Variations in MicroRNA-25 Expression Influence the Severity of Diabetic Kidney Disease

Abstract

Diabetic nephropathy is characterized by persistent albuminuria, progressive decline in GFR, and secondary hypertension. MicroRNAs are dysregulated in diabetic nephropathy, but identification of the specific microRNAs involved remains incomplete. Here, we show that the peripheral blood from patients with diabetes and the kidneys of animals with type 1 or 2 diabetes have low levels of microRNA-25 (miR-25) compared with those of their nondiabetic counterparts. Furthermore, treatment with high glucose decreased the expression of miR-25 in cultured kidney cells. In db/db mice, systemic administration of an miR-25 agomir repressed glomerular fibrosis and reduced high BP. Notably, knockdown of miR-25 in normal mice by systemic administration of an miR-25 antagomir resulted in increased proteinuria, extracellular matrix accumulation, podocyte foot process effacement, and hypertension with renin-angiotensin system activation. However, excessive miR-25 did not cause kidney dysfunction in wild-type mice. RNA sequencing showed the alteration of miR-25 target genes in antagomir-treated mice, including the Ras-related gene CDC42. In vitro, cotransfection with the miR-25 antagomir repressed luciferase activity from a reporter construct containing the CDC42 3' untranslated region. In conclusion, these results reveal a role for miR-25 in diabetic nephropathy and indicate a potential novel therapeutic target for this disease.

Keywords: blood pressure; cardiovascular; diabetic nephropathy; fibrosis; focal segmental glomerulosclerosis.

Figure 1.

Excessive miR-25 reverse albuminuria and fibrosis in diabetic nephropathy. (A) miR-25 levels in the sera of patients with type 1 diabetes (T1DM), type 2 diabetes (T2DM), or DN were reduced compared with the healthy controls (Con). (B and C) Similarly, miR-25 was reduced in the kidneys of type 1/2 diabetic (Dia [n=5] and db/db [n=5]) animals compared with controls (Con; n=5). (D) miR-25 expression levels in various tissues of normal C57BL/6 mice. (E) Quantification of 24-hour albumin excretion in miR-25 agomir–treated mice. (F) Representative images of H&E (row 1), PAS (row 2), Sirius Red (row 3), α-SMA (row 4), and TEM (row 5) staining from kidney sections of miR-25 agomir–injected mice. Green arrows indicate areas of fibrosis, an asterisk indicates the GBM, and a red arrow represents a foot process. (G) Quantification of the α-SMA–positive area within glomeruli from miR-25 agomir–treated kidney sections. (H) Quantification of GBM thickening after miR-25 agomir injection. (I) Quantification of foot process fusion from miR-25 agomir–treated db/db mice. (J) Measurement of BP from miR-25 agomir–injected db/db mice. Data are shown as the mean±SEM. **P<0.01; ***P<0.001.

Figure 2.

Overexpression of miR-25 inhibit RAS activation. (A) Representative images of KRI (row 1), angiotensin 1 (AT1; row 2), renin (row 3), and vWf (row 4) staining from miR-25 agomir–treated db/db mouse kidney sections (right panel represents a quantification of the images). (B) Quantification of an ELISA for serum renin, angiotensin I/II, and aldosterone (ALD) from miR-25 agomir– and antagomir–treated mice. Data are shown as the mean±SEM. Con, control. ***P<0.001.

Figure 3.

Inhibition of miR-25 resulted in proteinuria and kidney fibrosis in normal mice. (A and B) Quantification of 24-hour albumin excretion and urine albumin-to-creatinine ratio (ACR) in miR-25 antagomir mice. (C and D) Representative images of H&E, PAS, collagen I/IV, TEM, and PODXL staining from kidney sections of miR-25 antagomir–injected mice. Asterisks indicates mesangial expansion, and arrows indicate foot processes in the TEM images. (E and F) Quantification of the collagen I/IV–positive area within the glomeruli. (G) Quantification of GBM thickening after miR-25 antagomir injection. (H) Quantification of the PODXL-positive staining area within the glomeruli. Data are shown as the mean±SEM. Con, control. ***P<0.001.

Figure 4.

Decreased miR-25 cause high blood pressure and activate RAS. (A) Representative image of KRI by color Doppler ultrasound from miR-25 antagomir–treated mice, with quantification of the KRI in lower right panel. (B) Quantification of the BP from miR-25 antagomir–injected mice. (C–E) Representative images of renin, angiotensin 1 (AT1), and vWf staining of kidney sections from miR-25 antagomir–treated mice; lower right panels represent the quantification of renin-, AT1-, and vWf-positive areas. (F–I) Quantification of the ELISA for serum renin, angiotensin І/П, and aldosterone (ALD) from miR-25 antagomir–injected mice. Data are shown as the mean±SEM. Con, control. *P<0.05; **P<0.01; ***P<0.001.

Figure 5.

Overexpression of miR-25 does not cause kidney injury in normal mice. (A) miR-25 agomir transfection efficiency in the kidney. (B) Quantification of the 24-hour albumin excretion in miR-25 agomir–treated normal mice. (C) Representative images of H&E, PAS, collagen I, and TEM staining from kidney sections of miR-25 agomir–treated normal mice. (D) Representative images of KRI from an miR-25 agomir–treated mouse kidney. The right panel represents quantification of the KRI. (E) Representative images of vWf, renin, and angiotensin 1 (AT1) staining from kidney sections of miR-25 agomir–injected wild-type mice. (F) Quantification of the ELISA for serum renin, angiotensin І/П, and aldosterone (ALD) from miR-25 agomir–injected mice. Data are shown as the mean±SEM. Con, control. ***P<0.001.

Figure 6.

miR-25 positive regulate target gene alteration is responsible for kidney dysfunction. (A and B) Scatterplots representing the alteration of miR-25 target genes from miR-25 antagomir– or agomir–treated mice compared with controls. (C and D) Densitometry of the P values of miRNA target and control genes in the RNA-seq data from miR-25 antagomir– and agomir–injected mice, respectively. (E and F) The correlation between the number of miRNA-targeted mRNAs or agomir-injected mice. (G and H) Luciferase activity in HEK293A cells that were transfected with the indicated 3′-UTR reporter constructs showing binding of miR-25 to the 3′-UTR of CDC42. Data are shown as the mean±SEM. **P<0.01.