MicroRNA-21 orchestrates high glucose-induced signals to TOR complex 1, resulting in renal cell pathology in diabetes

Abstract

Hyperglycemia induces a wide array of signaling pathways in the kidney that lead to hypertrophy and matrix expansion, eventually culminating in progressive kidney failure. High glucose-induced reduction of the tumor suppressor protein phosphatase and tensin homolog deleted in chromosome 10 (PTEN) contributes to renal cell hypertrophy and matrix expansion. We identified microRNA-21 (miR-21) as the molecular link between high glucose and PTEN suppression. Renal cortices from OVE26 type 1 diabetic mice showed significantly elevated levels of miR-21 associated with reduced PTEN and increased fibronectin content. In renal mesangial cells, high glucose increased the expression of miR-21, which targeted the 3'-UTR of PTEN mRNA to inhibit PTEN protein expression. Overexpression of miR-21 mimicked the action of high glucose, which included a reduction in PTEN expression and a concomitant increase in Akt phosphorylation. In contrast, expression of miR-21 Sponge, to inhibit endogenous miR-21, prevented down-regulation of PTEN and phosphorylation of Akt induced by high glucose. Interestingly, high glucose-stimulated miR-21 inactivated PRAS40, a negative regulator of TORC1. Finally, miR-21 enhanced high glucose-induced TORC1 activity, resulting in renal cell hypertrophy and fibronectin expression. Thus, our results identify a previously unrecognized function of miR-21 that is the reciprocal regulation of PTEN levels and Akt/TORC1 activity that mediate critical pathologic features of diabetic kidney disease.

FIGURE 1.

Expression of fibronectin is associated with reduced PTEN abundance and increased miR-21 in OVE26 mice renal cortex. A, C, and E, renal cortical lysates from 3-month-old control FVB and OVE26 type 1 diabetic mice were immunoblotted with fibronectin, PTEN, phospho-Akt (p-Akt), Akt, and actin antibodies as indicated. B, D, and F are the quantification of the results shown in A, C, and E, respectively. C, control; D, OVE26 diabetic mouse. n = 3 in each group. *, p = 0.0286 versus control in B; *, p = 0.0497 versus control in D; *, p = 0.019 versus control in F. G, sequence complementarity between miR-21 and its target sites in the 3′-UTR of human (hsa), mouse (mmu), and rat (rno) PTEN mRNAs. H and I, total RNA from renal cortices of control and OVE26 mice was used in real time qRT-PCR to detect the pre-miR-21 (H) and mature miR-21 (I) levels as described under “Experimental Procedures.” n = 4 mice in each group. *, p = 0.002 versus control in H; *, p = 0.003 versus control in I. Means ± S.E. are shown in panels B, D, F, H, and I.

FIGURE 2.

High glucose increases miR-21 to target 3′-UTR of PTEN mRNA in mesangial cells. A, rat mesangial cells were transfected with PTEN 3′-UTR-Luc followed by incubation with 25 mm glucose (HG) and 5 mm glucose plus 20 mm mannitol (NG) for 24 h. The cell lysates were assayed for luciferase activity as described under “Experimental Procedures.” The mean ± S.E. of six measurements is shown. *, p = 0.0004 versus 5 mm glucose plus 20 mm mannitol. B–D, rat mesangial cells were incubated with 25 mm glucose (HG) for 24 h. Total RNAs were used to detect pre-miR-21 and mature miR-21 by real time qRT-PCR (B and D) as described under “Experimental Procedures.” n = 9 for both panels. *, p = 0.0001 versus 5 mm glucose plus 20 mm mannitol in B; *, p = 0.02 versus 5 mm glucose plus 20 mm mannitol in D. In C, the total RNA was used in Northern analysis to detect mature miR-21 as described under “Experimental Procedures.” The bottom panel shows quantification of the miR-21 band. n = 9. *, p = 0.0016 versus 5 mm glucose plus 20 mm mannitol. E, human mesangial cells were incubated with 25 mm glucose (HG) or 5 mm glucose plus 20 mm mannitol (NG) for 24 h. Total RNA was used to detect mature miR-21 as described under “Experimental Procedures.” n = 3. *, p = 0.0481 versus 5 mm glucose plus 20 mm mannitol.

FIGURE 3.

High glucose-stimulated Glut1 translocation to membrane of mesangial cells contributes to expression of miR-21. A, mesangial cells were incubated with 25 mm glucose (HG) for the indicated periods of time. As a control, 5 mm glucose plus 20 mm mannitol (NG) was used. Membrane fractions were prepared as described under “Experimental Procedures.” The membrane extracts were immunoblotted with Glut1 and actin antibodies. The histogram at the bottom shows quantification of the Glut1 protein. B, Glut1 siRNA (siGLUT1) reduces Glut1 protein expression. Mesangial cells were transfected with an siRNA pool against Glut1. The cell lysates were immunoblotted with Glut1 and actin antibodies. C, mesangial cells were transfected with an siRNA pool targeting Glut1 followed by incubation with 25 mm glucose for 24 h. Total RNA was used in real time qRT-PCR for detection of mature miR-21. The bottom panel shows down-regulation of Glut1 protein. In A, n = 4. *, p < 0.01 versus 5 mm glucose plus 20 mm mannitol. In C, n = 4. *, p < 0.01 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.05 versus 25 mm glucose.

FIGURE 4.

Expression of miR-21 mimics effect of high glucose on PTEN expression and on downstream signaling. A, miR-21 inhibits PTEN 3′-UTR reporter activity. Rat mesangial cells were cotransfected with the PTEN 3′-UTR-Luc reporter plasmid and pCMV-miR-21 (miR-21) or scrambled RNA expression vector (Scr) followed by incubation with 25 mm glucose (HG) and 5 mm glucose plus 20 mm mannitol (NG) for 24 h. The cell lysates were assayed for luciferase activity as described under “Experimental Procedures.” The mean ± S.E. of six measurements is shown. *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol. B–D, miR-21 inhibits the expression of PTEN, resulting in phosphorylation of Akt and GSK3β similar to that seen with high glucose. Mesangial cells were transfected with CMV-miR-21 expression vector followed by incubation with high glucose as described in A. The cell lysates were immunoblotted with PTEN, phospho-Akt (p-Akt), Akt, phospho-GSK3β (p-GSK3β), GSK3β, and actin antibodies as indicated in the different panels. The histogram at the bottom of each panel shows quantification of the protein bands. n = 6. *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol in B, C, and D; #, p < 0.01 versus 5 mm glucose plus 20 mm mannitol in B.

FIGURE 5.

Expression of miR-21 Sponge inhibits effect of high glucose on expression of PTEN and on downstream signaling. A, miR-21 Sponge prevents high glucose-induced down-regulation of PTEN 3′-UTR reporter activity. Rat mesangial cells were cotransfected with the PTEN 3′-UTR-Luc reporter plasmid and miR-21 Sponge expression vector followed by incubation with high glucose as described in the legend of Fig. 4A. The cell lysates were assayed for luciferase activity as described under “Experimental Procedures.” The mean ± S.E. of six measurements is shown. *, p < 0.05 versus 5 mm glucose plus 20 mm mannitol (NG); #, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.01 versus 25 mm glucose (HG). The bottom panel shows expression of GFP mRNA in one of the samples performed in parallel. B–D, expression of miR-21 Sponge prevents down-regulation of PTEN induced by high glucose, resulting in phosphorylation of Akt and GSK3β. Rat mesangial cells were transfected with miR-21 Sponge followed by incubation with high glucose as described in the legend of Fig. 4A. The cell lysates were immunoblotted with PTEN, phospho-Akt (p-Akt), Akt, phospho-GSK3β (p-GSK3β), GSK3β, and actin antibodies as indicated in the different panels. Expression of GFP mRNA is shown in the lysates prepared in parallel. The histogram at the bottom of each panel shows quantification of the protein bands. n = 6. *, p < 0.01 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.01 versus 25 mm glucose in B. *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.001 versus 25 mm glucose in C and D.

FIGURE 6.

miR-21 regulates phosphorylation of PRAS40 and S6 kinase. A and C, expression of miR-21 mimics the effect of high glucose on PRAS40 and S6 kinase phosphorylation. Rat mesangial cells were transfected with pCMV-miR-21 or vector expressing scrambled (Scr) RNA followed by incubation with high glucose (HG) as described in the legend of Fig. 4. The cell lysates were immunoblotted with phospho-PRAS40 (p-PRAS40), PRAS40, phospho-S6 kinase (p-S6K), S6 kinase (S6K), and actin antibodies as indicated. Histograms show quantification of the protein bands. n = 4. *, p < 0.01 versus 5 mm glucose plus 20 mm mannitol (NG); #, p < 0.001 versus 5 mm glucose plus 20 mm mannitol in A. *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; #, p < 0.01 versus 5 mm glucose plus 20 mm mannitol (n = 6) in C. B and D, miR-21 Sponge inhibits high glucose-induced phosphorylation of PRAS40 and S6 kinase. Mesangial cells were transfected with miR-21 Sponge or vector plasmid followed by incubation with high glucose (HG) as described in the legend of Fig. 4. The cell lysates were immunoblotted with phospho-PRAS40, PRAS40, phospho-S6 kinase, S6 kinase, and actin antibodies as indicated. Panels indicated by GFP and GAPDH show their mRNAs in samples prepared in parallel. The bottom histograms show quantification of the protein bands. n = 4. *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.001 versus high glucose in B. *, p < 0.05 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.05 versus high glucose (n = 6) in D. E and F, the PI 3-kinase/Akt axis regulates miR-21-mediated S6 kinase phosphorylation. Rat mesangial cells were transfected with CMV-miR-21 expression vector followed by incubation with PI 3-kinase inhibitor Ly294002 (25 μm; E) or Akt inhibitor MK-2206 (1 μm; F). The cell lysates were immunoblotted with phospho-S6 kinase, S6 kinase, and actin antibodies as indicated. Histograms in the bottom panels show quantification of the protein bands. In E, n = 4. *, p < 0.001 versus control; **, p < 0.001 versus miR-21-transfected. In F, n = 4. *, p < 0.001 versus control; **, p < 0.001 versus miR-21-transfected.

FIGURE 7.

mir-21 regulates high glucose-induced protein synthesis and hypertrophy of mesangial cells. Rat mesangial cells were transfected with pCMV-miR-21 or vector expressing scrambled (Scr) RNA (A and B) and miR-21 Sponge or vector (C and D) followed by incubation with high glucose (HG) for 24 h as described in the legend of Fig. 4. In A and C, incorporation of [³⁵S]methionine was determined as described under “Experimental Procedures.” In B and D, the cells were trypsinized and counted, and protein content was determined. The cell hypertrophy was expressed as a ratio of total protein per cell as described under “Experimental Procedures.” The mean ± S.E. of triplicate measurements is shown. In A, *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol (NG); #, p < 0.05 versus 5 mm glucose plus 20 mm mannitol. In B, *, p < 0.01 versus 5 mm glucose plus 20 mm mannitol; #, p < 0.05 versus 5 mm glucose plus 20 mm mannitol. In C, *, p < 0.05 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.05 versus high glucose. In D, *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.001 versus high glucose. E–H, the PI 3-kinase/Akt axis regulates miR-21-mediated protein synthesis and hypertrophy of mesangial cells. Rat mesangial cells were transfected with CMV-miR-21 expression vector followed by incubation with PI 3-kinase inhibitor Ly294002 (25 μm; E and F) and Akt inhibitor MK-2206 (1 μm; G and H), respectively, for 24 h. [³⁵S]Methionine incorporation (E and G) and cell hypertrophy (F and H) were determined as described above. The mean ± S.E. of triplicate measurements is shown for E–H. In E, *, p < 0.01 versus control; **, p < 0.01 versus miR-21-transfected. In F, *, p < 0.001 versus control; **, p < 0.001 versus miR-21-transfected. In G, *, p < 0.01 versus control; **, p < 0.01 versus miR-21-transfected. In H, *, p < 0.001 versus control; *, p < 0.001 versus miR-21-transfected.

FIGURE 8.

miR-21 regulates high glucose-stimulated fibronectin expression in mesangial cells. A and B, rat mesangial cells were transfected with pCMV-miR-21 or scrambled (Scr) RNA-expressing vector (A) and miR-21 Sponge or vector plasmid (B) followed by incubation with high glucose for 24 h as described in the legend of Fig. 4. The cell lysates were immunoblotted with fibronectin and actin antibodies as indicated. In B, the parts indicated by GFP and GAPDH show their mRNA expression in samples run in parallel. Bottom histograms show the mean ± S.E. of six independent experiments. In A, *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol (NG). In B, *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.001 versus high glucose (HG). C and D, mesangial cells were cotransfected with Fibro-Luc reporter plus pCMV-miR-21 or scrambled RNA expression vector (C) and Fibro-Luc plus miR-21 Sponge or vector plasmid (D). Transfected cells were treated with high glucose as described in the legend of Fig. 4A. The cell lysates were used for luciferase activity as described under “Experimental Procedures.” The mean ± S.E. of six measurements is shown. In C, *, p < 0.05 versus 5 mm glucose plus 20 mm mannitol. In D, *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.001 versus high glucose. The PI 3-kinase/Akt axis regulates miR-21-mediated fibronectin expression in mesangial cells. E and F, rat mesangial cells were transfected with CMV-miR-21 expression vector followed by incubation with PI 3-kinase inhibitor Ly294002 (25 μm; E) and Akt inhibitor MK-2206 (1 μm; F), respectively, for 24 h. The cell lysates were immunoblotted with fibronectin and actin antibodies as indicated. The histograms at the bottom show quantification of the protein bands. In E, n = 4. *, p < 0.001 versus control; **, p < 0.001 versus miR-21-transfected. In F, n = 4. *, p < 0.01 versus control; **, p < 0.05 versus miR-21-transfected.

FIGURE 9.

miR-21 regulates high glucose-induced down-regulation of PTEN and downstream signal transduction to induce renal PTE cell protein synthesis, hypertrophy, and fibronectin expression. A and B, high glucose stimulates miR-21 expression. PTE cells were incubated with 25 mm glucose (HG) or 5 mm glucose plus 20 mm mannitol (NG) for 24 h. Total RNAs were used to detect pre-miR-21 (A) and mature miR-21 (B) expression using real time qRT-PCR as described under “Experimental Procedures.” n = 6. *, p = 0.0111 versus 5 mm glucose plus 20 mm mannitol in A. *, p = 0.0008 versus 5 mm glucose plus 20 mm mannitol in B. C, high glucose inhibits PTEN 3′-UTR-Luc activity via miR-21 in PTE cells. Cells were cotransfected with PTEN 3′-UTR-Luc and miR-21 Sponge or vector plasmid followed by incubation with 25 mm glucose or 5 mm glucose plus 20 mm mannitol for 24 h. The cell lysates were assayed for luciferase activity as described under “Experimental Procedures.” The mean ± S.E. of triplicate measurements is shown. *, p < 0.05 versus 5 mm glucose plus 20 mm mannitol; #, p < 0.01 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.01 versus 25 mm glucose. D–H, miR-21 regulates PTEN down-regulation and downstream TORC1 signaling in PTE cells. Cells were transfected with miR-21 Sponge or vector plasmid followed by incubation with glucose as described above in C. The cell lysates were immunoblotted with PTEN, phospho-Akt (p-Akt), Akt, phospho-GSK3β (p-GSK3β), GSK3β, phospho-PRAS40 (p-PRAS40), PRAS40, phospho-S6 kinase (p-S6K), S6 kinase (S6K), and actin antibodies as indicated. Histograms at the bottom of each panel show quantification of protein bands. In D, *, p < 0.01 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.05 versus 25 mm glucose (n = 4). In E, *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.001 versus 25 mm glucose (n = 4). In F, *, p < 0.01 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.01 versus 25 mm glucose (n = 4). In G, *, p < 0.01 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.01 versus 25 mm glucose (n = 4). In H, *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.001 versus 25 mm glucose (n = 4). I and J, miR-21 regulates PTE cell protein synthesis and hypertrophy. PTE cells were transfected with miR-21 Sponge or vector plasmid followed by incubation with high glucose as described in the legend of Fig. 4. Protein synthesis and hypertrophy were determined as described under “Experimental Procedures.” The mean ± S.E. of triplicate measurements is shown. In I, *, p < 0.05 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.05 versus 25 mm glucose. In J, *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.001 versus 25 mm glucose. K and L, mir-21 regulates high glucose-stimulated fibronectin expression in PTE cells. K, cells were transfected with miR-21 Sponge or vector plasmid followed by incubation with high glucose as described in the legend of Fig. 4. The cell lysates were immunoblotted with fibronectin and actin antibodies, respectively, as indicated. The bottom histogram shows quantification of four independent experiments. *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.001 versus 25 mm glucose. L, cells were transfected with Fibro-Luc reporter plus miR-21 Sponge or vector plasmids followed by incubation with high glucose as described in the legends of Fig. 8, C and D. The cell lysates were used for luciferase activity as described under “Experimental Procedures.” The mean ± S.E. of triplicate measurements is shown. *, p < 0.001 versus 5 mm glucose plus 20 mm mannitol; **, p < 0.001 versus 25 mm glucose.

FIGURE 10.

PI 3-kinase/Akt axis regulates phosphorylation of Akt, protein synthesis, hypertrophy, and fibronectin expression in renal PTE cells. A and B, PTE cells were transfected with CMV-miR-21 followed by incubation with Ly294002 (25 μm; A) or MK-2206 (1 μm; B). The cell lysates were immunoblotted with phospho-S6 kinase (p-S6K), S6 kinase (S6K), and actin antibodies as indicated. The histograms at the bottom show quantification of the protein bands. In A, n = 4. *, p < 0.001 versus control; **, p < 0.001 versus miR-21-transfected. In B, n = 4. *, p < 0.001 versus control; **, p < 0.001 versus miR-21-transfected. C–F, PTE cells were transfected with CMV-miR-21 followed by incubation with Ly294002 (25 μm; C and D) or MK-2206 (1 μm; E and F). [³⁵S]Methionine incorporation (C and E) and cell hypertrophy (D and F) were determined as described under “Experimental Procedures.” In C–F, the mean ± S.E. of triplicate measurements is shown. In C, *, p < 0.01 versus control; **, p < 0.001 versus miR-21-transfected. In D, *, p < 0.001 versus control; **, p < 0.001 versus miR-21-transfected. In E, *, p < 0.01 versus control; **, p < 0.01 versus miR-21-transfected. In F, *, p < 0.001 versus control; **, p < 0.001 versus miR-21-transfected. G and H, PTE cells were transfected with CMV-miR-21 followed by incubation with Ly294002 (25 μm; G) or MK-2206 (1 μm; H). The cell lysates were immunoblotted with fibronectin and actin antibodies as indicated. The histograms at the bottom show quantification of the protein bands. In G and H, n = 4. *, p < 0.001 versus control; **, p < 0.001 versus miR-21-transfected.