Podocyte-selective deletion of dicer induces proteinuria and glomerulosclerosis

Abstract

Dicer is an enzyme that generates microRNA (miRNA), which are small, noncoding RNA that function as important regulators of gene and protein expression. For exploration of the functional roles of miRNA in glomerular biology, Dicer was inactivated selectively in mouse podocytes. Mutant mice developed proteinuria 4 to 5 weeks after birth and died several weeks later, presumably from kidney failure. Multiple abnormalities were observed in glomeruli of mutant mice, including foot process effacement, irregular and split areas of the glomerular basement membrane, podocyte apoptosis and depletion, mesangial expansion, capillary dilation, and glomerulosclerosis. Gene profiling revealed upregulation of 190 genes in glomeruli isolated from mutant mice at the onset of proteinuria compared with control littermates. Target sequences for 16 miRNA were significantly enriched in the 3'-untranslated regions of the 190 upregulated genes. Further suggesting validity of the in silico analysis, six of the eight top-candidate miRNA were identified in miRNA libraries generated from podocyte cultures; these included four members of the mir-30 miRNA family, which are known to degrade target transcripts directly. Among 15 upregulated target genes of the mir-30 miRNA, four genes known to be expressed and/or functional in podocytes were identified, including receptor for advanced glycation end product, vimentin, heat-shock protein 20, and immediate early response 3. Receptor for advanced glycation end product and immediate early response 3 are known to mediate podocyte apoptosis, whereas vimentin and heat-shock protein-20 are involved in cytoskeletal structure. Taken together, these results provide a knowledge base for ongoing investigations to validate functional roles for the mir-30 miRNA family in podocyte homeostasis and podocytopathies.

Figure 1.

NPHS2-Cre transgene efficiently deleted Dicer floxed region, leading to downregulation of mature miRNA and accumulation of precursor miRNA in podocytes of conditional knockout (mutant) mice. (A) A glomerulus of a control mouse after β-gal staining. (B) Two glomeruli from a 2-wk-old Rosa26^F/+:NPHS2-Cre mouse after β-gal staining. The arrows point to the blue spots depicting podocytes with deletion at Rosa26 allele locus in a single glomerulus. (C) Schematic representation of Dicer alleles: Wild-type, loxP floxed, and deleted. D1 and D3 are the primers for PCR. E20 and E21 depict exons 20 and 21 of Dicer gene. loxP sites are denoted with open arrowheads. (D) Electrophoretic agarose gel analysis of PCR products from glomerular genomic DNA of mice. Lane 1, Dicer^F/+ (control); lane 2, Dicer^F/F:NPHS2-Cre (mutant) without proteinuria; lane 3, Dicer^F/F:NPHS2-Cre (mutant) with proteinuria; lane 4, Dicer^F/+:NPHS2-Cre; lane 5, no DNA (negative control). Molecular weight (MW) 1 kb⁺ DNA ladder (Invitrogen). (E) Histogram shows the abundances of miR-30d and miR-23b in mutant mice without (−PU) and with (+PU) proteinuria relative to that in control mice. The bars represent average ± SD (n = 3). (F) Histogram shows the abundances of premiR-30a, 30c-2, and 30d in mutant without (−PU) and with (+PU) proteinuria relative to that in control mice. Bars represent average ± SD (n = 3).

Figure 2.

Dicer deficiency in podocytes resulted in progressive glomerular defects. (A) Kidneys from a Dicer^F/+:NPHS2-Cre mouse (control, left) and a Dicer^F/F:NPHS2-Cre mouse (mutant, right) that had severe proteinuria and was dying. (B) Periodic acid-Schiff (PAS) staining of a kidney section from a mutant mouse with massive proteinuria. Protein casts were present in most of tubular lumens. (C through F) Glomeruli from PAS-stained kidney sections of control (C) and mutant (D through F) mice. Glomeruli of mutant mice were sclerotic (D through F) with mesangial expansion (white arrow), extracellular deposition (white arrowhead), capillary dilation (▪), and reduced cellularity. In addition, tubulointerstitial fibrosis (black arrow), tubular dilation, and protein casts (⋄) are seen in kidney section of mutant mice (F). (G and H) Electronic microscopy examination showing FP effacement (white arrowhead), GBM irregularity (white arrow), and splitting (red arrow) of mutant mice (6-wk-old with proteinuria of 300 mg/L). Magnifications: ×2.5 in B; ×63 in C through E; ×20 in F.

Figure 3.

Synaptopodin and podocin expressions both were downregulated in podocytes of mutant mice with proteinuria (+PU). Different degrees of synaptopodin and podocin downregulations are shown in the middle panels (milder) and right panels (more severe). Magnification, ×40.

Figure 4.

Mutant podocyte underwent apoptosis, and podocyte number of mutant mice decreased as proteinuria progressed. (A through D) TUNEL assay showing an apoptotic podocyte in the glomerulus of a mutant mouse. WT1, green; TUNEL, red; and DAPI, blue. (E) Rates of apoptotic nuclei in four groups of mice: (1) control mice (n = 9), one apoptotic podocyte found in 930 glomeruli examined; (2) mutant mice without proteinuria (−; n = 4), no apoptotic podocyte in 379 glomeruli; (3) mutant mice with low-grade proteinuria (+; n = 6), three apoptotic podocytes in 590 glomeruli; (4) mutant mice with high-grade proteinuria (++; n = 7), 19 apoptotic podocytes in 874 glomeruli. (F) Podocyte number among four groups of mice as described in E shows podocyte numbers of mutant mice with proteinuria were reduced significantly. **P < 0.01.