The Tamm-Horsfall protein (THP; uromodulin), the dominant protein in normal urine, is produced exclusively in the thick ascending limb of Henle's loop. THP mutations are associated with disease; however, the physiological role of THP remains obscure. We generated THP gene-deficient mice (THP −/−) and compared them with wild-type (WT) mice. THP −/− mice displayed anatomically normal kidneys. Steady-state electrolyte handling was not different between strains. Creatinine clearance was 63% lower in THP −/− than in WT mice (P < 0.05). Sucrose loading induced no changes between strains. However, water deprivation for 24 h decreased urine volume from 58 ± 9 to 28 ± 4 μl·g body wt−1·24 h−1 in WT mice (P < 0.05), whereas in THP −/− mice this decrease was less pronounced (57 ± 4 to 41 ± 5 μl·g body wt−1·24 h−1; P < 0.05), revealing significant interstrain difference (P < 0.05). We further used RT-PCR, Northern and Western blotting, and histochemistry to study renal transporters, channels, and regulatory systems under steady-state conditions. We found that major distal transporters were upregulated in THP −/− mice, whereas juxtaglomerular immunoreactive cyclooxygenase-2 (COX-2) and renin mRNA expression were both decreased in THP −/− compared with WT mice. These observations suggest that THP influences transporters in Henle's loop. The decreased COX-2 and renin levels may be related to an altered tubular salt load at the macula densa, whereas the increased expression of distal transporters may reflect compensatory mechanisms. Our data raise the hypothesis that THP plays an important regulatory role in the kidney.
- gene-disrupted mice
- thick ascending limb
- renal concentrating mechanism
the tamm-horsfall protein (THP; uromodulin) is an 80-kDa glycoprotein synthesized exclusively in the thick ascending limb cells of Henle's loop (TAL) with exception of the macula densa (3, 4, 41). THP is the most abundant protein in the urine of normal mammals. THP production ranges from 30 to 60 mg/24 h in humans and from 0.3 to 0.5 mg·100 g body wt−1·24 h−1 in rats (2). Intracellularly, the protein resides along the basolateral membranes, in the subapical vesicle compartment, and to a lesser extent in or near the luminal plasma membrane (3, 38). Exocytosis and luminal secretion of THP are driven by a glycosylphosphatidylinositol (GPI) membrane anchor from which THP is cleaved and released into the tubular fluid (22). THP contains a COOH-terminal zona pellucida domain that allows the protein to polymerize and facilitates its gel-forming mucoid tendency (41). Interaction of THP with other proteins has thus far been reported only at the extracellular level and probably depends on the abundant oligosaccharide moieties of THP (48).
THP may be involved in the pathogenesis of cast nephropathy, urolithiasis, and tubulointerstitial nephritis (34, 36, 41, 49). The THP gene is mutated in persons with familial juvenile hyperuricemic nephropathy and autosomal dominant medullary cystic kidney disease, suggesting a role for the protein in renal urate handling and perhaps in renal development (9, 15, 31, 45). In affected persons, THP accumulates in the TAL epithelia, apparently owing to the retention of mutated THP in the endoplasmic reticulum and in a delayed export of the protein to the plasma membrane (31). Increased proximal tubular reabsorption of uric acid has been postulated possibly due to a decrease in tubular NaCl reabsorption in Henle's loop (9). Changes in the quantity of THP synthesis or urinary excretion have been assessed in pathological conditions such as diabetic nephropathy, polycystic kidney disease, and lupus nephritis (21, 32, 44). TAL transporter mutations leading to the antenatal variant of Bartter's syndrome feature a near complete to absolute deficiency of cellular and urinary THP production (16, 30, 40). THP has therefore been proposed as a renal disease marker (49). The fact that THP competes efficiently with urothelial cell receptors, such as uroplakins, in adhering to bacteria suggests that THP may protect the organism from ascending urinary tract infection (29). The THP −/− mouse indeed confirmed this hypothesis by demonstrating that THP serves as a soluble receptor for type 1 fimbriated Escherichia coli and helps to eliminate bacteria from the urinary tract (5, 24).
THP may also play a role in regulating renal function. Hypothyroid rats revealed a decreased THP synthesis along with impaired TAL function and enhanced vasopressin availability (38). Vasopressin reduces THP production (2, 18), whereas dietary salt loading and furosemide administration increase renal THP mRNA expression (47) but not urinary THP excretion (2). To test the notion that THP plays a regulatory role in renal salt and water excretion, we performed functional and morphological studies in THP gene-deficient (−/−) mice and their wild-type (WT; +/+) siblings.
MATERIALS AND METHODS
THP −/− and WT mice have been described (5). All mice were genotyped from tail tip-extracted DNA using polymerase chain reaction primers: 5′-TTGGGAAGACAATAGCAGGC-3′ (neomycin resistence gene) and 5′-GCAAATGCAGTATGACCGCC-3′ (THP intron) or the WT allele: 5′-ATGTGGATGAGTGCTCAGAG-3′ (THP coding sequence) and 5′-GCAAATGCAGTATGACCGCC-3′ (THP intron). The animals received standard mouse chow (SNIFF) and were allowed drinking water ad libitum. The local council on animal care approved the protocols. The standards correspond to the requirements of the American Physiological Society. For the physiological studies, routine parametric descriptive statistics were used. Groups were compared with two-way ANOVA and t-tests, Bonferroni corrected as appropriate. We also used t-tests to compare gene expression and Western blot quantification data. P < 0.05 was accepted as significant. Values are given as means ± SE.
Water balance and creatinine clearance.
THP −/− (total n = 14) and WT (n = 14) mice aged 8–13 wk were placed individually (10 AM) into metabolism cages for 24 h (Uno mouse metabolic cages, Uno, Zevenaar, The Netherlands). In the first protocol, we added sucrose (300 mmol/l) to the drinking water for 3 days. Mice received chow ad libitum. In the second protocol, mice received chow and water ad libitum for 2 days and on day 3, they were deprived of water but not chow. Urine volume, osmolality (freezing point depression), sodium, chloride, potassium, creatinine, uric acid, and total protein were determined by routine automated methods. The urine samples were checked for the presence of THP by Western blotting. Thereafter, the animals were killed under pentobarbital sodium anesthesia, and serum specimens and kidneys were obtained. Inter- and intrastrain kidney-to-body weight ratios were calculated. Serum sodium, chloride, and creatinine levels were determined; creatinine was measured by a modified Jaffé reaction. The creatinine clearance was determined in 12-wk-old mice and normalized for kidney weight. Plasma renin activity (PRA) was determined as the amount of formed ANG I (8).
RT-PCR and Northern blot analysis.
For RNA preparation, kidneys were rapidly dissected, homogenized, and total RNA was prepared using the RNeasy-total-RNA-kit (Qiagen). To perform semiquantitative RT-PCR, genomic DNA was digested by DNase. cDNA was synthesized by reverse transcription of 5 μg total RNA, using a cDNA synthesis kit (Invitrogen). Primers used for amplification of specific gene products are listed (Table 1). RT-PCR reactions were carried out in an automated thermal cycler (PerkinElmer, Boston, MA) using Taq polymerase (GIBCO). The number of cycles (between 23 and 35) was adapted for linear correlation between signal intensities of RT-PCR products and cDNA concentration. cDNA was adjusted to equal quantities by serial dilutions within linear range of these parameters. Reactions were controlled by PCR amplification of the housekeeping gene GAPDH. For Northern blot analysis, equal amounts of total RNA (30 μg per kidney as determined by UV at OD260 with Gene-Quant, Bio-Rad) were electrophoresed under denaturing conditions in a 1% agarose-formaldehyde gel at 40 V for 5 h. The separated RNA samples were then transferred in 10× SSC for 12 to 16 h onto a nylon membrane (Schleicher & Schull) by capillary blotting and cross-linked using a UV radiation chamber (Gene-Linker; Bio-Rad). A 383-bp mouse (furosimide/bumetanide-sensitive) Na+-K+-2Cl− transporter type 2 (NKCC2) cDNA fragment cloned into a in pBS vector and a 450 bp β-actin fragment were prepared for hybridization using asymmetric PCR. Probes were labeled with alkaline phosphatase (Alk Phos Systems and CDP Star reagent, Amersham); hybridization and chemiluminescence detection were done according to the manufacturer's protocol. After hybridization with the NKCC2 probe, the membrane was stripped in 0.5% (wt/vol) SDS at 60°C for 1 h and reprobed with the β-actin cDNA probe used as an internal standard.
Western blot analysis.
Methods were applied as described previously (37). Briefly, whole kidney homogenates (n = 6 per group) as obtained after cold homogenization in a sucrose-triethanolamine buffer using a tissue homogenizer were centrifuged at 1,000 g for 15 min at 4°C to remove whole cells, nuclei, mitochondria, and incompletely homogenized membrane fragments, and the supernatants were prepared for immunoblotting. Supernatants were solubilized, and 50 μg protein/lane as determined by a BCA protein assay reagent kit (Pierce) were run on 8 or 10% polacrylamide minigels. After electrophoretic transfer of the proteins to polyvinylidene fluoride membranes, equity in protein loading and blotting was verified by membrane staining using 0.1% Ponceau red. Coomassie staining was also performed to verify the equity in protein loading. Membranes were incubated with specific primary antibodies (Table 2) for 1 h at room temperature, following overnight incubation at 4°C, and then exposed to adequate horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Immunoreactive bands were detected by chemiluminescence, subsequently exposed to X-ray films, and the films were scanned and densitometrically evaluated.
Tissue preparation for morphology.
For morphological evaluation, male WT and THP −/− mice of different ages (total n = 12 per strain, age range 2 to 14 mo) were used. Mice were killed by in vivo perfusion fixation under pentobarbital sodium anesthesia. The kidneys were perfused retrogradely through the abdominal aorta using PBS adjusted to 330 mosmol/kgH2O with sucrose, pH 7.4, for 20 s. Next, 3% paraformaldehyde in PBS was infused for 5 min. The kidneys were removed and cut into slices. Slices were processed for embedding in Epon, LR white hydrophilic resin, or paraffin, or shock-frozen in liquid nitrogen-cooled isopentane for subsequent cryostat sectioning.
In situ hybridization.
In situ hybridization was performed on perfusion-fixed, paraffin-embedded, or frozen tissue as described (38). In brief, digoxygenin (DIG)-11-UTP-labeled riboprobes were synthesized by in vitro transcription (DIG RNA labeling kit [Sp6/T7]; Roche) using a 492-bp fragment from mouse THP cDNA (between position 1170 and 1662) cloned into a pCRII-TOPO vector, a 300-bp rat renin cDNA fragment cloned into a pGEM3 vector, and the 383-bp mouse NKCC2 cDNA fragment applied for Northern blotting. After linearization with the appropriate restriction enzymes, sense or antisense riboprobes were generated with T7 or SP6 RNA polymerases. For in situ hybridization, 6-μm-thick paraffin sections were treated with proteinase K, hybridized for 18 h with a probe concentration of 2.5 ng/μl of hybridization mix at 40°C, rinsed, and incubated with sheep anti-DIG-alkaline phosphatase-conjugated antibody (DAKO) diluted 1:50 in blocking medium. Signal was generated using 4-nitroblue tetrazolium chloride. For control, sense probes were applied in parallel with antisense probes. Slides were rinsed with PBS, coverslipped with PBS-glycerol, and viewed in brightfield microscopy.
For immunostaining, tissue sections were treated with 0.5% Triton X-100 in PBS for 30 min, washed in PBS, and blocked with 5% milk powder in PBS for 1 h at room temperature. Sections were then incubated with primary antibodies as listed (Table 2), diluted in blocking solution for 1 h at room temperature, followed by overnight incubation at 4°C. After being washed with PBS, appropriate secondary Cy3-conjugated (DIANOVA) or horseradish peroxidase-conjugated antibodies (DAKO) were incubated for 2 h. Slides were then rinsed in PBS, coverslipped with PBS-glycerol, and viewed with a Leica DMRB light microscope equipped with interference contrast optics and an HBO fluorescence lamp. Light microscopic images were obtained with a digital camera (Spot 32, Diagnostic Instruments) and processed with Meta View 3.6a software (Universal Imaging).
Ultrathin sections were viewed with an electron microscope. For ultrastructural immunogold localization of THP, LR white-embedded tissue was sectioned and incubated according to an established protocol (3).
For histochemical demonstration of nitric oxide synthase (NOS) tissue activity, the NADPH-diaphorase reaction was performed as described (6).
Quantification of NOS, cyclooxygenase-2, and renin content.
Cells of the macula densa and adjacent TAL portions that were histochemically positive for NADPH-diaphorase staining, NOS1-positive cells, or cyclooxygenase-2 (COX-2)-positive cells were counted, and renin-immunoreactive and renin mRNA-expressing arteriolar portions were quantified as detailed previously (6). Tissues from n = 4 mice per strain were used, and a total of eight coronary sections were evaluated for each parameter.
No significant differences between THP −/− and WT mice were observed in serum electrolyte, liver function, and lipid values at 3 mo. The absence of THP did not significantly interfere with water consumption, urine flow, osmolar excretion, sodium, potassium, and uric acid excretion between strains (Fig. 1 and Table 3). Interstrain kidney-to-body weight ratios showed no significant differences. However, creatinine clearance was 63% lower in THP −/− than in WT mice (P < 0.05; Table 3). Sucrose administration resulted in increased water intake that was similar in both groups. There was no significant effect on the excretion of any solute (Fig. 1). Under water deprivation, urine flow was decreased to 47.8% in WT (P < 0.05) but less so in THP −/− mice (72.3%; P < 0.05); interstrain differences were significant (P < 0.05). Three of 8 WT mice did not excrete measurable quantities of urine under deprivation due to low urine flow and related technical problems. They were excluded from the analysis, whereas eight of eight THP −/− mice were evaluated. Osmolar excretion under deprivation was significantly reduced in WT mice, but only numerically lower in THP −/− mice, compared with the hydrated state. Changes in sodium excretion were not significant in both strains (Fig. 1). Serum electrolytes (sodium and chloride) under water deprivation showed no differences between strains. These data suggest that the THP −/− mice could not conserve water as well as control mice.
Renal morphology and THP staining.
Kidneys and urinary tract epithelia were harvested from THP −/− and WT mice over an age range from 2 mo to over 1 yr. Tissues were screened for alterations induced by the gene disruption using light and electron microscopy and compared with age-matched WT controls. There were no obvious changes in the morphology of the renal tubules or the glomeruli. Sites of THP synthesis, i.e., the epithelium of the medullary and cortical TAL as well as tubular portions located downstream of TAL were apparently not altered by THP deficiency (Fig. 2). Ureter and bladder epithelia showed no major structural alterations either (not shown). Furthermore, there were no signs of epithelial or interstitial inflammation in THP −/− mice. Immunohistochemical staining revealed the total absence of THP from TAL epithelia in THP −/− compared with WT mice (Fig. 3, C and D). Fine structural immunogold THP staining of luminal and basolateral membranes, as well as of intracellular compartments of TAL cells, was absent (not shown). THP mRNA was also absent according to in situ hybridization (Fig. 3, A and B), although at high resolution an occasional remnant expression was evident. This finding may be due to nonfunctional, truncated mRNA still produced from the 3′-end of the targeted locus, probably owing to a partially ineffective stop site in the neo cassette of the transgenic construct. There was no generation of immunoreactive THP protein according to the immunohistochemical results using different polyclonal antibodies.
Major nephron transporters.
We found significant increases in mRNA in THP −/− mice compared with WT mice for distal nephron transporters including α-subunit of Na+-K+-ATPase (NKA; +36%), NKCC2 (+33%), Na+/H+ exchange type 3 (NHE3; +83%), barttin (+180%), K-type chloride channel (ClC-K2; +150%), ROMK (+260%), (thiazide-sensitive) Na+-Cl− cotransporter (NCC; +290%), and the α-subunit of (amiloride-sensitive) epithelial Na+ channel (ENaC; +80%) during steady state (Fig. 4). To confirm the increased NKCC2 mRNA expression as determined by semiquantitative RT-PCR, Northern blotting was used. An increase was found in THP −/− mice (+25%; P < 0.05; Fig. 4). GAPDH and β-actin mRNA expression levels were used as controls. There were no significant differences in GAPDH or β-actin signal intensities between strains (Fig. 4). Western blotting indicated less dramatic, albeit significant changes at the protein level with increases in THP −/− mice, compared with WT mice, in the α-subunit of NKA (+40%), NHE3 (+72%), NKCC2 (+60%), barttin (+29%), ROMK (+82%), and NCC (+30%) (Fig. 5). β-Actin was used as a control; there were no significant differences in β-actin signal intensities between individuals and strains (Fig. 5). Some of the Western blot differences were confirmed by histochemistry and in situ hybridization (Fig. 3, E–J). mRNA expression and/or Western blot signals of proximal tubular Na+-Pi cotransporter type IIa, aquaporin 1 (AQP1), and the mRNA coding for the β-subunit of NKA did not differ between strains (Figs. 4 and 5).
Juxtaglomerular paracrine parameters.
Semiquantitative evaluation of juxtaglomerular immunoreactive NOS1 and NADPH-diaphorase reactions revealed no strain differences during steady state (Figs. 6, E–H, and 7, A and B). COX-2 levels were significantly decreased in THP −/− mice compared with controls. Quantification of COX-2 mRNA by semiquantitative PCR from total kidney homogenate revealed a 51% decrease (P < 0.05; Fig. 4). Western blotting showed a 54% decrease in COX-2 (P < 0.05; Fig. 5) in the THP −/− mice. When the cells were counted, they showed a 46% decrease in immunoreactive COX-2-positive cells in THP −/− compared with controls (Figs. 6, I and J, and 7C). Renin expression from tissue homogenates, as well as from the numerical evaluation of renin-immunoreactive sites, was not different betweeen strains (Figs. 4, 5, 6, M and N, and 7E). However, juxtaglomerular renin mRNA levels were significantly reduced by 32% in the THP −/− mice (P < 0.05; Figs. 6, K and L, and 7D), and PRA levels were also numerically, although not significantly, reduced between the strains (Table 3C).
Our mouse model of THP deficiency revealed that kidneys were anatomically normal. This result implies that the only source of THP, namely, the TAL cells with their characteristic basolateral membrane interdigitation, densely packed flat mitochondria, and well-developed subapical vesicular compartment normally replete with immunoreactive THP, was left intact even in the absence of THP. Steady-state blood and urinary parameters were unchanged in THP −/− mice, even when fluid intake was increased by sucrose. However, three major findings were conspicuous. First, the glomerular filtration rate (GFR) in THP −/− mice as estimated by creatinine clearance was reduced. Second, the mice were less able to withstand water deprivation than WT mice. THP −/− mice could not concentrate their urine to the same degree under these circumstances and excreted higher urine volumes than the control mice. This increased urine output occurred without significantly elevated electrolyte losses. Third, the gene expression of paracrine/endocrine components of the juxtaglomerular apparatus and nephron-related ion transporters/channels was, in part, significantly altered in THP −/− mice, even at steady state.
The relationships of THP with ion transporters involved in the function of the TAL, chiefly with NKCC2, have been discussed in earlier studies (31, 38, 40). We speculated that more dramatic findings would be observed in THP −/− mice, such as NKCC2 dysfunction with a Bartter's-like syndrome, polyuria, dehydration, hypokalemia, nephrocalcinosis, and a stimulated prostaglandin synthesis (10, 16, 42). In Bartter's syndrome or after furosemide administration, the juxtaglomerular renin, COX-2, and NOS1 expression levels are strongly elevated along with high plasma renin concentrations (6, 14, 39). However, the phenotype we observed was much more innocuous than expected.
Instead, we observed that in THP −/− mice COX-2 was lowered and renin-related parameters were reduced, whereas NOS1 expression/activity was unchanged. COX-2 normally shows a coordinate regulation with renin, which has given rise to earlier considerations regarding a functional relationship between renin expression and COX-2. In contrast to the effects of furosemide, an enhanced tubular NaCl load at the macula densa and subsequently stimulated NKCC2-dependent transport mechanisms are believed to be responsible for reduced juxtaglomerular COX-2 expression and decreased renin biosynthesis (14, 39, 43, 46). Because THP is physiologically absent from the macula densa and therefore cannot interfere with ion transporters at this site (3, 4), the macula densa in THP −/− mice would be expected to function normally. Thus a direct influence of THP deficiency on the local paracrine system of macula densa cells is not to be expected. However, in the TAL the absence of THP may well have an effect on transport. Intracellular THP functions have been postulated and relationships of THP with ion transporters involved in TAL transport, chiefly with NKCC2, have been discussed (31, 38, 40).
We used biochemical and histochemical quantitative approaches to assess changes in the biosynthesis of products related to NaCl transport along the nephron in our mice (19, 25, 37). We found a significant upregulation of the major ion transporters/channels, namely the α-subunit of NKA, NHE3, NKCC2, ClCK-2, the β-subunit of ClC-K channels, barttin, ROMK, the α-subunit of ENaC, and NCC in THP −/− mice compared with WT at steady-state conditions. Their segmental role and nephron distribution have been studied and reviewed extensively in previous studies (1, 7, 12, 20, 26–28, 33, 39).
Although somewhat difficult to reconcile with our functional data, an upregulation of TAL transporters in THP −/− mice may be related to the absence of THP. THP mutations in humans cause malfunction of the renal concentrating mechanism (31). Others and we found that NKCC2 and THP are both localized in detergent-resistant membrane domains (DRM, also termed lipid rafts) normally involved in apical trafficking events, and both products appear to interact with one another (13, 41). Possibly, the absence of THP reduces the efficiency of NKCC2 or related local products involved in transepithelial Na-K-Cl transport, thereby altering their biosynthesis rate. An altered TAL-reabsorptive capacity would be consistent with some of our functional findings. First, the concentrating defect with water deprivation suggests malfunctioning of the TAL in mice with THP deficiency. Second, a resulting increase in tubular ion load could, in turn, downregulate the juxtaglomerular mechanisms. The reduced COX-2 and renin mRNA levels thus may be indicative of an elevated NaCl load reaching the macula densa and probably also the distal convolutions. The significantly reduced GFR in THP −/− mice would further support this interpretation, namely that an enhanced load at the macula densa activates TGF, which in turn reduces GFR (39). The fact that electrolyte excretion was not different in THP −/− and WT mice is not a contraction as steady state was achieved and the medullary collecting duct presumably compensated for any differences in delivery.
TAL malfunction may also be related to upregulation of channels and transporters in the distal convolutions and collecting ducts in THP-deficient mice with altered solute reabsorption. We found upregulation of distal convoluted tubule-specific NCC in the distal convolutions. This cotransporter may have increased its biosynthesis rate in response to an enhanced solute load, as was observed previously at the mRNA level in chronically furosemide-treated rats (11, 27). In portions located further downstream, namely in the late distal convoluted tubule, the connecting tubule, and the cortical collecting duct, the observed upregulation of the α-subunit of ENaC in THP knockouts may also be the result of altered tubular flow. Increased solute load has been shown to determine the expression of the channel (35). Of course, the higher expression levels may also depend on altered aldosterone release (23) that was not explored in our study. However, the suppression of the juxtaglomerular renin mRNA status we observed does not speak for increased mineralocorticoid availability. Taken together, there are signs of adaptive mechanisms in THP −/− mice during steady state that suggest an altered TAL-reabsorptive capacity. The failure to concentrate the urine to control levels in the absence of THP underscores this interpretation and may provide a hint at compensatory mechanisms in these mice.
Finally, our model provides some major inequities in terms of a human comparison. The human disease does feature a concentrating defect but also has clear morphological abnormalities that lead to interstitial nephritis with cyst formation (9, 15, 31, 45). These disease features did not occur in the mice. The human disease is further characterized by hyperuricemia, also not a feature of the mouse model. Mice are amply supplied with hepatic uricase (17), an enzyme that does not function in humans, so that disorders in urate handling are not to be expected. The human disease is characterized by a failure of transport of THP out of the cells in which it is synthesized. In the mouse, the gene was completely disrupted. The near complete absence of THP found in humans with the neonatal form of Bartter's syndrome is based on primary deficits of one of the essential NaCl transporters of TAL. Therefore, insufficient THP synthesis is probably not the initial event leading to the disease (16, 40, 42).
In sum, we believe that our findings are functionally and clinically relevant. We provide the first hard evidence that THP is important to regulatory physiology and actually participates in transporter function. The THP −/− model promises to be important in the evaluation of human disease ranging from myeloma cast nephropathy to familial juvenile hyperuricemic nephropathy. The fact that THP is relevant to epithelial transport regulation is an observation certainly deserving of further study.
We thank Dr. L. Bankir (Institut National de la Santé et de la Recherche Médicale Paris) for helpful advice in the evaluation of the urinary parameters, Dr. D. H. Ellison and Dr. T. Jentsch for providing the antibodies, and K. Riskowsky, P. Landmann, P. Schrade, and F. Serowka for expert technical assistence.
↵* S. Bachmann and K. Mutig contributed equally to this study.
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- Copyright © 2005 the American Physiological Society