MiR-27b regulates podocyte survival through targeting adenosine receptor 2B in podocytes from non-human primate

Abstract

MicroRNAs are a group of small non-coding RNAs that play key roles in almost every aspect of mammalian cell. In kidney, microRNAs are required for maintaining normal function of renal cells, disruption of which contributes to pathogenesis of renal diseases. In this study, we investigated the potential role of miRNAs as key regulators of podocyte survival by using a primary cell culture model from non-human primates (NHPs). Through microRNA profile comparison in glomeruli from mouse, rat and NHP, miR-27b was found to be among a list of glomeruli-enriched miRNA conserved across species. In NHP primary podocyte culture, significant downregulation of miR-27b was observed during treatment of puromycin aminonucleoside (PAN), a classic nephrotoxin. Overexpression of miR-27b enhanced PAN-induced apoptosis and cytoskeleton destruction in podocytes while its inhibition had a protective effect. Target identification analysis identified Adora2b as a potential direct target of miR-27b. Ectopic expression of miR-27b suppressed both Adora2b mRNA and protein expression, whereas inhibition of miR-27b increased the transcript and protein expression levels of Adora2B. Dual luciferase assay further confirmed Adora2b as a direct target of miR-27b. Furthermore, knockdown of Adora2b by siRNAs enhanced PAN-induced apoptosis, similar to the phenotypes we had observed with miR-27b overexpression. In addition, stimulating the adenosine signaling by an Adora2b agonist, NECA, improved podocyte survival upon PAN treatment. Taken together, our data identified a novel role of miR-27b-adora2b axis in primary podocyte survival upon injury and suggested a critical role of adenosine signaling pathway in podocyte protection.

Fig. 1. Identification and validation of glomeruli-enriched microRNAs.

a A group of microRNAs was identified to be conservatively expressed in glomeruli from mouse, rat, and NHP by TaqMan Microarray. b miR-24, 27b, and 126 were confirmed to be enriched in glomeruli of Sprague Dawley rats by in situ hybridization. FFPE sections of normal rat kidney tissues were stained with 40 nM microRNA probe, 1 nM U6 probe, and 40 nM Scramble control. Scale bar: 50 µm. c miR-24, 27b, and 126 were also enriched in NHP glomeruli. Glomeruli and tubule tissues from Sprague Dawley rats (n = 3) and NHP kidneys (n = 2) were collected for RNA extraction and qPCR analysis of miRNA expressions. Error bar represents data from 2 to 3 animals and two technical replicate samples per animal. Glo glomeruli; Tub ubule

Fig. 2. Isolation and characterization of primary podocytes from NHPs.

a The glomeruli from different mammalian species were isolated by classical sieving techniques. Scale bar: 250 µm. NHP glomeruli were isolated through 160 µm and 100 µm sieves. Rat glomeruli were isolated through 100 µm and 75 µm sieves, whereas mouse ones were isolated using 75 µm and 40 µm sieves. b Isolated glomeruli tissues have high expression of published glomerular marker genes. Marker genes were analyzed in isolated tissues, including glomeruli, tubule, kidney cortex, and medulla. Error bar represents data from duplicate wells from mixed samples of 2–3 animals. c Primary rat podocytes were positive for traditional podocyte markers. The glomeruli were isolated and plated onto collagen coated plates followed by an outgrowth of arborized mature podocytes that stain positive for well-characterized podocyte markers, including Wilms Tumor 1 (WT-1), Synaptopodin, and Nephrin. Small inserts represent nuclear staining by DAPI. Scale bar: 50 µm; d Primary NHP podocytes also stained positive for podocyte markers. Scale bar: 50 µm

Fig. 3. Use of primary podocytes from NHPs as an in vitro model for PAN-induced cytotoxicity.

a PAN-induced dose-dependent cell death in primary NHP and rat podocytes. NHP podocytes were treated with 10 µg/ml of PAN for 3–5 days before taking images. Rat podocytes were treated with 10 µg/ml of PAN for 3–5 days before taking images. Scale bar: 150 µm. b NHP podocytes responded to PAN in a dose-dependent manner. Cells were treated with PAN at different concentrations and collected at day 5 for cell viability test. Error bar represents data from eight biological replicates. c NHP podocytes responded to PAN in a time-dependent manner. Cells were treated with PAN at 50 or 10 µg/ml and collected at different time points for cell viability test. Error bar represents data from eight biological replicates. d Analysis of PAN-induced toxicity in NHP podocytes using Annexin V/7-AAD staining. NHP podocytes were treated with 10 or 50 µg/ml PAN for 3 days before collected for FACS analysis. PAN10:10 µg/ml, PAN50: 50 µg/ml. Error bar represents data from three technical replicates

Fig. 4. MiR-27b regulated PAN-induced apoptosis in NHP podocytes.

a miR-27b was significantly suppressed upon PAN treatment. Cells were exposed to 10 µg/ml PAN for 3–5 days before collected for RT-qPCR. Relative miR expression was calculated using U6 as normalization control. Error bar represents data from two independent experiments. b PAN-induced miR-27 change was also observed in primary rat podocytes. Cells were treated with PAN for 3 and 5 days before collected for miR-qPCR analysis. Error bar represents data from two independent experiments. c miR-27b was efficiently transfected into NHP podocytes. 50 nM miRNA mimic and siControl were transfected into NHP podocytes using Lipofectamine RNAiMax. Cells were collected at day 3 and day 5 post transfection and miRNA expression was analysis using miR-X kit (Clontech). Error bar represents data from two independent experiments. Data were normalized to U6. d Endogenous miR-27b was efficiently knocked-down by transfecting miR-27b hairpin inhibitor into NHP podocytes. miR inhibitor control and miR-27b inhibitor were transfected into NHP podocytes at the final concentration of 50 nM. Cells were collected for miRNA qPCR at day 3 and day 5 post transfection. Data were normalized to U6. Error bar represents data from two independent experiments. e miR-27b overexpression enhanced PAN-induced cell death while miRNA inhibition led to increased cell viability. Cells were transfected with 50 nM miRNA mimic or inhibitor and treated with 10 µg/ml PAN for 5 days before collected for cell viability test using CellTiter 96 kit (Promega). Relative cell viability was calculated by normalizing absorbance readings from PAN-treated samples to non-treated controls. Error bar represents data from three independent experiments with eight replicates. **p < 0.01 by Student's t-test. f miR-27b overexpression led to increased caspase3 activation. Cells were transfected with 50 nM miRNA mimic and treated with 50 µg/ml PAN for 36 h before fixation and immunostaining. Scale bar: 200 µm. g Cleaved caspase-3 expression was increased by miR-27b overexpression in NHP podocytes. Cells were treated with 50 µg/ml PAN for 48 h before collected for active caspase3 by western blotting. Actin was used as the loading control. h miR-27b overexpression led to increased caspase3/7 activation. Cells were treated with 50 µg/ml PAN for 36 h before collected for /7 glo assay (Promega). Error bars represent data from eight replicates. *p < 0.05 by Student's t-test. i miR-27b overexpression led to increased caspase3/7 activation. Cells were treated with 10 µg/ml PAN for 36 h before collected for /7 glo assay (Promega). Error bars represent data from eight replicates. *p < 0.05 by Student's t-test

Fig. 5. miR-27b regulated PAN-induced damage of cytoskeleton.

a miR-27b overexpression enhanced PAN-induced cytoskeleton destruction. Cells were transfected with 50 nM miRNA mimic and treated with 10 μg/ml PAN for 3 days before fixation and staining. Scale: 50 µm. b Protein expression of nephrin was decreased by miR-27b. Cells were transfected with 50 nM miRNA mimic and treated with 10 µg/ml PAN for 3–5 days before collected and protein expression of nephrin was analyzed by western blotting. GAPDH was used as the loading control. c Inhibit the function of miR-27b by sponge ameliorated cytoskeleton damage induced by PAN. Scale bar: 50 µm. d Protein expression of nephrin was reversed by miR-27b sponge. Error bars represent data from three independent experiments. *p < 0.05 by Student's t-test

Fig. 6. Adora2b is a direct target of miR-27b in NHP podocytes.

a Adora2b was induced upon PAN treatment in primary rat podocytes. Rat podocytes were treated with 10 µg/ml PAN for 3 and 5 days before collected for RT-qPCR. Several predicted miR-27b targets from TargetScan with multiple conserved miRNA targeting sites were selected for the analysis. Error bar represents data from two independent experiments. b Adora2b was specifically expressed in rat glomeruli. Primary tissues of glomeruli and tubule were isolated from two different rats. Total RNAs were extracted and expression of Adora2b was analyzed by RT-qPCR. Data were normalized to GAPDH. Error bar represents data from two technical replicates. c Adora2b was enriched in NHP glomeruli. Expression of Adroa2b was analyzed using primary tissues from NHP. Error bar represents data from two technical replicates. d Adora2b and miR-27b were confirmed to be expressed in same glomeruli of NHP by immunohistochemistry and in situ hybridization. FFPE sections of normal monkey kidney tissues were stained with anti-Adora2B and anti-Nephrin antibodies as well as 40 nM microRNA probe. The arrow indicates the same site of Adorae2b, Nephrin, and miR-27b. Scale bar: 25 µm. e Adora2b expression was strongly induced by PAN treatment in NHP podocytes. NHP podocytes were treated with 10 µg/ml PAN for 3–5 days before collected. Upper: protein expression was analyzed by western blotting. Lower: mRNA expression was analyzed by RT-qPCR. Error bar represents data from two independent experiments. f mRNA expression of Adora2b was suppressed by miR-27b in NHP podocytes. Cells were transfected with 50 nM of siRNAs such as siControl/miR-27b mimic, Inhibitor control/miR-27b inhibitors. Total RNAs were extracted for RT-qPCR analysis at 48 h after transfection. Error bar represents data from three independent experiments. *p < 0.05 by Student's t-test. g Protein expression of Adora2b was suppressed by miR-27b in NHP podocytes. Cells were transfected the same way as in f and protein expression of Adora2b was analyzed 48 h post transfection by western blotting. Tubulin was used as the loading control. h Adora2b is a direct target of miR-27b. Adora2b 3′-UTR was cloned into pmiR-glo luciferase reporter (Promega). Dual luciferase assay was done in 293T cells with both reporter and miRNA transfection. miRNA mimic or inhibitor were transfected at 50 nM for 2 days and luciferase activity was analyzed using Dual-Glo assay system (Promega). Error bar represents data from two independent experiments with eight replicates. *p < 0.05 by Student's t-test. i Seed region mutations abolish miR-27b’s suppression on Adora2b. Three point-mutations were introduced into the seed region of each miR-27b target site. These luciferase reporters were then transfected into 293T cells and analyzed in the same way as in g. Error bar represents data from two independent experiments with eight replicates

Fig. 7. Stimulating Adenosine signaling through Adora2b reversed PAN-induced injury in NHP podocytes.

a Adora2b was efficiently knocked-down by siRNAs in NHP podocytes. A total of 50 nM siRNAs were transfected into the cells using Lipofectamine RNAiMax (Life Technologies). Total RNAs were isolated for RT-qPCR at 48 h post transfection. Data were normalized to GAPDH. Error bar represents data from two replicates (Left panel). Protein expression of Adora2b was also significantly decreased in siA2B transfected cells (Right panel). Total proteins were isolated for western blotting at 48 h post transfection. Tubulin was used as the loading control. b Knockdown of Adora2b promoted PAN-induced injury in NHP podocytes. Cells were transfected with siControl, miR-27b mimic, and siA2B at the final concentration of 50 nM. PAN treatment (10 µg/ml) was initiated 1 day after transfection. Cells were subject to viability test using CellTiter-96 (Promega) at day 5 post PAN treatment. Relative cell viability was calculated by normalizing absorbance readings from PAN-treated samples to non-treated controls. Error bar represents data from two independent experiments with eight replicates. *p < 0.05 by Student's t-test. c Stimulating adora2b had a protective role in PAN-induced injury in NHP podocytes. Cells were stimulated with 5 µM NECA and PAN treatment (10 µg/ml) was initiated 1 day after. Cell viability test was the same as in b. Error bar represents data two independent experiments with eight replicates. *p < 0.05 by Student's t-test. d Adora2b was efficiently knocked-down by shRNAs in NHP podocytes. Total RNAs were isolated for RT-qPCR at 72 h post lentivirus infection. Data were normalized to GAPDH. Error bar represents data from two technical replicates. e Protein expression of Cleaved Caspase-3 was elevated in shRNA infected NHP podocytes cells. Cells were infected with shRNA and PAN treatment (50 µg/ml) was initiated 1 day after. Total proteins were isolated for western blotting at 48 h post PAN treatment. Actin was used as the loading control. f Quantification of cleaved caspase-3 expression. Relative quantification was performed by normalizing signal of cleaved caspase-3 to actin. g Overexpression of Adora2b by lentiviral vectors. Coding sequence of Adora2b was cloned into pCMV-MCS-EF1-copGFP vector (System Biosciences). Lentivirus transduced cells were spotted by GFP imaging. Scale: 100 µm. h Overexpression of Adora2b by lentiviral vectors. Total RNAs were isolated for RT-qPCR at 72 h post lentivirus infection. Data were normalized to GAPDH. Error bar represents data from two technical replicates. i Overexpression of Adora2b had protective effects against PAN-induced injury in NHP podocytes. Equal number of empty or Adora2b transduced cells were seeded in 96-well plates and treated with 10 µg/ml PAN and with or without NECA (5 µM). Data represent two independent experiments with eight replicates. **p < 0.01 by Student's t-test. j Protein expression of Cleaved Caspase-3 was modestly suppressed in pLenti-A2B infected NHP podocytes cells (Left panel). Cells were infected with empty or Adora2b and PAN treatment (50 µg/ml) was initiated 1 day after. Total proteins were isolated for western blotting at 48 h post PAN treatment. Actin was used as the loading control. Relative quantification was performed for western results (Right panel). k Stimulation of Adora2b by NECA decreased the expression of Cleaved Caspase-3 in NHP podocytes (Left panel). Cells were stimulated with 5 µM NECA and PAN treatment (50 µg/ml) was initiated 1 day after. Total proteins were isolated for western blotting at 48 h post PAN treatment. Actin was used as the loading control. Relative quantitation was performed for western results (Right panel)

Fig. 8. Model of miR-27b-mediated regulation of podocyte survival.

Expression of miR-27b is subject to a feedback regulation mechanism induced by PAN treatment in primary podocytes.Decreased expression of miR-27b leads to the released suppression of adenosine receptor 2b expression (A2B). Abundant A2B on the cell surface promotes intracellular pro-survival signaling pathways