Supplementary methods
Radiotelemetry
For measurements of arterial pressure, heart rate and locomotor activity animals where
instrumented with a radiotelemetric device (TA11PA-C40, DSI-Transoma Medical, St Paul, MN) under
ketamine/xylazine anesthesia. The abdomen was opened via a midline incision, the intestine was
mobilized and covered with gauze soked with isotonic saline and blood flow through the lower
abdominal aorta was temporarily interrupted with two vessel clamps. Then, the tip of the catheter
was placed into the aortic lumen and fixed with veterinary adhesive. Thereafter, the vessel clamps
were removed and blood flow through the lower abdominal aorta was reestablished. The
interruption of aortic blood flow never exceeded five minutes. Thereafter, the intestine was placed
back into the abdominal cavity and the wound was closed in layers with the transmitter fixed to the
abdominal wall. After a recovery period of seven days recordings were started. Data on arterial
pressure and heart rate were sampled every ten minutes for a 10 second time interval with a
sampling rate of 500 Hz during the entire 14 day protocol with lights on from 6 a.m. to 6 p.m. After
filtering for artifacts, 24h mean values were calculated for heart rate and mean arterial pressure
using an MS AccessTM routine.
Small vessel wire myography
Human and rat small artery segments were mounted in a model 410A small vessel wire myograph
(Danish Myotechnology, Aarhus, Denmark). Human small arteries were stretched until a wall tension
of 0.4-0.5 mN/mm was reached which resulted in a maximum K+-induced vasoconstrictor response.
In rat renal vessels, length-tension curves were obtained by stretching the vessels in 20 µm steps
each lasting 2 min after which the corresponding wall tension was recorded. Data on internal
circumference (IC) and corresponding wall tension (T) were fitted to a two-parametric exponential
1
curve T = T0exp[b(IC-IC0)/IC0] where T0 (mN/mm) is the wall tension at the transmural pressure
corresponding to the animals’ mean arterial pressure and b is a measure of vascular stiffness. IC0
(µm) is the internal circumference of the vessels at the animals’ mean arterial pressure. Experiments
were performed with the vessels stretched to an internal circumference corresponding to 90% of the
internal circumference at the respective mean arterial pressure values based on the arterial pressure
data obtained from the radiotelemetric measurements. Cumulative concentration-response curves
were obtained for vasoconstrictors and vasodilators. For investigations on endothelium-dependent
vasodilation care was taken that vessels from different experimental groups were pre-constricted to
the same degree. Pretreatment of isolated vessels with the kinase inhibitors sunitinib or SAR407899
always lasted for 15 min. All drugs were dissolved in isotonic saline with the exception of sunitinib
that was dissolved in dimethyl sulphoxide (DMSO). The DMSO concentration in the organ bath was
0.1%; control vessels were treated with DMSO only. Drug-induced increases in wall tension were
normalized to maximum K+-induced wall tension and drug-induced decreases in wall tension were
normalized to the wall tension in response to the agonist used to pre-constrict the vessel segments.
To avoid confounding effects that could be caused by different preconstrictions in control and
sunitinib-preincubated arteries when endothelium-dependent vasodilation was investigated, vessels
were preconstricted with 1 µmol/l phenylephrine under control conditions and with 3 µmol/l
phenylephrine after preincubation with sunitinib (1 µmol/l). The application of phenylephrine in
different concentrations resulted in similar wall tensions of 2.46 ± 0.21 and 2.30 ± 0.30 mN/mm in
the respective groups.
Animal instrumentation for acute renal function studies
For acute experiments on renal hemodynamics and renal sodium excretion, animals were
anesthetized with pentobarbital (60 mg/kg body weight, i.p.), paralyzed with pancuronium bromide
at 1 mg/(kg*h) and artificially ventilated. Catheters were implanted for intravenous fluid and
2
anesthetic supplementation, blood sampling and arterial pressure recordings [1, 2]. The animals
were infused with isotonic saline containing 4 mg/ml inulin and 10 mg/ml bovine serum albumin at
1.2 ml/(h*100 g body weight). A thin catheter with a bent tip was inserted into the left femoral
artery and advanced to the branching point of the renal artery for intra-arterial administration of
vasoactive compounds [1, 2]. Urine was collected via ureter catheters and renal blood flow (RBF) was
recorded with an ultrasound transit time flow meter (Transonic Systems, Ithaca, NY) from the left
renal artery [1, 2].
Calculation of parameters on renal function
RVR was calculated from simultaneously recorded mean arterial pressure and RBF. GFR was
determined as renal inulin clearance. Renal plasma flow (RPF) was calculated from RBF and
hematocrit. Filtration fraction (FF) was calculated from GFR and RPF. Fractional Na+ and Li+ excretions
were calculated from Na+ and Li+ renal ion clearances and GFR. The data were related to wet kidney
weight. Dose-response curves to acetylcholine, phenylephrine and angiotensin II represent maximum
changes in RVR relative to baseline.
Dye dilution technique
For plasma volume measurements, rats were anesthetized with pentobarbital (60 mg/kg body
weight, i.p.) and a small PE 10 catheter was inserted into the left femoral vein. Thereafter, 100 µl of
isotonic saline containing 0.5% Evans Blue were administered intravenously and the catheter was
flushed with another 100 µl of isotonic saline. Two minutes after the injection, blood was sampled by
orbital puncture from anesthetized animals for photometric determination of Evans Blue plasma
concentration[3].
Atomic absorption spectrometry
3
Endogenous plasma and urinary Li+ concentrations were determined with a highly sensitive graphite
furnace atomic absorption spectrometry method. We used a continuous source model (contrAA 700,
Analytik Jena, Germany) in combination with a pyrolytically coated platform tube. In order to
enhance the sensitivity of the Li+ absorbance, the tube was coated with tantalum according to the
method described by Sampson[4]. Fifty µl of an ammonium heptafluorotantalate(V) (Aldrich)
solution (50 mg/ml) were injected into the tube, dried and pyrolyzed for 10 s at 600 °C. This cycle was
repeated three times (about 5 mg Ta overall deposit) followed by annealing for 5 s at 2500 °C. The
coating was stable for at least 100 firings and was renewed when background noise or standard
deviation increased noticeably. Absorption readings at 670.8 nm were integrated over 4 s with a
delay of 0.6 s.
Frozen plasma samples were thawed and a 100 µl aliquot thereof was spiked with 100 µl 10% HNO3.
After centrifugation (10 min, 10,000 x g), 40 µl of the supernatant were subjected to the furnace
program (Table 1). All samples were measured in triplicate. For measurement of urine samples, a
standard addition method was employed. Frozen urine samples were thawed and an aliquot thereof
was diluted by a factor of 2 to 20 with nitric acid (final concentration 5% HNO3). After centrifugation
(10 min, 10,000 x g), 20 µl of the supernatant were subjected to the same furnace program. The
sample was once measured as it was and further spiked with 4, 8, 12, 16 and 20 µl of a 10 µg/l
standard Li+ solution.
The methods applied have been validated with respect to linearity/range, limit of quantitation,
precision as well as recovery after sample preparation. Rat plasma and urine samples were spiked
with different amounts of dilutions obtained from a standard Li+ solution (1000 mg Li+/l, Certipur®,
Merck). For all dilutions and standard preparations, deionized water (AnalaR Normapur, VWR BDH
Prolabo) was used. The calibration curves were linear up to 2 µg/l for plasma and up to 10 µg/l for
urine. The limits of quantitation were 0.20 µg/l and 0.54 µg/l for plasma and urine, respectively. For
estimation of the precision, six replicates with low and high added Li+ concentrations were measured.
The relative standard deviation was always less than 3%. The recovery was determined at two
4
varying Li+ levels by analyzing three independently spiked samples. For low spike levels, recovery was
103.6 ± 9.8% and 91.4 ± 8.4% for plasma and urine, respectively. For high spike levels, recovery was
91.3 ± 1.8% and 87.8 ± 4.4% for plasma and urine, respectively.
NOS1 and NOS3 mRNA abundance
NOS1 and NOS3 abundance in small renal arteries was determined real-time RT-PCR[2, 5]. Total RNA
was isolated with the RNAqueous®-4PCR Kit (Ambion). To remove residual genomic DNA, total RNA
was treated with DNase I and subsequently concentrated by ammonium acetate precipitation
(Ambion). Reverse transcription was performed with approximately 1 µg RNA per reaction and
random hexamer primers using the High-Capacity cDNA Reverse transcription Kit (Applied
Biosystems, Foster City, CA). NOS1 and NOS3 cDNA abundances were analyzed using real-time
quantitative PCR with the ready-to-use TaqMan Universal PCR Master Mix (Applied Biosystems,
Foster City, CA) and a GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City,
CA). Relative cDNA abundance was determined by the ∆∆Ct method using the geometric mean of the
content of Gapdh (glyceraledehyde 3-phosphate dehydrogenase) and Ywhaz (tyrosine 3monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) as endogenous
references for normalization. Oligonucleotide primers and fluorescent probes used for amplification
are specified in Table 2.
Histology
Whole renal slices including cortex and medulla were cut, immersion fixed in buffered formalin,
dehydrated and embedded in paraffin. From this 3 µm slices were cut with a microtome and stained
with hematoxylin eosine and periodic acid-Schiff (PAS). Histological examinations were performed
independently by two investigators (K.E. and M.E.) blinded to the experimental design with a Nikon
microscope (Eclipse 80i) equipped with a Nikon digital camera (DS-2Mv) and a digital imaging system
(NIS Elements F 4.00.06).
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References
1 Mahler N, Freyer M, Kauschke R, Schluter T, Steinbach AC, Oswald S, et al. Kidney-specific deletion
of multidrug resistance-related protein 2 does not aggravate acute cyclosporine A nephrotoxicity in
rats. Pharmacogenet Genomics 2012; 22:408-20.
2 Schluter T, Rohsius R, Wanka H, Schmid C, Siepelmeyer A, Rettig R, et al. Amiloride lowers arterial
pressure in cyp1a1ren-2 transgenic rats without affecting renal vascular function. J Hypertens 2010;
28:2267-77.
3 Grisk O, Heukaufer M, Steinbach A, Gruska S, Rettig R. Analysis of arterial pressure regulating
systems in renal post-transplantation hypertension. J Hypertens 2004; 22:199-207.
4 Sampson B. Determination of low concentrations of lithium in biological samples using
electrothermal atomic absorption spectrometry. Journal of Analytical Atomic Spectrometry 1991;
6:115-118.
5 Grisk O, Schluter T, Reimer N, Zimmermann U, Katsari E, Plettenburg O, et al. The Rho kinase
inhibitor SAR407899 potently inhibits endothelin-1-induced constriction of renal resistance arteries. J
Hypertens 2012; 30:980-9.
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Table 1. Furnace program for determination of Li+ in rat plasma and urine.
stage
temperature (°C)
ramp (°C/s)
hold (s)
drying #1
70
6
0
drying #2
90
1
40
drying #3
110
5
5
pyrolysis
600
50
20
gas adjustment*
600
0
5
atomization*
2350
1500
5
cleaning
2450
1500
3
Purge gas: 2l/min argon, * gas stop
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Table 2. Primer sequences and TaqMan probes*
Gene
Forward primer (5’- 3’)
Reverse primer (5’- 3’)
Probe (5’- 3’)
GATCGGCGTCCGTGACT
AC
AGCAATGTTGATCTCCA
CCAGT
TGACAACTCTCGATACA
ACATCC
CATGGAAAGGAAGTGC
AGCA
AGCTGCTGTGCGTAGCT
CT
CATACAGGATAGTCGCC
TTCACACGC
CATCTGCAACGACGTAC
TGTCTCT
GACTGGTCCACAATTCC
TTTCTTG
ACTACTACCGCTACTTG
GCTGAGGTTGCTG
(Acc. No.)
NOS1
(NM_052799.1)
NOS3
(NM_021838.2)
Ywhaz
(NM_013011.3)
Gapdh
(NM_017008.4)
TTGTCAGCAATGCATCCT CGGCATGTCAGATCCAC
GC
AAC
TCTGGAAAGCTGTGGCG
* Probes were labeled at the 5' end with FAM as reporter fluorophore and at the 3' end with TAMRA
as the quencher).
8