Online supplement

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1 Online supplement

The role of distal tubule and collecting duct sodium reabsorption in sunitinib-induced hypertension

Jeannine WITTE, Josephine LAMPE, Anna KOENEN, Ines URBANECK, Antje STEINBACH, Rainer RETTIG, Olaf GRISK

Institute of Physiology, University of Greifswald, Greifswald, Germany

Correspondence: Prof. Dr. Olaf Grisk

Institute of Physiology, University of Greifswald Greifswalder Str. 11c, 17495 Karlsburg

Germany

E-mail: [email protected] Phone: 49-3834-8619300

Fax: 49-3834-8619310

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2 Calculation of glomerular filtration rate (GFR), fractional sodium excretion (FENa) and fractional sodium reabsorption (FRNa)

GFR was determined by inulin clearance (Cinulin) which is calculated according to

: urine flow; [Uinulin]: urinary inulin concentration; [Pinulin]: plasma inulin concentration

FENa was calculated according to

[UNa]: urinary sodium concentration; [PNa]: plasma sodium concentration

FRNa was calculated according to

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3 Fig. S1. Relative renal ENaC subunit mRNA abundances in control and four-day sunitinib- treated rats. Data on ENaC mRNA are normalized to Ywhaz (tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide).

100 kDa 75 kDa 65 kDa 48 kDa 35 kDa

28 kDa

65 kDa 48 kDa

35 kDa

28 kDa

-ENaC in cortex

β-actin

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4 Fig. S2. Whole Western blots on renal cortical -ENaC and -actin in control and four-day sunitinib-treated rats. Starting with a sample obtained from a control animal on the left, the sequence of bands alternates between controls and sunitinib-treated rats.

Fig. S3. Whole Western blots on renal medullary -ENaC and -actin in control and four-day sunitinib-treated rats. Starting with a sample obtained from a control animal on the left, the sequence of bands alternates between controls and sunitinib-treated rats from left to right.

100 kDa 75 kDa 65 kDa 48 kDa

35 kDa

28 kDa

65 kDa 48 kDa 35 kDa

28 kDa

-ENaC in medulla

β-actin

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5 Fig. S4. Relative renal ENaC subunit mRNA abundances in control and 14-day sunitinib- treated rats. Data on ENaC mRNA are normalized to Ywhaz (tyrosine 3-

monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide).

OSOM: outer stripe of the outer renal medulla; ISOM inner stripe of the outer renal medulla IM: inner medulla

Fig. S5. Relative renal cortical NCC mRNA abundance in control and 14-day sunitinib-treated rats. Data on NCC mRNA are normalized to Ywhaz (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide).

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6 Fig. S6. Whole Western Blots for cortical -ENaC and -actin obtained from animals treated with sunitinib for 14 days (S) and in respective controls (C).

100 kDa 75 kDa 63 kDa

-ENaC, cortex

48 kDa 35 kDa

25kD a

-actin, cortex

100 kDa 75 kDa 63 kDa 48 kDa 35 kDa

25kD a

S C S C S C S C S C S C S C S C S

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7 Fig. S7. Whole Western Blots for -ENaC and -actin in the outer stripe of the outer renal medulla (OSOM) obtained from animals treated with sunitinib for 14 days (S) and in respective controls (C).

100 kDa 75 kDa 63 kDa

-ENaC, OSOM

48 35 kDa kDa

25kD a

-actin, OSOM

100 kDa 75 kDa

63 kDa 48 35 kDa kDa

25 kDa

S C S C S C S C S C S C S C S C S

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8 Fig. S7. Whole Western Blots for -ENaC and -actin in the inner stripe of the outer renal medulla (ISOM) and the inner renal medulla (IM) obtained from animals treated with sunitinib for 14 days (S) and in respective controls (C).

100 kDa 75 kDa 63 kDa

-ENaC, ISOM + IM

48 35 kDa kDa

25kDa

-actin, ISOM + IM

100 kDa 75 kDa

63 kDa 48 35 kDa kDa

25 kDa

S C S C S C S C S C S C S C S C S

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9 Fig. S9. Western blot of -ENaC using the Biorbyt catalogue-No. orb389322 primary antibody. Left panel without blocking peptide, right panel with blocking peptide. Only the

~80-kDa band disappeared in the presence of the blocking peptide.