Supporting InformationKakoki et al. 10.1073/pnas.1302641110SI Materials and MethodsBlood Pressure and Heart Rate Measurement. We measured bloodpressure and heart rate with the tail-cuff method (1) or the directaortic measurement with telemetry devices. For continuous bloodpressure recording, the PA-C20 telemetric device (Data Sciences)was implanted via the left carotid artery into the aorta as de-scribed elsewhere(2).

Quantitative Reverse Transcription-PCR. Total RNA was extractedfrom different tissues and the mRNAs were assayed by quanti-tative reverse transcription-PCR as previously described (3). Theprimers and the probes used to measure the mRNAs are shownin Table S5.

Measurement of Biological Parameters. Plasma glucose levels weredetermined with the glucose oxidase method (Wako Chemical).Plasma insulin levels were determined with ELISA (Crystal Chem).Plasma urea nitrogen concentrations were determined with theVitros 250 Chemistry system (Ortho-Clinical Diagnostics). Plasmatotal cholesterol (Wako Chemical) and triglyceride (Stanbio Lab-oratory) were measured with enzymatic colorimetric methods.Plasma creatinine levels were studied with liquid chromatographytandemmassspectrometry(LC-MS/MS)aspreviouslydescribed(4).Plasma and urine electrolytes were determined with the Vitros 250Chemistry system (Ortho-Clinical Diagnostics). Active renin con-centration was measured by FRET-based assay kits (SensoLyte 520Mouse Renin Assay kit; AnaSpec). Plasma Ang II levels werestudied with ELISA (Human/Rat Ang II ELISA; BioVendor).Plasma adrenocorticotropic hormone (ACTH) levels were studiedwith a chemiluminescence immunoassay Mouse/Rat ACTH UltraSensitive lumELISA kit (Calbiotech). Plasma levels of vasopressin,corticosterone, metanephrine, normetanephrine, triiodothyronine,thyroxine, and TGFβ1 were studied with ELISA (Arginine Vaso-pressin EIA, Corticosterone EIA; Cayman) (Metcombi PlasmaELISA, IBL; Mouse/Rat T3 and T4 EIA; Calbiotech) (QuantikineMouse/Rat/Porcine/Canine TGFβ1 Immunoassay; R&D Systems).Plasma aldosterone concentrations were determined in HormoneAssay and Analytical Services Core, Vanderbilt Diabetes Researchand Training Center. Plasma nitrite/nitrate levels were studied withthe Griess method (Nitrite/Nitrate Colorimetric Assay; Cayman).Metabolic balance studies were performed using metabolic cages(Solo Mouse Metabolic Cage; Tecniplast). Rectal temperature wasrecorded with a TH-8 thermometer (Physitemp Instruments).

Histology.After the inferior vena cavawas cut, the left ventriclewaspunctured by a 23-gauge needle and perfused with PBS for 3 minand with 4% (wt/vol) paraformaldehyde for 5 min. Thereafter thetissues were dissected out and put in 4% paraformaldehyde for atleast 3 d. These were then paraffin embedded and sectioned. Thestained sections were prepared by Center for GastrointestinalBiology and Diseases Histology Core and imaged using a NikonIX700 microscope.

Measurement of Plasma Volume.Evans blue (0.3mg/mL, 50 μL) wasinjected via the tail vein. Fifteen, 30, and 60min after the injection,65 μL of blood was withdrawn from the retroorbital sinus usingheparinized micro-hematocrit capillary tubes (Fisher Scientific).Plasma was separated by centrifugation of the blood at 5,000 × gfor 5 min. Absorbance was read at 620 nm and plasma volume wasdetermined by using the dilution factors of Evans blue.

Measurement of Glomerular Filtration Rate. Glomerular filtrationrate (GFR) was studied by the single bolus injection of FITC-inulinas described elsewhere (5) with slightmodification. Briefly, dialyzedFITC-inulin (3.74 μL/g body wt) was injected via the tail vein.Approximately 20 μL of blood was withdrawn from the retroorbitalsinus at 3, 7, 10, 15, 35, 55, and 75 min after the injection, usingheparinized micro-hematocrit capillary tubes (Fisher Scientific).Plasmawas separated by centrifugation of the blood at 5,000× g for5 min. Fluorescence was determined using a POLARstar fluo-rometer (BMG Labtech) with 485-nm excitation and 538-nm emis-sion. Plasma fluorescence data were fit to a two-phase exponentialdecay curve using nonlinear regression (GraphPad Prism).

Metabolic Studies.Mice were placed individually in chambers of anOxymax system (Columbus Instruments) for 96 h, and readingswere taken 24 h after acclimation. Measures of food and waterintake, calorie production, and physical activity were obtained.Data were collected via Oxymax Windows software version 2.3.Measurement and settle timeswere set at 60 and 120 s, respectively.

Immunoprecipitation/Western Blot. The tissue was homogenated inimmnoprecipitation buffer (50 mM Tris·HCl, pH 7.4, 150 mMNaCl, 50 mM β-glycerophosphate, 30 mM NaF, 2 mM EDTA,2mMEGTA, 30mMNa4P2O7, 2mMNa3VO4, 1%Triton X-100)and protease inhibitors (Complete; Roche). The homogenate wascentrifuged at 1,000 × g to pellet the nucleus and debris. Equalamounts of protein were incubated with an appropriate antiserumand protein G agarose for 16 h at 4 °C. The proteins (100 μg) werefractionated by SDS/PAGE (Criterion; Bio-Rad), transferred toPVDF membranes, incubated with an appropriate antibody, anddetected by chemiluminescence (Supersignal West Pico, Thermo).Western blot was performed with a phosphorylated (Ser465/467)Smad2 antibody (rabbit mAb; Cell Signaling), a phosphorylated(Ser1177) eNOS antibody (rabbit mAb; Cell Signaling), aneNOS antibody (rabbit mAb; Cell Signaling), a phosphorylated(Ser473) Akt antibody (rabbit mAb; Cell Signaling), an Akt an-tibody (rabbit mAb; Cell Signaling), or an Na+-K+ ATPase (NK)α-subunit antibody (mouse mAb; Cell Signaling) as previously de-scribed (6). Thereafter, membranes were stripped and incubatedwith a GAPDH (14C10) antibody (rabbit mAb; Cell Signaling) asloading control. NK was immunoprecipitated from each sample(3 mg protein) using a NK antibody (5 μg). The immunoisolatedmaterial was separated on SDS/PAGE. Western blot was per-formed using a specific antibody raised against the phosphorserineresidue (Sigma-Aldrich). The signals were quantified using ImageJsoftware (http://rsb.info.nih.gov/ij/).

Determination of NK Activity.NK activity was measured as ouabain(200 μM)-sensitive generation rate of inorganic phosphate (Pi)in the presence of abundant ATP using kidney homogenate in50 mM Tris·HCl (pH 7.4) using EnzChek Phosphate Assay kit(Invitrogen). Briefly, 2-amino-6-mercapto-7-methylpurine (0.2 mM)is converted in the presence of Pi into ribose 1-phosphate and2-amino-6-mercapto-7-methyl-purine (absorbance at 360 nm) bypurine nucleoside phosphorylase (1 unit/mL) in a buffer con-taining 3 mM Na2ATP, 100 mM NaCl, 20 mM KCl, 6 mMMgCl2, 0.1 mM EDTA.

Determination of Epithelial Sodium Channel Activity. The epithelialsodium channel (ENaC) activity in the WT and L/L mice wasassayed in freshly isolated split-opened cortical collecting ductswith patch-clamp method (7). Briefly, mice were killed by CO2administration followed by cervical dislocation; the kidneys

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were removed immediately. Kidneys were cut into thin slices (<1mm) with slices placed into ice-cold physiologic saline solutionbuffered with Hepes (pH 7.4). The cortical collecting ducts weremechanically isolated from cortical sections of kidney slices bymicrodissection using watchmaker forceps under a stereomicro-scope. Isolated cortical collecting ducts were attached to cover-glasses coated with poly-L-lysine and placed in a perfusionchamber mounted on an inverted Nikon Eclipse Ti microscopeand perfused with room temperature Hepes-buffered (pH 7.4)saline solution (1.5 mL/min). Cortical collected ducts were splitopened with two sharpened micropipettes, controlled with differ-ent micromanipulators, to gain access to the apical membrane.The tubules were used within 1–2 h of isolation. ENaC activity inprincipal cells was determined in cell-attached patches on theapical membrane made under voltage-clamp conditions (−Vp =−60 mV). Typical bath and pipette solutions were: 150 mm NaCl,5 mm KCl, 1 mm CaCl2, 2 mm MgCl2, 5 mm glucose, and 10 mmHepes (pH 7.4); and 140 mm LiCl, 2 mm MgCl2 and 10 mm

Hepes (pH 7.4), respectively. Gap-free single-channel current datafrom gigaohm seals were acquired (and subsequently analyzed)with an Axopatch 200B (Axon Instruments) patch-clamp amplifierinterfaced via a Digidata 1440 (Axon Instruments) to a PC runningthe pClamp 10.2 suite of software (Axon Instruments). Currentswere low pass filtered at 100 Hz with an eight-pole Bessel filter(Warner Instruments.). ENaC activity was assessed over a span of60–120 s for each experimental condition. Functional ENaC ex-pression on the apical membrane (fN) was determined as theproduct of the number of active channels per patch, N, and theprobability of observing patches with active channels, f = numberof patches with active channels/total number of patches).

Statistical Analysis.Data are expressed asmeans± SE. To comparegroups, we used one- or two-factor ANOVA. Post hoc pairwisecomparisons were performed by Tukey–Kramer honestly signifi-cance differences (HSD) test (JMP 8.0; SAS Institute).

1. Krege JH, Hodgin JB, Hagaman JR, Smithies O (1995) A noninvasive computerized tail-cuff system for measuring blood pressure in mice. Hypertension 25(5):1111–1115.

2. Tsai YS, et al. (2004) Hypertension and abnormal fat distribution but not insulinresistance in mice with P465L PPARgamma. J Clin Invest 114(2):240–249.

3. Kim HS, Lee G, John SW, Maeda N, Smithies O (2002) Molecular phenotyping foranalyzing subtle genetic effects in mice: Application to an angiotensinogen genetitration. Proc Natl Acad Sci USA 99(7):4602–4607.

4. Takahashi N, Boysen G, Li F, Li Y, Swenberg JA (2007) Tandem mass spectrometrymeasurements of creatinine in mouse plasma and urine for determining glomerularfiltration rate. Kidney Int 71(3):266–271.

5. Qi Z, et al. (2004) Serial determination of glomerular filtration rate in conscious miceusing FITC-inulin clearance. Am J Physiol Renal Physiol 286(3):F590–F596.

6. Sjöström M, et al. (2007) SIK1 is part of a cell sodium-sensing network that regulatesactive sodium transport through a calcium-dependent process. Proc Natl Acad Sci USA104(43):16922–16927.

7. Staruschenko A, Pochynyuk O, Vandewalle A, Bugaj V, Stockand JD (2007) Acuteregulation of the epithelial Na+ channel by phosphatidylinositide 3-OH kinasesignaling in native collecting duct principal cells. J Am Soc Nephrol 18(6):1652–1661.

( )

3 4 5 6 7 8 9

1 2 3 PActb PHprt

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hrGFPn cDNA

A.

B.

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0

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200

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400

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ce (

% T

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Fos Tgfb1 bGH

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**

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Fig. S1. In vitro comparison of the effects of 3′-untranslated regions (3′-UTR) on gene expression. (A) Single copy transgene strategy for testing the effects of3′ regions on the expression of a humanized Renilla green fluorescence protein with a nuclear localization signal (hrGFPn) driven by the β-actin promoter(PActb). (A, line A) The target locus, into which the transgene is introduced by homologous recombination, is the hypoxanthine-guanine phosphorybosyltransferase (Hprt) gene of E14TG2a embryonic stem cells; it lacks the promoter and exons 1 and 2 (ΔHprt). (A, line B) The targeting vector into which different3′-UTRs with their GU/U-rich sequence elements (GRE) are introduced. (A, line C) The resulting locus after homologous recombination. The Hprt gene hasa promoter (PHprt) and all nine exons and the locus now contains a single copy transgene, which can be selected in hypoxanthine-aminopterin-thymidinemedia. (B) Green fluorescence of the colonies. Fluorescence with the 3′-UTR and GRE of bGH, Tgfb1, and Fos is shown. (C) Mean fluorescence levels of the singlecells with the 3′-UTR and GRE of bGH, Tgfb1, and Fos from different colonies. **P < 0.01, ‡P < 10−5 vs. Tgfb1.

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L/L L/+ WT H/+ H/H

9.8 kb

7.1 kb7.6 kb

Fig. S2. Southern blots of NcoI-digested genomic DNA from mice of the five genotypes, Tgfb1L/L (L/L), Tgfb1L/+ (L/+), WT, Tgfb1H/+ (H/+), and Tgfb1H/H (H/H)using a probe (P in Fig. 1A) from a region 5′ to the targeting construct.

WTL/L L/+

H/+ H/H

Fig. S3. Hematoxylin and eosin staining of the adrenal gland. The normal structure of the adrenal cortex is retained in the five genotypes for Tgfb1. (Scalebar, 100 μm.)

0

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ca b

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Fig. S4. Excretion of electrolytes in the feces of 12-wk-old WT and L/L male mice. (A) Fecal sodium excretion. (B) Fecal potassium excretion. (C) Fecal chlorideexcretion. No significant differences are detected between WT and L/L.

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*

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0

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lyte

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Eq/m

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Fig. S5. Electrolytes in the dorsal hair of 12-wk-old WT and L/L male mice. *P < 0.05, **P < 0.01 vs. WT.

0

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XT+YT XA+YA Z0

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Fig. S6. Increased locomotive activity and heat generation in L/L mice. **P < 0.01, †P < 0.001 vs. WT. (A) Locomotive activity of 12-wk-old male mice. XT + YT,total number of x- and y-axis IR-beam breaks; XA + YA, number of ambulatory x- and y-axis IR-beam breaks; Z, number of vertical motions. (B) Body coretemperature of 12-wk-old male mice. (C) Heat generation of 12-wk-old male mice.

WTL/L L/+

H/+ H/H

Fig. S7. Periodic acid Schiff (PAS) with hematoxylin staining of the renal cortex. The histology of the kidneys of the five genotypes for Tgfb1 is comparable.(Scale bar, 100 μm.)

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0

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Fig. S8. Glomerular filtration rate (GFR). The GFRs of mice of the five genotypes are not significantly different.

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ls (%

WT)

(n = 6) (n = 8) (n = 6)

Fig. S9. Renal mRNA levels for water and sodium transporters and their related genes in WT, L/+, and L/L male mice: Sodium-hydrogen antiporter 3 (Slc9a3);Sodium/glucose cotransporter (Slc5a1); Electrogenic sodium bicarbonate cotransporter (Slc4a4); Sodium/phosphate cotransporter (Slc34a1); Na-K-Cl co-transporter 2 (Nkcc2); Sodium-chloride symporter (Slc12a3); Epithelial sodium channel subunit alpha (Scnna); Epithelial sodium channel subunit beta (Scnnb);Epithelial sodium channel subunit gamma (Scnng); Sodium/potassium-transporting ATPase subunit alpha 1 (Atp1a1); Sodium/potassium-transporting ATPasesubunit beta 1 (Atp1b1); Sodium/potassium-transporting ATPase gamma chain (Fxyd2); Aquaporin 2 (Aqp2); WNK lysine deficient protein kinase 1 (Wnk1); andWNK lysine deficient protein kinase 4 (Wnk4).

Table S1. Baseline characteristics of WT, L/+, and L/L male mice at age 12 wk

Characteristic WT L/+ L/L

No. of mice 7 8 10Body weight (BW), g 29.2 ± 1.2 29.7 ± 0.6 20.2 ± 1.2††

Kidney weight (right), mg 155 ± 8 179 ± 8 130 ± 7†

Kidney weight/BW, ‰ 5.34 ± 0.19 6.02 ± 0.20* 6.51 ± 0.35*Heart weight, mg 128 ± 8 123 ± 9 87 ± 5**Heart weight/BW, ‰ 4.40 ± 0.23 4.13 ± 0.26 4.39 ± 0.16Adrenal weight (right), mg 2.72 ± 0.22 2.84 ± 0.22 2.99 ± 0.27Adrenal weight/BW, ‰ 0.082 ± 0.007 0.094 ± 0.008 0.155 ± 0.014*Hematocrit, % 49.5 ± 0.7 49.3 ± 0.6 47.4 ± 1.7Plasma glucose, mmol/L 10.66 ± 0.78 11.78 ± 0.92 10.18 ± 0.36Plasma insulin, pmol/L 67.5 ± 8.3 98.5 ± 11.3 83.9 ± 6.4Plasma urea nitrogen, mmol/L 8.52 ± 0.46 9.06 ± 0.42 8.46 ± 0.58Plasma creatinine, μmol/L 7.05 ± 0.35 7.21 ± 0.58 7.53 ± 0.57Plasma sodium, mEq/L 151.4 ± 1.3 150.9 ± 1.0 153.1 ± 0.9Plasma potassium, mEq/L 5.7 ± 0.1 5.5 ± 0.1 4.9 ± 0.1††

Plasma chloride, mEq/L 127.1 ± 1.9 125.6 ± 2.1 122.9 ± 1.2Plasma osmolarity, mOsm 319.4 ± 1.8 319.1 ± 3.0 316.9 ± 2.7

Body length, a distance between tip of nose and base of tail. *P < 0.05, **P < 0.01, †P < 0.001, ††P < 10−4 vs. WT.

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Table S2. Baseline characteristics of H/+ and H/H male mice atage 12 wk

Characteristic H/+ H/H

Number of mice 7 9Body weight (BW), g 29.0 ± 1.7 30.3 ± 0.7Kidney weight (right), mg 155 ± 8 179 ± 8Kidney weight/BW, ‰ 5.34 ± 0.19 6.02 ± 0.20*Heart weight, mg 128 ± 8 123 ± 9Heart weight/BW, ‰ 4.40 ± 0.23 4.13 ± 0.26Adrenal weight (right), mg 1.82 ± 0.22 1.91 ± 0.15Adrenal weight/BW, ‰ 0.064 ± 0.008 0.069 ± 0.006Hematocrit, % 48.7 ± 0.6 49.3 ± 0.8Plasma glucose, mmol/L 9.43 ± 0.65 9.79 ± 1.10Plasma insulin, pmol/L 53.6 ± 9.4 65.0 ± 7.7Plasma urea nitrogen, mmol/L 8.40 ± 0.38 8.60 ± 0.47Plasma creatinine, μmol/L 6.88 ± 0.24 7.37 ± 0.19Plasma sodium, mEq/L 153.8 ± 0.7 155.3 ± 2.6Plasma potassium, mEq/L 5.4 ± 0.3 5.4 ± 0.2Plasma chloride, mEq/L 127.7 ± 0.9 127.9 ± 1.5Plasma osmolarity, mOsm 311.8 ± 7.2 314.1 ± 9.4

*P < 0.05, **P < 0.01, †P < 0.001, ††P < 10−4 vs. WT.

Table S3. P values for the interactions between factors affectingsystolic tail-cuff blood pressure in WT and L/L male mice at age12 wk

Factors P of interaction

Genotype and Aliskiren 0.0016Genotype and Losartan 0.0096Genotype and Spironolactone <0.0001Genotype and Amiloride <0.0001Genotype and Furosemide 0.1951Genotype and NAME 0.0921

Table S4. Primers and probes for determination of genotypes with real-time quantitative PCR

Genes for detection Sequences

Tgfb1 wild-type 3′-UTR(Fwd primer) 5′-TACCCACAGGAAGGAATCCT-3′(Rev primer) 5′-CTTGGTAGCCTTCCTCTGAA-3′(Probe) 5′-FAM-ATGCCTGTGGATCCTGGCAGTGAG-Tamra-3′

Tgfb1 mutant 3′-UTR(Fwd primer) 5′-TGAAGAGCGTGCCCTACTTC-3′(Rev primer) 5′-AGGGACAGATTGTGGCGAAT-3′(Probe) 5′-FAM-TCCAGCCCGCCGAGCTGTTGCT-Tamra-3′

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Table S5. Primers and probes for quantification of mRNA with real-time quantitative reversetranscription-PCR

Gene symbol Primer/probe Sequences

Tgfb1 Fwd primer 5′-TGACGTCACTGGAGTTGTACGG-3′Rev primer 5′-GGTTCATGTCATGGATGGATGGTGC-3′

Probe 5′-FAM-TTCAGCGCTCACGTCTCTTGTGACAG-Tamra-3′Tgfb2 Fwd primer 5′-GATGCTGCCTACTGCTTTAG-3′

Rev primer 5′-TTCCATCCAAGATCCCTCTTA-3′Probe 5′-FAM-CAGGATAATTGCTGCCTTCGCCCTC-Tamra-3′

Tgfb3 Fwd primer 5′-ATTACTGCTTCCGCAACCTG-3′Rev primer 5′-CATTTCCAGCCTAGATCCTG-3′

Probe 5′-FAM-AGAACTGCTGTGTACGCCCCCTTTA-Tamra-3′Agt Fwd primer 5′-GATGAGAGGTTTCTCTCAGC-3′

Rev primer 5′-TTGCCAGTGCCGGAGATCAT-3′Probe 5′-FAM-ACACCGAGATGCTGTTGTCCACCC-Tamra-3′

Ace Fwd primer 5′-TCAGATGCCCTGGATGCCAA-3′Rev primer 5′-ACTGATTCTCTGGCCAGCCT-3′

Probe 5′-FAM-TCTGCTCTTCCAGCCACTGGCTGA-Tamra-3′Ren1c Fwd primer 5′-ACAGTATCCCAACAGGAGAGACAAG-3′

Rev primer 5′-GCACCCAGGACCCAGACA-3′Probe 5′-FAM-TGGCTCTCCATGCCATGGACATCC-Tamra-3′

Atr1a Fwd primer 5′-GCTTGGTGGTGATCGTCACC-3′Rev primer 5′-GGGCGAGATTTAGAAGAACG-3′

Probe 5′-FAM-CATGAAGCTGAAGACTGTGGCCAGT-Tamra-3′Cyp11b1 Fwd primer 5′-AGAGAACTCCGTGGCCTGG-3′

Rev primer 5′-GCTGCAGTCGGTCGAAGTA-3′Probe 5′-FAM-CGTGGTGTGTTCTTGCTAAATGGGCC-Tamra-3′

Cyp11b2 Fwd primer 5′-AGAGAAAGGTTTCTCTCAGA-3′Rev primer 5′-CCGCAGTCGGTTGAGACG-3′

Probe 5′-FAM-CGTGGTGTGTTCTTGCTAAATGGGCC-Tamra-3′Slc12a1 Fwd primer 5′-AGGCTCTGTCCTATGTGAGT-3′

Rev primer 5′-CATGGGTCCGCCTGTTAAG-3′Probe 5′-FAM-TAGACAACGCTCTGGAATTAACCACAG-Tamra-3′

Slc9a3 Fwd primer 5′-TCCTATCTGACCTCCGAGAT-3′Rev primer 5′-CTTCACGTACTTCTGACAGC-3′

Probe 5′-FAM-CCATCCTGGCCATCACCTTTTGCG-Tamra-3′Slc34a1 Fwd primer 5′-GGAGAAGCTATCCAGCTCAT-3′

Rev primer 5′-AGCGGGTACCACAGTAGGAT-3′Probe 5′-FAM-TGCCCTCTGCCACTTCTTCTTCAACA-Tamra-3′

Slc5a1 Fwd primer 5′-GCCATTTTCTGCAAGAGAGTC-3′Rev primer 5′-GTGATCATACGGGAGATTCC-3′

Probe 5′-FAM-AAGCCCAGGATCAGTCCCCAGAAG -Tamra-3′Slc4a4 Fwd primer 5′-CTCACTTAACGGTGTGCAGT-3′

Rev primer 5′-ATGAAGTCGGGCTGATGCTT-3′Probe 5′-FAM-TGGACCGTCTCAAGCTGCTGCTGA-Tamra-3′

Atp1a1 Fwd primer 5′-GAGATGAGGGAGAAATAGAG-3′Rev primer 5′-CTCAGATGCATTTGGGTTCT-3′

Probe 5′-FAM-AGATTCCCTTCAACTCCACCAACAAG-Tamra-3′Atp1b1 Fwd primer 5′-GGACGACATGATTTTCGAGG-3′

Rev primer 5′-CTCTCCTCGTTCGTGATTGA-3′Probe 5′-FAM-TGTGGCAATGTTCCCAGTGAACCCA-Tamra-3′

Fxyd2 Fwd primer 5′-TCCCTTCGAGTACGACTATG-3′Rev primer 5′-TGAGGAGGCCCACGACGAA-3′

Probe 5′-FAM-CCGTCCGCAAAGGAGGCCTGATC-Tamra-3′Slc12a3 Fwd primer 5′-GGCTGGCTCCTACAATCTG-3′

Rev primer 5′-GGGGCGGTAGTTCTTGATG-3′Probe 5′-FAM-TCGTTGAGGCCCACGGAGTAGCTCA-Tamra-3′

Scnn1a Fwd primer 5′-AGCGCGTCTTCCAGTGTAC-3′Rev primer 5′-GATTTGTTCTGGTTGCACAGT-3′

Probe 5′-FAM-CAACAATCCCCAAGTGGACAGGAAGG-Tamra-3′Scnn1b Fwd primer 5′-CAACAGCAGCAACCCGGC-3′

Rev primer 5′-CTGGTGAAGTTCCGCAAGG-3′Probe 5′-FAM-AGTTCCATTGGCACTGCACAGCCTC-Tamra-3′

Scnn1g Fwd primer 5′-CACTGGTCGGAAGCGGAAA-3′Rev primer 5′-GCACAGTCAGAGGTGTCATT-3′

Probe 5′-FAM-ACAAGGCTTCTAATGTCATGCACGTTC-Tamra-3′

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Table S5. Cont.

Gene symbol Primer/probe Sequences

Aqp2 Fwd primer 5′-TACGTGGCTGCCCAGCTG-3′Rev primer 5′-GGCTGTTGCATTGTTGTGGA-3′

Probe 5′-FAM-CATGAGATTACCCCTGTAGAAATCCGC-Tamra-3′Nppa Fwd primer 5′-GAGAAGATGCCGGTAGAAGA-3′

Rev primer 5′-AAGCACTGCCGTCTCTCAGA-3′Probe 5′-FAM-ATGCCCCCGCAGGCCCGG-Tamra-3′

Wnk1 Fwd primer 5′-TACCAAGCCTTTCTGCTCCA-3′Rev primer 5′-CAGCCTGGCTACTCACTGTT-3′

Probe 5′-FAM-CAAGGGACCAGCAGCACAAATACCG-Tamra-3′Wnk4 Fwd primer 5′-GCAGCTGCCATGGTATACAA-3′

Rev primer 5′-CGCTGGATAATCTCCCGGAT-3′Probe 5′-FAM-TCATCCTGCCCTCGGAGCGAGATG-Tamra-3′

Actb Fwd primer 5′-CTGCCTGACGGCCAAGTC-3′Rev primer 5′-CAAGAAGGAAGGCTGGAAAAGA-3′

Probe 5′-FAM-CACTATTGGCAACGAGCGGTTCCG-Tamra-3′Star Fwd primer 5′-CCGGAGCAGAGTGGTGTCA-3′

Rev primer 5′-ACTTCCAGCCAGTGGATGAA-3′Probe 5′-FAM-CAGAGCTGAACACGGCCCCACC-Tamra-3′

Hsd3b1 Fwd primer 5′-CCATCCTAGATGTCAATCTGA-3′Rev primer 5′-AAGATGAAGGCTGGCACACT-3′

Probe 5′-FAM-ACCCAGAACCTATTGGAGGCCTGTG-Tamra-3′Cyp21a1 Fwd primer 5′-AAGTTCTGGCCAGATCGCTT-3′

Rev primer 5′-TGCCCCACAGCCAAAGGAT-3′Probe 5′-FAM-TGGAACCTGGGAAGAATCCCAGAACA-Tamra-3′

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