MicroRNA-195a-5p regulates blood pressure by inhibiting NKCC2A

Abstract

BACKGROUND:

Previous studies showed that miR-195a-5p was among the most abundant miRNAs expressed in the kidney.

METHODS:

Lentivirus silencing of TNF was performed in vivo and in vitro. Luciferase reporter assays confirmed that NKCC2A mRNA is targeted and repressed by miR-195a-5p. Radiotelemetry was used to measure mean arterial pressure (MAP).

RESULTS:

TNF up-regulates mmu-miR-195a-5p, and −203 and down-regulates mmu-miR-30c and −100 in the medullary thick ascending limb (mTAL) of male mice. miR-195a-5p was more than 3-fold higher in the renal outer medulla of mice given an intrarenal injection of murine recombinant TNF, while silencing TNF inhibited miR-195a-5p expression by approximately 51%. Transient transfection of a miR-195a-5p mimic into mTAL cells suppressed NKCC2A mRNA by approximately 83% while transfection with Anti-miR-195a-5p increased NKCC2A mRNA. Silencing TNF in mTAL cells prevented increases in miR-195 induced by 400 mosmol/kg H₂O medium, an effect reversed by transfection with a miR-195a-5p mimic. Expression of pNKCC2 increased 1.5-fold in mTAL cells transfected with Anti-miR-195a-5p and a miR-195a-5p mimic prevented the increase, which was induced by silencing TNF in cells exposed to 400 mosmol/kg H₂O medium after osmolality was increased by adding NaCl. Intrarenal injection of TNF suppressed NKCC2A mRNA while injection of miR-195a-5p prevented the increase of NKCC2A mRNA abundance and pNKCC2 expression when TNF was silenced. Intrarenal injection with miR-195a-5p markedly attenuated MAP after renal silencing of TNF in mice given 1% NaCl.

CONCLUSIONS:

The study identifies miR-195a-5p as a salt-sensitive and TNF inducible miRNA that attenuates NaCl-mediated increases in blood pressure by inhibiting NKCC2A.

Graphical Abstract

High salt intake increases TNF production and NKCC2A expression by the TAL. TNF increases miR-195a-5p expression, which decreases NKCC2A mRNA thereby increasing NaCl excretion and attenuating blood pressure.

INTRODUCTION

Hypertension affects approximately 25% of the adult population in industrialized societies and is an independent, heritable, modifiable risk factor for cardiovascular and renal diseases, which are leading causes of death^1,2,3,4. Previous studies have shown that regulation of fluid reabsorption by the kidney is essential for maintaining proper blood pressure and the thick ascending limb of Henle’s loop TAL (TAL) has been linked to the development of salt-sensitive hypertension^{2,5 6-9}. The transport of NaCl across the apical membrane of the TAL occurs mainly via a bumetanide-sensitive Na⁺⁻K⁺-2Cl⁻cotransporter (NKCC2), which is highly expressed along the TAL and is the molecular target of loop diuretics^10-12. The Slc12a1 gene encodes NKCC2, and differential splicing of pre-mRNA, which is influenced by changes in salt conditions, yields several isoforms that vary in their function and localization along the nephron^13-16.

A class of small noncoding RNAs, termed microRNAs (miRNAs), has been identified in the regulation of targeted gene expression via several mechanisms including interaction with complementary sequences in the 3′ untranslated regions (3’-UTR) of their mRNA targets^17-19. In addition, miRNAs may be expressed in a cell- or tissue-specific manner and regulate the level of their target proteins by additional mechanisms including controlling the level of transcription and alternative pre-mRNA splicing ^18,20-22. Some miRNAs that are particularly abundant in the kidney play important roles in regulating renal function^23,24. We recently demonstrated that miR-133a is a salt-sensitive and tumor necrosis factor-alpha (TNF) inducible miRNA that inhibits expression of angiotensinogen (AGT) in the proximal tubule (PT)²⁵. In the present study, we addressed the hypothesis that a TNF inducible miRNA in the TAL may contribute to a mechanism involving NKCC2 that regulates blood pressure in response to elevated sodium intake.

NKCC2 functional diversity is augmented by the production of alternative splice variants of this integral membrane protein, which are expressed axially along the nephron and exhibit characteristics that facilitate the precision-tuning of its function^6,13,26-30. The alternatively spliced ABF cassette exons, three mutually exclusive 96-bp cassettes in exon 4 of the Slc12a1 gene, encode a transmembrane segment of the protein that is strictly conserved for about half the residues and includes regularly spaced variability at the 3' end of the exon. Previous studies have shown that high salt conditions increase expression of the NKCC2 isoform A (NKCC2A)^31-33. We previously showed that TNF produced by renal epithelial cells inhibits NKCC2A as part of a mechanism that attenuates increases in blood pressure in response to elevated sodium intake^34,35. However, the mechanisms by which TNF inhibits NKCC2A isoform expression and NKCC2 activity along the TAL remain to be determined. In the present study, we identified TNF-dependent miR-195a-5p expression as part of a negative feedback mechanism that limits NKCC2A-mediated increases in blood pressure.

METHODS

A detailed description of the methods can be found in the online Supplement. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Materials and reagents:

Antibodies for β-actin were obtained from Abcam, and anti-phosphorylated (p)NKCC2 was a gift from Dr. Kerim Mutig. Lipofectamine reagents, TRIzol, and SDS polyacrylamide bis-Tris gels were from Invitrogen. Silencing of TNF mRNA was accomplished using the lentiviral vector psiLv-U6 (GeneCopoeia) and the lentivirus purification kit from Takara Bio USA. Recombinant mouse TNF was obtained from PharMingen. Polyvinylidene difluoride membranes were obtained from Amersham. The Nonidet P-40 lysis buffer contained protease inhibitors (Roche Diagnostics) for anti-pNKCC2 analysis. Tissue culture media were obtained from Life Technologies (Grand Island, NY). The luciferase assay kit was from Promega (Madison, WI). All other chemical reagents were purchased from Sigma.

Tissue samples and Cell preparation:

All samples were immediately snap-frozen in liquid nitrogen following surgery and stored in liquid nitrogen until further use. Human embryonic kidney (HEK)-293T cells were maintained in Eagle's minimum essential medium supplemented with 10% heat-inactivated FBS and 4 mM l-glutamine. mTAL tubules and primary mTAL cells (90–95% purity) were isolated from mice as previously described³⁶. For details, refer to the online Supplement.

Cell culture and transfection:

HEK293-T cells were grown in 20-cm² flasks to generate lentivirus as previously described^35,36. Primary cultures of murine mTAL cells were then cultured to 70–80% confluence in 6-well plates with membrane inserts (BD Biosciences) then transfected using Lipofectamine 2000 (Invitrogen) as previously described^35,36. Cells were harvested and used for different analyses as detailed in the online Supplement.

Luciferase Reporter Constructs:

Reporter gene vectors, containing the 3′-UTR of a miRNA target gene, were constructed according to the manufacturer's protocol (ThermoFisher Scientific) as previously described²⁴. Briefly, a synthetic, double-stranded oligonucleotide spanning a region of the murine 3′ UTR of NKCC2A mRNA containing the putative miR-195 binding site was cloned downstream of the firefly luciferase reporter gene in the pMIR-report TM Expression Reporter Vector System (ThermoFisher Scientific), thereby generating pMIR-NKCC2A- luciferase reporter vector (NKCC2A-3′-UTR). Site-directed mutagenesis was performed using QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene), following the protocol suggested by the company to generate mutated pMIR-NKCC2A luciferase reporter vector (mut-NKCC2A-3′-UTR). Sequence analysis confirmed proper cloning and sequence of the inserts.

Luciferase Activity Analysis:

Luciferase activity was measured using a luciferase assay system (Promega, Fitchburg, WI) according to the manufacturer’s instructions. Briefly, to detect suppression by miR-195a-5p, HEK293T cells were co-transfected with the Lenti-miR-195a-5p or Anti-miR-195a-5p and indicated NKCC2A 3′ UTR luciferase reporter (NKCC2A-3′-UTR, 200ng) or mut-NKCC2A-3′-UTR (200ng) containing a single point mutation (G to C at position 4133) in the binding site with miR-195a-5p. A renilla luciferase expression plasmid (pRL-SV40, Promega) served as a transfection efficiency control. The luciferase activities of cell extracts were determined 24 h after transfection by using the Dual-Luciferase Reporter Assay System (Promega) and activity calculated as relative light units from firefly luciferase normalized to Renilla luciferase values. All results were expressed as the means ± SE for the independent cultures.

Lenti-miR-195a-5p and Anti-miR-195a-5p:

Constructs for Lenti-miR-195a-5p and Anti-miR-195a-5p were designed, generated, amplified, and verified by DNA sequencing using standard cloning procedures according to the manufacturer’s instructions (GeneCopoeia) and previous studies^25,37,38. Primer sequences are provided in the online Supplement.

shRNAs and Other Constructs:

The lentivirus constructs encoding shTNF or shNKCC2A were designed and constructed using a short hairpin (sh)RNA-expressing construct targeting exon 4 of murine TNF (U6-TNF-ex4) or exon 4 of murine NKCC2A (U6-N2A-ex4), respectively. The U6-shRNA (U6) was used as a negative control for the experiments. pLKO.1, psPAX2 and pMD2.G plasmids were purchased from Addgene (MIT, Cambridge, MA). All constructs were generated using standard cloning procedures and verified by restriction enzyme analysis and DNA sequencing as previously described^32,35,39. The details of cloning procedures are provided in the online Supplement.

Lentivirus Preparation and Administration in vivo:

Generation of lentiviral supernatants was performed using psPAX2, pMD2.G (Addgene), and pLKO.1 or psiLV plasmids as previously described³⁹. Purified lentivirus administration in vivo was via injection into both kidneys using established procedures; approximately 50 μL filter-purified lentivirus including U6, U6-TNF-ex4, U6-N2A-ex4, Lenti-miR-195a-5p, Anti-miR-195a-5p, as well as murine recombinant TNF or vehicle control were used in various experiments³⁵.

Isolation of total RNA and small-RNA fractions:

Total RNA was extracted from tissue or cells using TRIzol (Thermo Fisher) and small RNAs were further purified using the miRVANA kit (Ambion) as previously described^25,40; see online Supplement.

Validation of Selected miRNAs by Luminex Bead Arrays:

The differential expression of 10 selected miRNAs was determined using the quantitative method of single-channel Luminex bead arrays as previously described⁴¹. Briefly, each of the tested miRNAs was selected and 21-25 nt of its complementary sequence was synthesized with 5′-amino-modified oligonucleotides with a 6-carbon linker as the capture probes. Probes were conjugated to carboxylated xMAP beads (Luminex Corporation) in 96-well plates, following the manufacturer's protocol. For each probe set, 3μl of every probe-bead conjugate was mixed into 1 ml of 1.5 × TMAC Hybridization Buffer (4.5 M tetramethylammonium chloride, 0.15% sarkosyl, 75 mM Tris-HCl pH 8.0, 6 mM EDTA). Small RNAs (targets) were adaptor-ligated sequentially on the 3′-end and 5′-end using T4 RNA ligase (Amersham Biosciences). After reverse-transcription using adaptor-specific primers, products were PCR-amplified using a 3′-primer, 5′-TACTGGAATTCGCGGTTA-3′, and 5′ primer, 5′-biotin-CAACGGAATTCCTCACTAAA-3′ or a 5′-Alexa-532-modified primer for the bead detection array according to standard procedures. Hybridization was carried out overnight at 50 °C with 33 μl of the bead mixture and 15 μl of labelled material. Beads were spun down, resuspended in 1 × TMAC containing 10 μg ml⁻¹ streptavidin-phycoerythrin (Molecular Probes) and incubated at 50 °C for 10 min. Samples were hybridized in a 96-well plate, with two mock PCR samples (using water as template) in each plate as a background control. The fluorescence intensity value was detected by the Bio-Plex^™ (Luminex^® 100^™, Bio-Rad Laboratories) system.

Quantitative Real-Time Polymerase Chain Reaction Analysis:

The total RNAs or miRNAs from each sample were used to generate cDNA by using Super Script II reverse transcriptase per manufacturer's specifications (Invitrogen Life Technologies Inc). The amplification of cDNA fragments, TaqMan MicroRNA Assays, and quantitative real-time polymerase chain reaction analysis were performed using PCR or CYBR Green Chemistry using an Applied Biosystems real-time polymerase chain reaction instrument. The transcript quantities were compared by using the relative Ct method, where the amount of target is normalized using the housekeeping gene β-actin. Relative mRNA or miRNA expression was calculated by the 2(^−ΔΔCT) method^25,40.

LSC analysis:

Laser-scanning cytometry (LSC; iCys; CompuCyte, Cambridge, MA) was used to measure expression of pNKCC2 as described⁴⁰. The integrated value of green fluorescence representing pNKCC2 immunofluorescence was measured by LSC^42,43; additional details are provided in the Supplement.

Daily Measurements:

Electrolyte excretion was determined in urine from mice as previously described³⁵. Mice placed in metabolic cages were used for studies to evaluate the effects of renal miR-195a-5p on electrolyte excretion.

Measurements of MAP:

Telemetry transmitters (PA-C10; Data Science International, St Paul, MN) were implanted surgically under aseptic conditions and anesthesia with ketamine-xylazine anesthesia with the catheter inserted into the aorta through right common carotid artery. Mean arterial pressure (MAP) was measured by radiotelemetry as described previously³⁵.

Western Blot Analysis:

Analysis of phospho-NKCC2 expression was performed as described previously³⁴. For details, refer to the online Supplement.

Animal protocol:

C57BL/6 mice (male, B.W. 20-22g) were maintained on a normal-salt diet containing 0.4% NaCl and tap water ad libitum. All animal experiments were performed in accordance with New York Medical College Institutional Animal Care and Use Committee approval and international guidelines for the welfare of animals (animal welfare assurance nos. A3362–01 or A5848–01, Office of Laboratory Animal Welfare, Public Health Service, National Institutes of Health).

Statistical Analysis:

Data were analyzed using the GraphPad Prism software (version 9.3.1). Student’s t test was used for comparisons between 2 groups, and One-way ANOVA (followed by Tukey or Dunnett post hoc test) was used for comparisons among groups. Radiotelemetry data obtained on consecutive days were analyzed by repeated-measures ANOVA. Data were presented as the mean ± SE. For all analysis, P values <0.05 were considered statistically significant.

RESULTS

TNF regulates miRNA Expression in the outer medulla (OM) and mTAL-

miRNA analysis was performed in OM obtained 7 days after an intrarenal injection into both kidneys of murine recombinant TNF (5ng/g body weight) or saline (control). The 10 candidate miRNAs in Figure 1 were selected because their levels were highly expressed in the OM; sequences were based on data from previous studies ^24,44 and miRbase (available online at https://mirbase.org). A detailed description of oligo sequence design and preparation is provided in Methods and Supplemental TableS1 and S2. For ten selected miRNAs, oligonucleotide probes containing the active sequence were designed to analyze miRNA expression using miRNA Luminex bead arrays. The data indicate that TNF up-regulates mmu-miR-195a-5p, and −203 and down-regulates mmu-miR-30c and −100 in the OM (Fig.1A). We also used quantitative real-time RT-PCR to compare the relative abundance of the ten selected miRNA 7days after injection of TNF into both kidneys. Importantly, the differences of miRNA expression were confirmed when the differential expression of miRNAs was validated by two independent methods for miRNA quantitation: Luminex bead arrays (Fig.1A) and real-time qRT-PCR (Fig.S1). The remainder of the study was directed towards determining if TNF-inducible miR-195a-5p expression regulates NKCC2A since the target prediction databases TargetScan and miRDB revealed a potential binding site for miR-195a-5p, but not miR-203, within the 3’-untranslated region (3’UTR) of NKCC2A (Fig.1B). Accordingly, qRT-PCR analysis of mTAL tubules microdissected from the OM of mice given an intrarenal injection of TNF for 7 days exhibited a 4-fold increase in miR-195a-5p abundance (Fig.1C). In contrast, renal-specific silencing of TNF following intrarenal injection of lentivirus U6-TNF-ex4 into both kidneys for 7 days suppressed miR-195a-5p expression (Fig.1D). Moreover, miR-195a-5p and TNF are expressed at higher levels in mice given 1% NaCl in the drinking water for 7 days compared with mice receiving tap water (Fig.1 E&F). Collectively, the data suggest that TNF and high salt intake increase miR-195a-5p in the mTAL.

Figure 1: Effects of TNF on miRNA expression in the OM and mTAL tubules.

(A) miRNA analysis was performed using Luminex Bead Arrays in renal outer medulla (OM) 7 days after a single intrarenal injection into both kidneys of murine recombinant TNF (5ng/g body weight) or saline (control). The 10 candidate miRNAs were selected because the levels of these miRNAs were highly expressed in OM. (B) Schematic diagram showing a putative miR-195a binding site within the 3’-untranslated region (3’UTR) of NKCC2A; coding sequence (CD), and microRNA response element (MRE). (C) miR-195a-5p levels in freshly isolated mTAL tubules were analyzed by qRT-PCR after administration of either murine recombinant TNF or (D) lentivirus U6-TNF-ex4 for renal-specific lentivirus silencing of TNF by intrarenal injection into both kidneys for 7 days. (E) TNF mRNA and (F) miR-195a-5p expression were analyzed after mice were given normal tap water or 1% NaCl in the drinking water for 7 days. Data are shown as mean ± SE; n=6.

NKCC2A Is a Direct Target of miR-195a-5p-

The molecular mechanisms by which TNF modulates the effects of high salt conditions on NKCC2A isoform expression are unknown. Accordingly, a luciferase reporter analysis was performed to evaluate the interaction between miR-195a-5p and the NKCC2A 3′-UTR region. The corresponding vector construct including position 624-632 in the 3’-UTR of NKCC2A was designed and linked to a reporter gene. The 8-base sequence in the mouse 3′-UTR that is complementary to the region of miR-195a-5p is present in the 3′-UTR reporter (Fig.1B). The data indicate that overexpression of miR-195a-5p in HEK293 cells using Lenti-miR-195a-5p significantly reduces luciferase activity compared with a control scrambled miRNA mimic (Fig.2A). In contrast, the Lenti-miR-195a-5p mimic did not reduce luciferase activity in cells transfected with a mutated NKCC2A-3’UTR construct (Fig.2B). These data are supported by experiments in which the NKCC2A-3’UTR or Mut-NKCC2A-3’UTR constructs were co-transfected with Anti-miR-195a-5p into HEK293 cells. The data indicate that suppression of miR-195a-5p significantly increases luciferase activity for the wild type NKCC2A 3′-UTR reporter (Fig.S2 A), an effect that was not observed in cells expressing mutated NKCC2A 3′-UTR (Fig.S2 B).

Figure 2: miR-195a-5p interacts with target 3’-UTR NKCC2A and inhibits NKCC2A mRNA and pNKCC2 expression.

(A) 3′-UTR luciferase reporter analysis was performed to directly evaluate the interaction between miR-195a-5p and the NKCC2A 3′-UTR. The NKCC2A-3’UTR construct, including position 4129-4136 according to the binding site, was co-transfected with miR-195a-5p or scrambled control into HEK293 cells. (B) The corresponding mutant sequence (Mut-NKCC2A-3’UTR construct) with a single point mutation in the miR-195 binding site was designed and co-transfected with miR-195a-5p or scrambled control into HEK293 cells. (C) NKCC2A mRNA was analyzed by qRT-PCR and (D) expression of pNKCC2 was measured by LSC in primary mTAL cells transfected with scrambled control of Lenti-miR-195a-5p to overexpress miR-195a-5p; then mTAL cells were exposed for 2 h to 400 mosmol/kg H₂O medium (HS) after osmolality was increased by adding NaCl. Data are shown as mean ± SE; n=8.

We previously showed that NKCC2A mRNA increases in mTAL cells exposed to high salt conditions, however the molecular mechanisms that regulate NKCC2A are not well understood ^32,36. Overexpression of miR-195a-5p inhibited NKCC2A mRNA accumulation in mTAL cells that were challenged with hypertonic saline (400 mosmol/kg H₂O) for 2h (Fig.2 C). Laser-scanning cytometry detected a 56% decrease in pNKCC2 expression in mTAL cells overexpressing miR-195a-5p (Fig. 2D). Complementary experiments were then performed in which miR-195a-5p was blocked with Anti-miR-195a-5p. The data indicate that Anti-miR-195a-5p increased NKCC2A mRNA expression in mTAL cells exposed to 400 mosmol/kg H₂O medium (Fig. S3). Laser-scanning cytometry detected a 1.5-fold increase in pNKCC2 protein expression in mTAL cells transfected with Anti-miR-195a-5p in the cells exposed to 400 mosmol/kg H₂O medium (Fig. S4). Taken together, these results indicate that miR-195a-5p expression in the mTAL increases in response to elevated NaCl conditions, inhibits NKCC2A mRNA accumulation, and contributes to a concomitant decrease in pNKCC2 expression.

Expression of miR-195a-5p is TNF-dependent and inhibits NKCC2A mRNA-

The role of TNF in the NaCl-induced increase in miR-195a-5p expression was evaluated by qRT-PCR in mTAL cells exposed to 400 mosmol/kg H₂O medium (HS). Levels of miR-195a-5p decreased in response to HS in mTAL cells that were transfected with lentivirus U6-TNF-ex4 for 24h to inhibit TNF production (Fig. 3A). As the bioinformatic and luciferase reporter data suggest that miR-195a-5p directly regulates NKCC2A mRNA, the relative abundance of mRNA levels for NKCC2A in mTAL cells exposed to 400 mosmol/kg H₂O medium was determined after mTAL cells were transfected with a mixture of lentivirus U6-TNF-ex4 and Lenti-miR-195a-5p for 24h. The data show that overexpression of miR-195a-5p suppressed increases of NKCC2A mRNA induced by silencing TNF in mTAL cells (Fig. 3B). Treatment of mTAL cells with TNF increases miR-195a-5p expression (Fig.3C), and transfection of Anti-miR-195a-5p into mTAL cells reversed the inhibitory effect of TNF on NKCC2A mRNA accumulation (Fig.3D). Collectively, these findings support in vivo data showing that TNF inhibits NKCC2A expression in response to high NaCl intake via a miR-195a-5p-dependent mechanism and confirms that miR-195a-5p targets the 3′-UTR of NKCC2A mRNA.

Figure 3: miR-195a-5p mediates inhibition of NKCC2A mRNA by TNF in mTAL cells.

(A) miR-195a-5p expression and (B) NKCC2A mRNA abundance in primary mTAL cells were measured after cells were transfected with lentivirus construct U6-TNF-ex4 or the mixture of U6-TNF-ex4 and Lenti-miR-195 for 24 h, then mTAL cells were exposed to 400 mosmol/kg H₂O medium (HS) after osmolality was increased by adding NaCl for 2h. (C) qRT-PCR analysis of miR-195 expression and (D) NKCC2A mRNA abundance in mTAL cells exposed to 400 mosmol/kg H₂O medium (HS) for 2 h were performed after cells were incubated with TNF or the mixture of TNF and anti-miR-195 for 24 h. Data are shown as mean ± SE; n=8.

Effects of renal miR-195a-5p on electrolyte excretion and blood pressure regulation-

We previously demonstrated that silencing TNF in the kidney modulates changes in NKCC2A expression and NKCC2 activity thereby attenuating the diuretic and natriuretic responses to NaCl ³⁵. Presently, we determined the effects of renal miR-195a-5p on TNF-mediated inhibition of NaCl excretion in mice given 1% NaCl in the drinking water. The data show that silencing renal TNF using U6-TNF-ex 4 decreases urine volume, sodium, and chloride excretion (Fig.4 A-C)³⁵. Notably, intrarenal injection of Lenti-miR-195a-5p reverses the effects on urine volume, sodium, and chloride excretion observed in mice in which TNF was silenced (Fig.4 A-C). These data suggest that renal miR-195a-5p enhances the diuretic and natriuretic response to 1% NaCl intake by inhibiting NKCC2A expression and NKCC2 activity.

Figure 4: Effects of miR-195a-5p on electrolyte excretion and blood pressure.

(A-C) mice (n=12) were switched from tap water to 1% NaCl in the drinking water for 7 days after the injection of U6, U6-TNF-ex4, or a mixture of U6-TNF-ex4 and Lenti-miR-195a-5p for 3 days, and urine for electrolyte excretion analysis was collected for 24 h. (D) Daily and (E) summation of MAP measurements for mice (n=8) implanted with radiotelemetry probes, injected with either a mixture of U6-TNF-ex4 and scramble or a combination of U6-TNF-ex4 and miR-195a-5p into both kidneys for 3 days, and then given 1% NaCl in the drinking water for 7 days. Data are shown as mean ± SE.

The effect of renal-specific overexpression of miR-195a-5p on blood pressure responses to an increase in NaCl intake was determined in mice that received intrarenal injections of either TNF silencing lentivirus (U6-TNF-ex4) and scramble control, or a mixture of U6-TNF-ex4 and Lenti-miR-195a-5p. The data show that MAP measured in mice injected with U6-TNF-ex4 (TNF silencing) and Lenti-miR-195a-5p was markedly reduced compared to mice that received a scrambled miRNA control and U6-TNF-ex4 (Fig.4D). Similarly, MAP averaged over 7 days of 1% NaCl intake was lower in mice that received both U6-TNF-ex4 and lenti-miR-195 compared to mice in which only TNF was silenced (Fig.4E).

Renal miR-195a-5p regulates NKCC2A mRNA and pNKCC2 protein expression in response to high NaCl intake-

Since the preceding data indicate that miR-195a-5p regulates NKCC2A in mTAL cells, we determined whether renal miR-195a-5p also regulates NKCC2A in vivo. Compared with control group, Lenti-miR-195a-5p increased expression of miR-195a-5p approximately 3.5-fold (Fig.5A). Meanwhile, intrarenal administration of Lenti-miR-195a-5p prevented the increase of NKCC2A mRNA (Fig.5B) and pNKCC2 expression (Fig.5C and Fig.S5). Moreover, administration of Anti-miR-195a-5p reversed the inhibition of NKCC2A mRNA by TNF in mice given 1% NaCl in the drinking water for 7 days (Fig.5D). We also determined if renal miR-195a-5p regulates NaCl-dependent changes in blood pressure in the absence or presence of NKCC2A silencing, achieved as previously described³⁵. Mice received murine recombinant TNF and a mixture of either Anti-miR-195a-5p and U6 control (−) or Anti-miR-195a-5p and U6-N2A-ex4 to silence NKCC2A. After 3 days mice were given 1% NaCl in the drinking water for an additional 7 days. The data show that MAP averaged over 7 days of 1% NaCl intake was lower in mice that received the combination of TNF, Anti-miR-195a-5p and U6-N2A-ex4 compared to mice with the mixture of TNF, Anti-miR-195 and U6 control (Fig.5E). These results indicate that NKCC2A is part of the mechanism by which renal miR-195a-5p regulates NaCl-dependent changes in blood pressure.

Figure 5: miR-195a-5p inhibits NKCC2A mRNA and pNKCC2 protein expression in vivo.

(A) The expression of miR-195a-5p was detected by qRT-PCR when mice were switched from tap water to 1% NaCl in the drinking water for 7 days after the injection of Lenti-miR-195a-5p or scramble (control group) for 3 days (n=8). (B) The relative abundance of NKCC2A mRNA (n=6) and (C) pNKCC2 protein expression (n=4) were determined in the OM from mice injected Lenti-miR-195a-5p or control for 3 days and then given 1% NaCl in the drinking water for 7 days. (D) NKCC2A mRNA abundance (n=6) was analyzed when mice injected with scramble or a mixture of TNF and scramble as well as a combination of TNF and Anti-miR-195a-5p into both kidneys for 3 days, and then given 1% NaCl in the drinking water for 7 days. (E) Mice (n=8) given a combination of TNF and Anti-miR-195a-5p also received an intrarenal injection of either a U6 control (−) or U6-N2A-ex4 to silence NKCC2A for 3 days, then given 1% NaCl in the drinking water for an additional 7 days. Data are shown as mean ± SE.

TNF inhibits NKCC2A mRNA and pNKCC2 expression via miR-195a-5p in vivo-

The contribution of miR-195a-5p to the inhibitory effects of TNF on NKCC2A mRNA and pNKCC2 expression was determined in mice that received intrarenal injections of either TNF silencing lentivirus (U6-TNF-ex4) or control U6. The data show that miR-195a-5p expression decreases when mice receiving 1% NaCl in the drinking water were given intrarenal injections of TNF silencing lentivirus (U6-TNF-ex4) for 7 days compared with control (Fig.6A). A mixture of U6-TNF-ex4 and Lenti-miR-195a-5p or scrambled miR-195a-5p was injected to evaluate NKCC2A mRNA in mice ingesting 1% NaCl in the drinking water for 7 days. Compared with the scramble control, Lenti-miR-195a-5p significantly downregulated NKCC2A mRNA that was increased in response to TNF silencing (Fig.6B). Similarly, western blot analysis showed that pNKCC2 expression was lower in mice that received a mixture of U6-TNF-ex4 and Lenti-miR-195a-5p vs the scramble group (Fig.6C and Fig.S6). Collectively, these results and those using primary mTAL cells confirm that miR-195a-5p is part of the mechanism by which renal TNF inhibits NKCC2A mRNA and pNKCC2 protein expression.

Figure 6: In vivo inhibition of NKCC2A by TNF is miR-195a-5p-dependent.

(A) The relative abundance of miR-195a-5p expression was determined in OM from mice that received intrarenal injections of either TNF silencing lentivirus (U6-TNF-ex4) or control U6 for 3 days, and then given 1% NaCl in the drinking water for 7 days (n=8). (B) NKCC2A mRNA abundance (n=8) and (C) pNKCC2 protein expression (n=6) were analyzed when mice injected with scramble (control group) or a mixture of U6-TNF-ex4 and scramble as well as a combination of U6-TNF-ex4 and Lenti-miR-195a-5p into both kidneys for 3 days, and then given 1% NaCl in the drinking water for 7 days. Data are shown as mean ± SE.

DISCUSSION

This study identifies miR-195a-5p as a salt- and TNF-inducible miRNA that inhibits NaCl-mediated increases in blood pressure, at least in part, by inhibiting NKCC2A along the TAL segment of the nephron. A tissue type-specific analysis was used to confirm miR-195a-5p expression in the renal OM, freshly isolated mTAL tubules, and primary cultures of polarized mTAL cells, whereby in vivo and in vitro lentivirus targeting approaches demonstrated that TNF production in the TAL induced renal miR-195a-5p in response to elevated NaCl conditions. Since expression of NKCC2A is limited to the TAL segment of the kidney, the present findings reveal a mechanism in the kidney by which TNF increases an miRNA that interacts within the 3’-UTR of NKCC2A to modulate increases of NKCC2A induced by elevated NaCl, thereby facilitating NaCl excretion and attenuating increases in blood pressure. Overall, this study suggests that TNF-induced miR-195a-5p expression along the TAL regulates salt and water homeostasis in response to changes in NaCl intake as part of an intratubular TNF system that contributes to blood pressure regulation via suppression of NKCC2A.

miRNAs contribute to many physiological and pathological processes and control diverse functions in the kidney, liver, and cardiovascular system ^45-47. However, specific targets for most miRNAs remain unclear. In the present study, the mechanism by which miR-195a-5p exerts its effect in the TAL was explored. Since the mechanisms by which TNF regulates NKCC2 have not yet been defined, we addressed the role of miRNA on the accumulation of NKCC2A mRNA as miRNA have been shown to play important roles in hypertension, chronic kidney disease, and cardiorenal syndrome^48,49. Potential target genes of miR-195a-5p in the kidney were evaluated using the multiple target prediction databases TargetScan and miRDB and identified NKCC2 as a candidate gene. Subsequently, in vivo and in vitro experiments showed that TNF increases miR-195a-5p expression in the TAL, while lentivirus silencing of TNF reduces its expression. Furthermore, we demonstrated that miR-195a-5p directly regulates NKCC2A expression by performing 3′-UTR luciferase reporter experiments that utilized both knockdown and overexpression approaches as well as wild type and mutated 3’-UTR constructs. Accordingly, increases in luciferase activity in HEK293 cells transfected with Anti-miR-195a-5p supports data in primary mTAL cells suggesting that the expression of NKCC2A is limited by a negative feedback mechanism involving miR-195a-5p. The data suggest that an important function of miR-195a-5p relates to targeting NKCC2A along the TAL and support a previous study using TNF knockout mice in which TNF was proposed as an endogenous inhibitor of NKCC2³⁴.

Recent studies have addressed the diverse effects of miRNAs on renal function via regulation of their target genes in a manner that either facilitates or protects against the development of cardiovascular and renal diseases. For instance, miRNA-192-5p was shown to regulate renal transporters and protect against increases in blood pressure via targeting ATP1B1 (β1 subunit of Na⁺/K⁺-ATPase) in the kidney ⁴⁸. Other miRNA such as miRNA-214–3p play a role in the development of hypertension by targeting eNOS in the kidney ²³ while miR-379 may mediate Klotho deficiency-associated cardiomyocyte apoptosis by targeting Smurf1, which is required for miR379-induced apoptotic cell death ⁵⁰. It was reported that up-regulation of miR-195 contributes to cardiac hypertrophy-induced arrhythmia by targeting calcium and potassium channels⁵¹. In contrast, miR-195 improved symptoms of diabetic nephropathy, possibly by inhibiting proliferation and promoting apoptosis of macrophages in association with decreased expression of TLR4 and the NF-κB pathway in macrophages⁵². In our study, miR-195a-5p in renal epithelial cells reversed the inhibitory effects of TNF silencing on urine volume, sodium, and chloride excretion by targeting NKCC2A. Thus, a given miRNA may be involved in the regulation of genes that can elicit either a net protective or detrimental effect related to cardiovascular and renal physiology as well as diverse pathophysiological processes. Similarly, miR-204 attenuates pulmonary hypertension and damage to the kidney caused by hypertension or diabetes, yet it promotes hypertension and endothelial dysfunction ⁵³. Collectively, these findings illustrate the importance of understanding the cell-type specific mechanisms by which miRNAs contribute to renal physiology and blood pressure regulation via alterations of target mRNA ^22,54,55.

Previous studies showed that differential splicing of NKCC2 pre-mRNA results in the formation of 3 functionally distinct isoforms, NKCC2A, NKCC2B, and NKCC2F that are important for the fine-tuning of TAL function^14,15,56. While little is known regarding the molecular mechanisms that regulate NKCC2 isoform expression, changes in salt conditions cause a shift in isoform expression in vivo and in vitro, possibly by altering NKCC2 pre-mRNA splicing as a means of adaptation in TAL cells ^33,57. While the high salt conditions used in the present study favor the induction of the NKCC2A isoform ³² they also induce expression of both TNF and miR-195a-5p, which modulates the effects of high salt conditions on isoform expression. In the absence of either of these molecules, the expression of NKCC2A increases. Thus, miR-195a-5p overexpression and Anti-miR-195a-5p approaches in vitro and in vivo both yield data showing that miR-195a-5p inhibits NKCC2A mRNA, pNKCC2 protein expression, increases in NaCl reabsorption, and blood pressure. While the molecular mechanisms by which miR-195a-5p regulates NKCC2A will require additional studies, the regulatory effects of miRNAs go beyond their effects in the 3’-UTR as they have been shown to function at multiple hierarchical levels of gene regulatory networks, from targeting hundreds of effector genes to controlling the levels of global regulators of transcription and alternative pre-mRNA splicing ¹⁸. Thus, miR-195a-5p may function at several levels or in combination with other TNF-inducible miRNA to achieve the downregulation of NKCC2A mRNA in the mTAL. Overall, these findings suggest that miRNA-195a-5p is part of the mechanism by which TNF limits NKCC2A-dependent increases in sodium retention and blood pressure following an increase in salt intake.

TNF exhibits pro-hypertensive and pro-inflammatory effects in the kidney and regulates several transport pathways along the nephron in a context-dependent manner ⁵⁸. TNF also is part of a regulatory mechanism that limits increases in blood pressure in response to increases in salt intake. For instance, we previously showed that high salt intake increases TNF production in the kidney and renal-specific silencing of TNF unmasks salt-dependent increases in blood pressure via a mechanism involving NKCC2A ^32,35. The present study reveals that intrarenal TNF increases miR-195a-5p expression in the outer medulla, mTAL tubules, and primary cultures of mTAL cells. In each model the inhibitory effects of TNF on NKCC2A mRNA accumulation were dependent on miR-195a-5p. To our knowledge no miRNA has yet been described to play a role in the regulation of NKCC2. Indeed, the NaCl-mediated increase in miR-195a-5p was TNF-dependent and essential to the ability of this cytokine to limit NKCC2A expression under high salt conditions. TNF has been shown to increase miRNA-133a in the proximal tubule and alter the expression of additional miRNAs in extrarenal tissues²⁵. For instance, TNF increases the expression of miR-155 in osteosarcoma cells, regulates miRNAs in endothelial cells as part of a regulatory network, induces miR-98 in fibroblast-like synoviocytes, and inhibits bone marrow stem cell proliferation/differentiation by reducing expression of miR-34a ^59-62. The signaling pathways and TNF receptor subtypes responsible for increases in the expression of various miRNAs is likely to involve cell type-specific molecular mechanisms and warrants further investigation.

The roles of miRNAs in different models of hypertension and tissue injury have been reported ^2,46,63, however, a role for miR-195a-5p as a regulator of blood pressure homeostasis has not been described. The present data show that renal silencing of TNF increased MAP when mice ingest high salt, an effect that was abolished by concomitant intrarenal administration of miR-195a-5p. Moreover, intrarenal silencing of TNF increased both NKCC2A mRNA and pNKCC2 expression, effects that were prevented when a miR-195a-5p overexpression lentivirus was co-administered. This suggests that miR-195a-5p plays an important role in blood pressure regulation by mediating the effects of TNF on NKCC2, thus maintaining sodium homeostasis ^32,35. Previous findings revealed that prominent differences between the NKCC2 isoforms include the designation of NKCC2A as a high-capacity transporter whose expression is induced by high NaCl concentration in several cell models^36,57,64. As renal-derived TNF is an inhibitor of NKCC2A mRNA accumulation it is important to note that the functional changes observed in this, and a previous study are consistent with the elegant studies performed using NKCC2A knockout mice ^34,65. Collectively, the present data suggest that increases of NKCC2A, pNKCC2, and MAP induced by NaCl, after silencing of TNF in the kidney, are dependent on a decrease of miR-195a-5p expression; effects that are reversed by overexpression of miR-195a-5p. The present findings are the first to suggest that induction of NKCC2A expression by high salt conditions can be modulated by a miRNA-dependent mechanism that limits the increase in blood pressure when TNF is silenced in the kidney.

Perspectives-.

-Blood pressure homeostasis in response to high NaCl intake is regulated by a miRNA-195a-5p dependent mechanism that regulates NKCC2A mRNA.

-The present findings suggest that TNF orchestrates an epigenetic mechanism along the TAL that modulates NKCC2A expression induced by high salt intake via a mechanism involving miRNA-195a-5p.

Pathophysiological Novelty and Relevance-.

What is new?

-This is the first study to show that NKCC2A mRNA accumulation is modulated by a miRNA-dependent mechanism.

-The data are the first to demonstrate that miRNA-195a-5p expression in the kidney is upregulated by high salt intake in a TNF-dependent manner.

What is Relevant?

-The present study is the first to demonstrate a role for miR-195a-5p in blood pressure regulation via electrolyte excretion involving NKCC2A.

Clinical/Pathophysiological Implications?

-A more comprehensive understanding of renal mechanisms involving miRNA and TNF that regulate sodium transporters may provide the rationale for developing novel therapies to treat hypertension.

Nonstandard Abbreviations and Acronyms:

TAL

the thick ascending limb of Henle’s loop

mTAL

medullary thick ascending limb

OM

outer medulla

NKCC2

bumetanide-sensitive Na⁺-K⁺-2Cl⁻ cotransporter

NKCC2A

bumetanide-sensitive Na⁺-K⁺-2Cl⁻ cotransporter isoform A

pNKCC2

phosphorylated Na⁺-K⁺-2Cl⁻cotransporter

miRNAs

microRNAs

TNF

tumor necrosis factor-alpha

AGT

angiotensinogen

PT

proximal tubule

3’UTR

3’-untranslated region

NKCC2A-3′-UTR

pMIR-NKCC2A- luciferase reporter vector

mut-NKCC2A-3′-UTR

pMIR-NKCC2A luciferase reporter vector

U6-TNF-ex4

a short hairpin (sh)RNA-expressing construct targeting exon 4 of murine TNF

U6-N2A-ex4

a short hairpin (sh)RNA-expressing construct targeting exon 4 of murine NKCC2A

LSC

Laser-scanning cytometry

ATP1B1

β1 subunit of Na⁺/K⁺-ATPase