular ROS as total than control cells when exposed to extracellular H₂O₂. (F and G) Quantitative analyses confirmed that the intracellular generation of H₂O₂ (F) and superoxide anion (G) was inhibited in Hb-overexpressing cells (▪) more than in control cells (□; P < 0.05). *P < 0.05; **P < 0.01.

Figure 6.

Transfection with the globin-expressing vector improved cell viability against oxidative stress. SV40-MES13 viability when exposed to H₂O₂ of 0, 250, 500, or 1000 μM for 24 h as extracellular oxidative stress was evaluated by the LDH release assay. The assay showed that SV40-MES13 cells transfected with the globin-expressing vector (▪) were more resistant against H₂O₂ stimuli of 500 and 1000 μM for 24 h than cells transfected with the control vector (□). *P < 0.05; **P < 0.01.