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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2012 Nov 8;23(12):1941–1948. doi: 10.1681/ASN.2012030321

MicroRNAs Regulate Renal Tubule Maturation through Modulation of Pkd1

Vishal Patel *,✉, Sachin Hajarnis *, Darren Williams *, Ryan Hunter *, Donovan Huynh *, Peter Igarashi *,†
PMCID: PMC3507362  PMID: 23138483

Abstract

MicroRNAs (miRNAs) contribute to the regulation of early kidney development, but their role during later stages of renal tubule maturation is not well understood. Here, we found that ablation of the miRNA-processing enzyme Dicer from maturing renal tubules produces tubular and glomerular cysts in mice. Inactivation of Dicer is associated with downregulation of miR-200, a kidney-enriched miRNA family, and upregulation of the polycystic kidney disease gene Pkd1. Inhibition of miR-200 in cultured renal epithelial cells disrupted tubulogenesis and led to upregulation of Pkd1. Using bioinformatic and in vitro approaches, we found that miR-200b/c/429 induce post-transcriptional repression of Pkd1 through two conserved binding sites in the 3′-Untranslated regions of Pkd1. Overexpression of PKD1 in renal epithelial cells was sufficient to disrupt tubulogenesis and produce cyst-like structures. In conclusion, miRNAs are essential for the maturation of renal tubules, and Pkd1 is a target of miR-200. These results also suggest that miRNAs may modulate PKD1 gene dosage and play a role in the initiation of cystogenesis.

MicroRNAs (miRNAs) are short noncoding RNAs that negatively regulate gene expression. miRNAs are transcribed from the genome as long precursor transcripts that are sequentially processed by the enzymes Drosha and Dicer to generate 19–25 nucleotide mature miRNAs. Nucleotides 2–8 at the 5′ end of a mature miRNA are referred to as the seed sequence. Watson-Crick base-pairing between the mature miRNA seed sequence and 3′-Untranslated regions (UTRs) of target mRNAs results in gene silencing. In this manner, miRNAs function as sequence-specific inhibitors of post-transcriptional gene expression.1–3 miRNAs are implicated in a wide range of biologic processes, including early stages of kidney development.4–6 Removal of the miRNA-processing enzyme Dicer from nephron progenitors leads to premature termination of renal vesicle (RV) formation, whereas inactivation of Dicer from ureteric buds (UBs) leads to premature termination of UB branching.7,8 Hoxb7/cre-mediated inactivation of Dicer produces hydroureter, hydronephrosis, cortical cysts, and renal dysplasia.9 The HoxB7 promoter drives cre expression in the UB tips and UB stalks as early as embryonic day (E) 11.5. Therefore, these phenotypes may arise due to defects in UB branching and RV induction. The role of miRNAs during later stages of renal tubule maturation subsequent to RV formation and UB branching is not well understood.

To examine the role of miRNAs in kidney tubule maturation, we generated Ksp/cre; Dicer F/F (Dicer mutant) transgenic mice. The Ksp-cadherin promoter drives cre expression in the maturing renal tubules and UB stalks but not in the UB tips (until E17.5) or nephron progenitor cells.10,11 Thus, this approach permitted targeted deletion of Dicer from the maturing renal tubules and collecting ducts without affecting RV formation or UB branching. PCR analysis detected the recombined allele of Dicer in genomic DNA from kidneys of Dicer mutant mice, confirming cre-mediated recombination (Figure 1A). Quantitative RT-PCR (qRT-PCR) analysis showed that the expression of Dicer mRNA transcripts was decreased by approximately 70% in Dicer mutant kidneys compared with control kidneys (Figure 1B).

Figure 1.

Figure 1.

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Characterization of Dicer mutant mice. (A) PCR products obtained after amplification of genomic DNA from kidneys of 2-day-old Ksp/Cre; Dicer +/+ and Ksp/Cre; Dicer F/F mice. Arrows indicate the wild-type (WT) and recombined alleles (Del). (B) qRT-PCR analysis showed that the expression of Dicer was decreased in kidneys from 2-day-old Dicer mutant mice compared with control mice. Error bars indicate SD. *P<0.001. (C) Kaplan–Meier survival curves of control mice (n=13) and Dicer mutant mice (n=5). P<0.01, Log-rank (Mantel–Cox) test. (D) Gross and histologic images of kidneys and ureters from control mice (upper panel) and Dicer mutant mice (lower panel). Ureters of control mice (a) were normal, whereas bilateral hydroureter (arrows) was present in Dicer mutant mice (e). H&E staining demonstrated normal histology of kidneys (b) and ureters (c) from control mice, whereas hydronephrosis (f) and hydroureter (g) were observed in Dicer mutant mice. Staining with an antibody against α-smooth muscle actin (green) showed similar expression in smooth muscle cells in ureters of control mice (d) and Dicer mutant mice (h). Nuclei were counterstained with DAPI. (E) Summary of phenotypes observed in Dicer mutant mice at various ages. All age-matched control littermates demonstrated normal morphology and histology of kidneys and ureters. Scale bars, 100 µm in b and f; 50 µm in c, d, g, and h. H&E, hematoxylin and eosin; DAPI, 4',6-diamidino-2-phenylindol.

Dicer mutant mice were born at normal Mendelian ratios, but survival analysis revealed that 60% of the mutant mice died 1–2 weeks after birth (Figure 1C). No deaths were observed in control mice. Gross and histologic examinations did not demonstrate abnormalities in kidneys or ureters of control mice (Figure 1D). In contrast, 74% (n=14 of 19) of newborn Dicer mutant mice developed hydroureter and hydronephrosis (Figure 1, D and E). The percentage of mutant mice exhibiting hydronephrosis declined with age, suggesting that hydronephrosis was the cause of death (Figure 1E). Because the Ksp-cadherin promoter drives cre expression in the developing ureter,10 hydroureter may result from Dicer inactivation in precursors of urothelial cells. Future studies will be needed to determine the mechanism of hydroureter and hydronephrosis.

Twenty-six percent (n=5 of 19) of Dicer mutant mice did not develop hydroureter and hydronephrosis and exhibited normal kidney morphology and histology at birth (Figure 1E). This result indicated that Ksp/cre-mediated inactivation of Dicer did not perturb RV induction or UB branching in these mice. At postnatal day (P) 10, 75% (n=9 of 12) of Dicer mutant mice exhibited numerous kidney cysts (Figure 1E). By P35, the cysts had increased in size and number, and focal glomerular cysts were also observed (Figure 2, B and D). Eighty-nine percent (n=8 of 9) of Dicer mutant mice that were aged ≥35 days developed kidney cysts (Figure 1E). Seventy-seven percent (n=20 of 26) of Dicer mutant mice developed kidney cysts without hydronephrosis, indicating that the cysts did not arise secondary to hydronephrosis. In addition to kidney cysts, adult Dicer mutant mice also developed tubulointerstitial fibrosis (Figure 1E and Supplemental Figure 1).

Figure 2.

Figure 2.

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Tubular and glomerular cysts in Dicer mutant mice. H&E staining of the kidneys from 35-day-old control littermates demonstrated normal tubular and glomerular histology (A and C), whereas Dicer mutant mice contained numerous tubular cysts (B) and glomerular cysts (D). (E–H) To determine the lineage of cyst epithelial cells, kidney sections from control and Dicer mutant mice were stained with an antibody against GFP that also recognizes EYFP. EYFP expression was observed in renal tubules (E) and Bowman’s capsule of glomeruli (G) of control mice confirming Ksp/cre-mediated recombination. All cells lining the tubular cysts (F, arrows) and glomerular cysts (H, arrows) also expressed EYFP, indicating that the cysts were composed of recombined epithelial cells. (I–L) To identify the origins of the cysts, kidney sections from control mice (I and K) and Dicer mutant mice (J and L) were stained with markers of specific nephron segments. Staining with DBA (red; I and J) and anti-THP antibody (red; K and L) revealed that the cysts originated from the collecting ducts (J) and the loops of Henle (L). Scale bars, 100 µm in A and B; 50 µm in C, D, E, F, I, and J; 40 µm in G, H, K, and L. H&E, hematoxylin and eosin; cy, cyst; GFP, green fluorescent protein; gl, glomeruli; DBA, Dolichos biflorus agglutinin; THP, Tamm-Horsfall protein.

To determine the lineage of cyst epithelial cells, an enhanced yellow fluorescent protein (EYFP) reporter gene that is activated by cre/loxP recombination was introduced in the cross. Kidney sections from Dicer mutant mice were stained with an antibody against green fluorescent protein, which also detects EYFP. All cells lining the tubular cysts (Figure 2F) and majority of cells lining the glomerular cysts expressed EYFP (Figure 2H). EYFP expression indicates that cre/loxP recombination has occurred but does not necessarily imply that both alleles of Dicer were recombined. Staining with specific nephron segment and collecting duct markers showed that the majority of the cysts were derived from collecting ducts (Figure 2J). Some cysts were also derived from loops of Henle (Figure 2L). No proximal tubule-derived cysts were observed despite cre-mediated recombination in proximal tubule of Dicer mutant mice (Supplemental Figure 2).

To identify specific miRNAs that contribute to cyst formation in Dicer mutant mice, miRNA microarray analysis was performed on kidneys of P13 Dicer mutant and control mice. Four miRNAs with signals >500 arbitrary units (AU) were downregulated and four miRNAs with signals higher than 500 AU were upregulated in the Dicer mutant kidneys compared with the control kidneys (Figure 3A and Supplemental Table 1). Two of the four downregulated miRNAs, miR-200a and miR-429, belong to the miR-200 miRNA family. Accordingly, we studied the miR-200 family in greater detail (see Supplemental Figure 3 for more information). Northern blot analysis of tissues from wild-type mice revealed that miR-200 family members are highly expressed in the kidneys and lungs compared with other organs (Figure 3C). qRT-PCR analysis confirmed that the expression of miR-200a, miR-200b, miR-200c, and miR-429 was decreased in kidneys from Dicer mutant mice compared with control mice (Figure 3B). To determine the function of miR-200s in the kidney, the expression of miR-200a, miR-200b, and miR-200c was inhibited in murine inner medullary collecting duct (mIMCD3) cells using locked nucleic acid (LNA)–modified anti-miRs. qRT-PCR analysis showed that transfection with LNA–anti-miRs against miR-200a, miR-200b, and miR-200c resulted in a >95% decrease of target miRNA expression compared with mock-transfected and scramble-transfected cells. Expression of the unrelated miRNA miR-30d did not change, indicating high specificity of LNA–anti-miRs (Supplemental Figure 4). Immunostaining showed that the expression of epithelial markers E-cadherin and HNF-1β was comparable between miR-200–deficient cells and control cells, suggesting that miR-200–deficient mIMCD3 cells retain epithelial characteristics (Supplemental Figure 4). miR-200–deficient cells were grown in three-dimensional (3D) collagen gels in the presence of hepatocyte growth factor (HGF) to stimulate tubulogenesis. HGF-dependent tubulogenesis was decreased by 65% in miR-200–deficient cells compared with mock-transfected cells (Figure 3, D and E). Scramble-transfected cells showed no differences in tubule formation compared with mock-transfected cells. Taken together, these results suggest that miR-200s are required for tubulogenesis and that downregulation of miR-200s may underlie kidney cyst formation in Dicer mutant kidneys.

Figure 3.

Figure 3.

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Downregulation of miR-200 in Dicer mutant mice. (A) Heatmap of differentially expressed miRNAs between kidneys from 13-day-old control mice and Dicer mutant mice (n=2). (B) qRT-PCR analysis showed that the expression of miR-200a, miR-200b, miR-200c, and miR-429 was decreased in the Dicer mutant kidneys compared with control kidneys at P10 (n=4 for each group). *P<0.001. (C) Northern blot analysis in wild-type mice showed that miR-200s are enriched in the kidney and lung compared with other organs. (D and E) Tubule formation is decreased in cells transfected with LNA–anti-miRs to miR-200s compared with mock-transfected cells. Tubule formation was unchanged between mock-transfected cells and scramble-transfected cells (n=3). #P<0.05; ns, P>0.05. Error bars represent SD in all graphs. C, control; M, mutant; ctrl, control; mt, mutant; ns, not significant.

The transcription factors Zeb1 and Zeb2 are repressors of E-cadherin gene expression and are known miR-200 targets.12,13 qRT-PCR analysis showed that neither Zeb1 nor Zeb2 was upregulated in kidneys of Dicer mutant mice compared with control mice (Supplemental Figure 5A). Moreover, immunostaining and Western blot analysis showed that the expression of E-cadherin was unchanged in cyst epithelial cells compared with normal tubule epithelial cells (Supplemental Figure 5, B and C). The expression of other potential miR-200 targets, Snai1 and Snai2, was also unchanged (Supplemental Figure 5A). These results suggest that Zeb1 and Zeb2-independent mechanisms drive cyst formation in Dicer mutant mice.

Kidney cysts are observed in several genetic disorders including polycystic kidney disease (PKD), nephronophthisis (NPHP), the Bardet–Biedl syndrome (BBS), and renal cysts and diabetes (RCAD).14 To further explore the mechanisms that underlie cyst formation in Dicer mutant mice, we determined whether genes that are mutated in these disorders are miR-200 targets. We combined expression profiling with 3′-UTR analysis to identify candidate miR-200 targets. qRT-PCR analysis was performed to compare the expression of cystic kidney disease genes between Dicer mutant kidneys and control kidneys and between miR-200 knockdown cells and control cells (Supplemental Table 2). We used the TargetScan (http://www.targetscan.org/),15 PicTar (http://pictar.mdc-berlin.de/),16 and miRanda-mirSVR (http://www.miRNA.org)17,18 bioinformatic tools to identify miR-200 binding sites in the 3′-UTRs of the differentially expressed genes. Two criteria were used to identify putative miR-200 targets: (1) expression of the target gene must be increased in Dicer mutant kidneys and miR-200–deficient cells compared with their respective controls, and (2) the 3′-UTRs of the putative target must contain conserved miR-200 binding site(s).

The autosomal dominant PKD (ADPKD) gene Pkd1 and the RCAD gene Hnf-1β fulfilled both criteria. qRT-PCR analysis showed that expression of Pkd1 was increased in Dicer mutant kidneys and miR-200 knockdown cells (Figure 4A). In situ hybridization showed that expression of Pkd1 was specifically increased in cyst epithelial cells of Dicer mutant mice (Figure 4B). TargetScan and miRanda-mirSVR predicted that the 3′-UTR of mouse Pkd1 contains two miR-200b/c/429 binding sites. The first binding site (B1) is conserved in mammals, whereas the second binding site (B2) is not conserved (Figure 4C). To test whether the binding sites are functional, the 3′-UTR of Pkd1 was cloned into a luciferase reporter plasmid. mIMCD3 cells were cotransfected with the luciferase reporter plasmid and plasmids encoding miR-200a and miR-200b or a negative control vector. The overexpression of miR-200a and miR-200b was verified by qRT-PCR (Supplemental Figure 6). Reporter assays revealed that overexpression of miR-200 repressed the 3′-UTR of Pkd1 (Figure 4D). Individual mutations of B1 or B2 in the Pkd1 3′-UTR did not abolish miR-200–dependent repression. However, miR-200–dependent repression was abrogated by mutations of both B1 and B2 (Figure 4D). These results indicate that miR-200 family members induce post-transcriptional repression of Pkd1 through base-pairing at B1 and B2. To determine whether overexpression of Pkd1 is sufficient to stimulate cyst formation, control and PKD1-overexpressing mIMCD3 cells were grown in 3D culture in the presence of HGF. Tubulogenesis was decreased in PKD1-overexpressing cells and numerous cyst-like structures were observed (Figure 4, E and F). Expression of the RCAD gene Hnf-1β was also increased in Dicer mutant kidneys and miR-200 knockdown cells (Supplemental Table 2). However, reporter assays did not demonstrate miR-200–dependent repression of the Hnf-1β 3′-UTR (Supplemental Figure 7).

Figure 4.

Figure 4.

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Pkd1 is a miR-200 target. (A) qRT-PCR analysis showed that the expression of Pkd1 was increased in Dicer mutant kidneys (n=5) compared with control kidneys (n=5) and in miR-200 knockdown cells compared with control cells. *P<0.01. Error bars represent SD. (B) In situ hybridization showed that expression of Pkd1 was increased in cyst epithelial cells of Dicer mutant mice compared with renal tubules of control mice. * indicates cysts. (C) The 3′-UTR of Pkd1 contains two consensus evolutionarily conserved miR-200b/c/429 binding sites. The seed sequences are shown in red. To determine whether the putative miR-200 binding sites are functional, the 3′-UTR of Pkd1 was cloned in a luciferase reporter plasmid. (D) Reporter assays in mIMCD3 cells showed miR-200–dependent repression of wild-type Pkd1 3′-UTR. Individual mutations of the two miR-200 binding sites, B1 or B2, did not affect repression. MiR-200–dependent repression was abrogated by mutations of both B1 and B2. *P<0.01; ns, P>0.05. Error bars represent SD. WT indicate wild-type 3′-UTR. B1mt indicates Pkd1 3′-UTR containing mutations of B1, B2mt indicates Pkd1 3′-UTR containing mutations of B2 and B1+B2mt indicates Pkd1 3′-UTR containing mutations of both B1 and B2. To determine whether overexpression of Pkd1 can contribute to cyst formation, control and mIMCD3 cells overexpressing PKD1 were grown in 3D cultures. (E) Cyst-like structures were observed in PKD1-overexpressing cells. (F) Quantification revealed that tubule formation was significantly decreased in PKD1-overexpressing cells compared with control cells. n=3 experiments, each performed in triplicate. *P<0.01. Error bars represent SD. (G) Model in which Dicer and miR-200–mediated regulation of Pkd1 affects renal tubule maturation. ctrl, control; mt, mutant; KD, knockdown.

We found that kidney-specific inactivation of Dicer produces hydroureter and hydronephrosis, a finding that is consistent with previous studies.8,9 Both Ksp/cre and HoxB7/cre transgenic mice express cre recombinase in the developing ureter, explaining the similar phenotypes. A major difference compared with the previous studies is the presence of numerous kidney cysts in the Ksp/cre; Dicer F/F mice in the absence of hydronephrosis. The cysts were derived from recombined cells and were first observed at P10. These results directly implicate miRNAs in normal renal tubule maturation. Cyst formation is associated with downregulation of miR-200s and knockdown of miR-200 in mIMCD3 cells is sufficient to disrupt tubulogenesis in 3D culture, providing a potential explanation for cyst formation. Expression of miRNAs is also upregulated in Dicer mutant kidneys (Figure 3A and Supplemental Table 1). Increased miRNA expression would not be expected in a Dicer knockout and may represent upregulation in cells unaffected by the Ksp-cre driver. These cells could impact cyst growth in a cell nonautonomous manner.

To understand direct consequences of Dicer inactivation and subsequent miR-200 downregulation, we determined whether genes involved in cyst pathogenesis are miR-200 targets. miR-200 and the ADPKD gene Pkd1 exhibit a reciprocal pattern of expression, and miR-200 binds to the Pkd1 3′-UTR and represses its expression. An independent study showed that miR-429, a miR-200 family member, physically interacts with Pkd1 3′-UTR in vivo.19 Therefore, our finding that Pkd1 is a miR-200 target is unlikely to be coincidental. Expression of BBS and NPHP genes is decreased in Dicer mutant kidneys, providing another explanation for cyst formation (Supplemental Table 2). Downregulation of these genes likely occurs due to secondary effects of Dicer inactivation because mRNA targets are not typically downregulated in the absence of miRNAs.

Our studies provide a new hypothesis for cyst formation involving miRNAs and regulation of PKD1 gene dosage. Whereas the majority of ADPKD patients carry inactivating mutations of PKD1, accumulating evidence points toward a role for PKD1 gene dosage in cyst initiation.20–22 Two-fold or higher expression of Pkd1 causes kidney cysts in mice.23,24 Knockdown of Pkd1 disrupts tubulogenesis in 3D culture.25 In this study, we found that overexpression of Pkd1 is also sufficient to disrupt tubulogenesis. These studies suggest that PKD1 expression must be tightly regulated for normal renal tubule maturation. Rather than producing dramatic changes in gene expression, miRNAs primarily act to fine-tune changes in gene expression.26 Consistent with this notion, our results suggest a model in which miR-200s function to modulate Pkd1 gene dosage in kidney (Figure 4G). In addition to the miR-200s, another miRNA that is downregulated in Dicer mutant mice is miR-20b (Figure 3A), which is also predicted to target PKD1. This raises the possibility that several miRNAs function in concert to regulate PKD1 gene dosage in the kidney.

Concise Methods

Generation and Characterization of Transgenic Mice

Kidney-specific Dicer mutant mice were produced by breeding Ksp/Cre mice10 with Dicer F/F mice27 that contain loxP sites inserted in the genomic DNA flanking exon 23 of the Dicer gene. Exon 23 encodes an essential RNase III domain; therefore, excision of exon 23 by cre/loxP recombination leads to the loss of pre-miRNA processing by Dicer. Age-matched littermates with Dicer F/F or Ksp/Cre; Dicer +/+ genotypes were used as controls. Genotyping was performed using previously described protocols.27 All experiments involving animals were performed under the auspices of the University of Texas Southwestern Institutional Animal Care and Research Advisory Committee.

miRNA Microarray Analyses

Microarray analysis was performed by LC Sciences (Houston, TX). The small RNA fraction (<300 nt) was extracted from total kidney RNA and hybridized on a µParaflo Microfluidic chip containing detection probes for all mouse miRNAs in the miRBase version 14 (http://microrna.sanger.ac.uk/sequences). The hybridized microarray chips were labeled with fluorescent dyes and laser scanned to obtain fluorescent images. The data were then analyzed as previously described.28 The signal values for each sample were derived by background subtraction and normalization. The signal values for the two controls and mutants were averaged, and fold-change and P values (t test) were calculated. The differentially detected signals are those with P value <0.1. miRNAs with signals values <500 AU are considered to be expressed with low abundance (see Supplemental Table 1).

Northern Blot Analyses

Twenty micrograms of total RNA from various tissues was loaded on a denaturing 20% acrylamide gel and transferred to a Zeta probe GT membrane (Bio-Rad). The RNAs were ultraviolet cross-linked and hybridized for 18 hours in Rapid-Hyb buffer (GE Healthcare) at 39°C with 32P-labeled antisense probes directed against the mature sequences of miR200a, miR200b, miR200c, and miR429. U6 RNA was utilized as a loading control.

Real-Time PCR

Total RNA from mice kidneys or mIMCD3 cells was extracted using the miRNeasy mini kit (Qiagen). First-strand cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) or the Universal cDNA Synthesis Kit (Exiqon). Real-time PCR was performed in triplicate using an iCycler and SYBR green Supermix reagents from Bio-Rad Laboratories or Exiqon. We used 18S and 5S RNA as the control genes for normalization of mRNA and miRNA expression, respectively. Data were analyzed using IQ software (Bio-Rad Laboratories).

Antibodies, Immunostaining, and Western Blots

The following antibodies and dilutions were used: FITC-conjugated anti-green fluorescent protein that cross-reacts with EYFP (1:200; Rockland), α-smooth muscle actin (1:1000; Santa Cruz), and Tamm-Horsfall protein (1:300; Biomedical Technologies). Secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 594 (1:400; Molecular Probes). Lectins used were Dolichos biflorus agglutinin (1:400; Vector Laboratories) and Lotus tetragonolobus agglutinin (1:400; Vector Laboratories). Tissue sections were stained as previously described.29 The slides were examined using an LSM 510 META confocal laser scanning microscope (Zeiss), and images were reconstructed using Imaris software (Bitplane AG). Protein extraction and Western blot analysis were performed as previously described.30

Cell Culture

miR-200 expression was inhibited using LNA–anti-miRs to miR-200a, miR-200b, and miR-200c were obtained from Exiqon (Denmark). We transfected 1.5×105 mIMCD3 cells with 10 nM each of miR-200a, miR-200b, and miR-200c anti-miRs using Lipofectamine 2000. Mock-transfected and mIMCD3 cells transfected with 30 nM of a scrambled anti-miR were used as controls. PKD1-overexpressing cells were generated by transfecting mIMCD3 cells with 1 µg of plasmid, which expresses the full-length human PKD1 cDNA (pcDNA3-hPkd1). Two days after transfection, the cells that expressed the human PKD1 transgene were selected by supplementing the medium with 0.5 mg/ml of G418. After 10–14 days of antibiotic treatment, the surviving cells were expanded. Mock-transfected cells were used as controls.

Tubulogenesis Assays

miR-200–deficent and PKD1-overexpressing mIMCD3 were trypsinized to form a single-cell suspension and mixed with 90 µl of neutralization buffer (2× Hanks balanced salt solution, 50 mM HEPES, pH 7.4) and 430 µl of Cellmatrix collagen matrix in a total volume of 1 ml per well. The collagen gels were allowed to polymerize at 37°C for 4 hours and 1 ml of media containing 10% FBS was added above the gel. Tubule formation was induced by HGF (50 ng/ml), and tubulogenesis was quantified as previously described.31

Luciferase Assays

mIMCD3 cells were plated in six-well dishes (2×105 cells/well) and transfected with 0.8 μg of pLS-Renilla-3′-UTR plasmids and 0.2 µg of miRNA expression plasmids (pCMV-mir200a and pCMV-mir200b) or 0.4 µg of the empty pCMV plasmid as a negative control. Cells were transfected using Lipofectamine 2000 (Invitrogen), and 0.08 μg of the pGL3-control plasmid encoding firefly luciferase was cotransfected to control for differences in transfection efficiency. After growth for 48 hours, the cells were lysed in 250 μl of passive lysis buffer (Promega Corp, Madison, WI). Forty microliters of the cell lysate was added to 96-well plates, and firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega Corp), according to the manufacturer’s directions.

Statistical Analyses

Data shown are mean ± SD. The significance of differences between the means was calculated using the t test. ANOVA was used for multiple comparisons followed by Dunnett’s test to detect differences between specific pairs of groups. Log-rank (Mantel–Cox) test was used to compare differences in survival between control and Dicer mutant mice. P<0.05 was considered statistically significant.

Disclosures

None.

Supplementary Material

Supplemental Data
supp_23_12_1941__index.html (772B, html)

Acknowledgments

We thank Patricia Cobo-Stark for helpful discussions, and Rajiv Parmar and Taelor Oppliger for technical assistance.

Work from the authors' laboratory is supported by grants from the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (KO8 DK084311-01 to V.P. and 5RC1DK086887-02 to P.I.) and the University of Texas Southwestern O'Brien Kidney Research Core Center (NIH P30DK079328).

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

See related editorial, “Small RNAs Have a Big Effect on Polycystic Kidney Disease,” on pages 1909–1910.

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