Endothelin-1 (ET-1) was first described as a vasoconstrictor peptide (1). Evidence that it also plays a role in the progression of renal disease has since been accumulated. ET-1 is a potent growth factor for mesangial cells (2), and progressive renal damage has been observed in transgenic animals overexpressing ET (3). Urinary excretion of ET-1 is correlated with the severity of renal disease in human subjects (4,5,6). Many, but not all, studies demonstrated beneficial effects of ET-1 receptor (ET-R) antagonists in slowing progressive renal damage in experimental models, even when BP was not modified (7,8,9,10).
The diverse actions of ET-1 are mediated via at least two receptor subtypes, i.e., ET-RA and ET-RB. The former subtype mediates cell proliferation and vasoconstriction, and the latter presumably mediates vasodilation via nitric oxide stimulation, natriuresis, and ET clearance (11,12,13,14).
In normal human kidneys, ET-1 and ET-R are confined mainly to vascular tissue and to a lesser extent to glomerular structures (15,16,17,18,19). ET-RB are also observed in tubular epithelial cells (20). In human subjects, increased ET-1 expression was observed in several renal diseases, e.g., allograft nephropathy (21,22) and lupus nephritis (23). These results correspond to findings in several animal models of renal disease in which the ET system is activated. Extrapolation from animal models to human disease is somewhat problematic, however, because of some species-specific differences in the ET system. Under basal conditions, more prominent expression of ET-1 is observed in rat kidneys than in human kidneys. A preponderance of ET-RA is noted in rat kidneys, compared with human kidneys, although ET-RB expression is also markedly elevated in rat kidneys under pathologic conditions (24,25). These considerations emphasize the importance of examining human disease.
Of particular interest is the possibility of coactivation of ET-1 and ET-RB in proteinuric models of renal damage (24,25), suggesting activation of the ET system when tubular epithelial cells are exposed to protein overload. To date, however, expression of the components of the renal ET system has not been examined in detail among patients with proteinuric renal disease. These considerations prompted us to examine the expression of ET-1, ET-RA, and ET-RB in renal biopsies from patients with varying levels of proteinuria.
Materials and Methods
Patient Data
Study 1: Reverse Transcription-PCR for Patients with Different Renal Diseases. Twenty-six patients were included in the study (18 men and 8 women; median age, 50 yr; age range, 29 to 72 yr). At the time of the study, treated or untreated systolic BP was 133 ± 3.3 mmHg; diastolic BP was 81 ± 1.9 mmHg. The patients exhibited IgA nephropathy (n = 7), lupus nephritis (n = 5), membranous glomerulonephritis (GN) (n = 4), nephrosclerosis (n = 3), amyloidosis (n = 2), mesangiocapillary GN (n = 1), postinfectious GN (n = 1), focal segmental GN (n = 1), interstitial nephritis (n = 1), or diabetic nephropathy type 2 (n = 1). The mean protein excretion was 3.4 ± 0.6 g/24 h, and the creatinine clearance was 75.0 ± 5.0 ml/min. Some patients (18 of 26 patients) were receiving antihypertensive medication, and 11 of those 18 patients were receiving more than one drug. Eight patients received angiotensin-converting enzyme (ACE) inhibitors, eight received calcium channel blockers, three received β-blockers, and 14 received diuretics.
Control biopsy samples were obtained from nine patients under-going tumor nephrectomy (seven men and two women; median age, 60 yr; age range, 54 to 66 yr; systolic BP, 133 ± 2.2 mmHg; diastolic BP, 84 ± 2.8 mmHg). Renal biopsies were obtained at the time of surgery, from sites remote from renal cell carcinoma-bearing tissue. Patients had not undergone thromboembolization of the renal artery before surgery.
Study 2: Immunohistochemical Analyses in Patients with IgA Nephropathy. Sixteen patients (10 men and six women; median age, 35 yr; age range, 19 to 71 yr) with biopsy-proven IgA nephritis were included. The mean protein excretion was 2.3 ± 0.5 g/24 h, and the creatinine clearance was 95.0 ± 13.0 ml/min. Systolic BP was 133 ± 3.5 mmHg, and diastolic BP was 81 ± 2.9 mmHg. Only two patients were receiving antihypertensive treatment (a calcium channel blocker for one patient and a calcium channel blocker plus a diuretic for the other patient); none of the patients was receiving an ACE inhibitor.
Control biopsy samples were obtained from five patients undergoing tumor nephrectomy (three men and two women; median age, 57 yr; age range, 50 to 63 yr; systolic BP, 129 ± 2.0 mmHg; diastolic BP, 82 ± 3.2 mmHg).
Biopsy Technique
Patients underwent biopsies for diagnostic purposes. Written informed consent was obtained before the biopsies. The study was approved by the local ethics committee. Biopsies were performed as described elsewhere (26). In brief, samples were obtained from the left lower pole under ultrasonographic guidance (Toshiba Sonolayer; Toshiba Medical Systems, Neuss, Germany), using a Biopty system (Radiblast AB, Uppsala, Sweden) and an 18-gauge (1.2-mm) needle. Control samples were obtained immediately after ligation of the renal artery (<10 min of warm ischemia time). The Biopty needle was directed perpendicularly to the surface, so that sampling conditions were similar to those for standard percutaneous renal biopsies. Samples were immediately placed in sterile reaction tubes (Eppendorf, Hamburg, Germany), shock-frozen in liquid nitrogen, and stored at -80°C until further analysis.
Measurement of Serum Creatinine Concentrations, Creatinine Clearance, and Urinary Protein Excretion
Serum and urinary creatinine levels were measured with the Jaffé method, using an Hitachi autoanalyzer (Hitachi, Frankfurt, Germany). Twenty-four-hour protein excretion was measured by using a commercial kit (Roche, Basel, Switzerland) based on the biuret method.
RNA Isolation and Reverse Transcription
The Trizol (Life Technologies, Gaithersburg, MD) method was used for RNA isolation, according to the recommendations of the manufacturer. Selected biopsy specimens were checked for degradation of total RNA on 1% agarose gels. RNA concentrations were determined by spectrophotometric measurements at wavelengths of 260/280 nm. Reverse transcription was performed as described elsewhere (27). For each biopsy, reverse transcription was performed three times and the resulting cDNA was pooled.
Quantitative PCR Assays
Quantification of specific mRNA was performed essentially as described by Paul et al. (28) and Wagner et al. (27). For each gene, a DNA deletion mutant was cloned (29). These mutants had the same sequences as the wild-type genes (with identical primer binding sites) but with deletions of a maximum of 20%, resulting in shorter amplification products. Reverse-transcribed RNA (0.1 μg) was used for amplification in the presence of defined concentrations of DNA deletion mutants, as an internal standard. The concentration of standard DNA was selected to allow comparable degrees of amplification of wild-type and mutant genes. Primers used were as follows: prepro-ET-1, 5′-TGGCTTTCCAAGGAGCTCC-3′ (374 to 392 bp, sense) and 5′-GCTTGGCAGAAATTCCAGC-3′ (692 to 710 bp, antisense); ET-RA, 5′-AGCTCAGCTTCCTGGTTACC-3′ (615 to 634 bp, sense) and 5′-AATTCCCTGAACACGACTCC-3′ (1053 to 1072 bp, antisense); ET-RB, 5′-TTGGAGCTGAGATGTGTAAGC-3′ (763 to 783 bp, sense) and 5′-CAGTGAAGCCATGTTGATACC-3′ (1367 to 1387 bp, antisense) (30).
The PCR mixture contained 0.25 mM dNTP (Promega, Madison, WI), 2.5 mM MgCl2, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 80 nM levels of sense and antisense primers (Life Technologies), and 1 U of Taq polymerase (Life Technologies). The thermal profile used consisted of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, performed 30 times for ET-1 and ET-RB and 32 times for ET-RA. In all experiments, possible contamination with genomic DNA was excluded by PCR amplification in the absence of reverse transcriptase. Amplification products were separated by agarose gel electrophoresis and then digitized by using a gel documentation system (Intas, Göttingen, Germany) and Scion Image software (National Institutes of Health, Bethesda, MD). The ratio of the OD of the endogenous cDNA to the OD of the mutant DNA [OD ratio (ODR)] was determined. Each sample was measured in triplicate, in individual PCR assays, for each gene.
ET-1 and ET-RB Immunohistochemical Studies
Staining Protocol. Primary antibodies 1H11 and BH2B10 (a gift from Schering Company, Berlin, Germany) were raised in mice against human ET-1 and ET-RB, respectively. The anti-ET-1 antibody demonstrated 100% cross-reactivity with ET-1, ET-2, and sarafotoxin-6b and 50% cross-reactivity with ET-3. The anti-ET-RB antibody did not cross-react with ET-RA.
Cryostat sections (3-μm thickness) were air-dried for 2 h and fixed in acetone for 10 min at 4°C. Nonspecific binding of avidin or biotin to the tissue was prevented with the use of avidin and biotin blocking solutions (Vector Laboratories, Burlingame, CA). Sections were overlaid with 50 μl of primary mouse antibody solution (diluted 1:10) for 24 h at 4°C. Phosphate-buffered saline was used for solutions and washings (10 min). Subsequent incubations were performed at 20°C for 30 min, in a moist chamber. The primary antibody was washed off, and sections were incubated with 50 μl of secondary (link) antibody (biotinylated goat anti-mouse Ig, 1:40). After washing, sections were incubated with streptavidin-peroxidase complex (1:100). Binding of the primary antibody was observed with freshly prepared 3-amino-9-ethylcarbazole and hydrogen peroxide solution. Sections were counterstained with Mayer's hemalum and mounted under glass coverslips. Negative control experiments were performed by incubating control sections with phosphate-buffered saline or normal mouse serum.
Semiquantitative Grading. Immunostaining was graded essentially as described elsewhere (31), as follows: 0, no staining; 1, mild staining; 2, moderate staining; 3, intense staining. The localization of the reaction, i.e., glomeruli or proximal tubule epithelial cells, was recorded. The intensity and extent of staining were taken into account and graded by two separate investigators, in a blinded manner.
Statistical Analyses
Data are expressed as mean ± SEM or median and range as indicated. Data were analyzed by using the nonparametric Mann-Whitney test, linear regression, or multivariate ANOVA, as indicated. The null hypothesis was rejected at P < 0.05.
Results
Study 1: Reverse Transcription-PCR for Patients with Different Renal Diseases
ET-1 mRNA Expression. When patients receiving ACE inhibitor treatment were excluded from analysis, ET-1 mRNA expression among the remaining 18 patients was significantly higher for patients with creatinine clearances of ≤80 ml/min. The wild-type/mutant mRNA ODR for patients with creatinine clearance values of ≤80 ml/min (n = 8) was 1.06 ± 0.26, whereas that for patients with creatinine clearance values of >80 ml/min (n = 10) was 0.47 ± 0.06 (P < 0.05) (Figure 1a). No significant correlation between creatinine clearance and ET-1 ODR was noted by ANOVA, however. Patients (n = 10) with higher-grade proteinuria (≥2 g protein/24 h, 4.5 ± 2.4 grs/24 h, mean ± SD) exhibited significantly higher levels of ET-1 mRNA expression (ODR, 1.00 ± 0.21), compared with patients (n = 8) with lower-grade proteinuria (<2 g protein/24 h, 0.8 ± 0.6 grs/24 h, mean ± SD) (ODR, 0.40 ± 0.04; P < 0.01) and control subjects (ODR, 0.55 ± 0.07; P < 0.05) (Figure 1b). There was a modest correlation between 24-h protein excretion rate and ET-1 expression (ODR) (r2 = 0.24, P < 0.04). When patients receiving ACE inhibitors were analyzed, ET-1 gene expression was significantly lower among patients with higher-grade proteinuria who were receiving ACE inhibitors (n = 5), compared with patients who were not (P < 0.05) (Figure 1c).
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Figure 1: Expression of prepro-endothelin-1 (prepro-ET-1) mRNA as a function of creatinine clearance (a) and protein excretion rate (b) among patients not receiving angiotensin-converting enzyme (ACE) inhibitors and expression among patients with higher-grade (≥2 g/24 h) proteinuria, with or without ACE inhibitor treatment (c). ODR, OD ratio (wild-type versus mutant cDNA). Individual values and medians are presented. * P < 0.05, ** P < 0.01, by Mann-Whitney test.
ET-RA mRNA Expression. mRNA levels tended to be higher among patients with higher-grade proteinuria, but the difference did not reach statistical significance (Figure 2b). No significant differences in ET-RA gene expression were noted between patients with creatinine clearances of ≤80 or >80 ml/min and control subjects (Figure 2a) or between patients receiving ACE inhibitors and those not receiving ACE inhibitors (Figure 2c).
Figure 2: Expression of ET receptor type A (ET-RA) mRNA as a function of creatinine clearance (a) and protein excretion rate (b) among patients without ACE inhibitor treatment and expression among patients with higher-grade proteinuria, with or without ACE inhibitor treatment (c). Individual values and medians are presented. * P < 0.05, ** P < 0.01, by Mann-Whitney test.
ET-RB mRNA Expression. ET-RB mRNA expression tended to be higher both for patients with creatinine clearances of >80 ml/min and for those with creatinine clearances of ≤80 ml/min, compared with non-nephritic control subjects, but this difference did not reach statistical significance (Figure 3a). Receptor expression was significantly higher for patients with higher-grade proteinuria, compared with patients with lower-grade proteinuria (ODR, 1.81 ± 0.30 versus 0.63 ± 0.13; P < 0.01) or control subjects (ODR, 0.57 ± 0.09; P < 0.01) (Figure 3b). There was some correlation between urinary protein excretion and ET-RB mRNA expression (ODR) (r2 = 0.28, P < 0.03). The correlation between proteinuria and ET-RB mRNA expression did not reach statistical significance by ANOVA, by a narrow margin (P = 0.08). Among patients with higher-grade proteinuria, gene expression by subjects who were receiving ACE inhibitors was significantly lower than that by subjects without ACE inhibitor treatment (P < 0.05) (Figure 3c).
Figure 3: Expression of ET-RB mRNA as a function of creatinine clearance (a) and protein excretion rate (b) among patients without ACE inhibitor treatment and expression among patients with higher-grade proteinuria, with or without ACE inhibitor treatment (c). Individual values and medians are presented. * P < 0.05, ** P < 0.01, by Mann-Whitney test.
Study 2: Immunohistochemical Analyses for Patients with IgA Nephropathy
Immunohistochemical Staining for Immunoreactive ET. A negative control assay for ET-1 demonstrated no significant nonspecific staining in a biopsy from a patient with IgA nephropathy and high-grade proteinuria (Figure 4A). Control sections demonstrated no or only very weak immunoreactivity in glomeruli (endothelial and capsular epithelial cells), in the vasculature (endothelial and vascular smooth muscle cells), and in proximal tubular epithelial cells (Figure 4C). Among patients with IgA nephropathy, staining was markedly increased in proximal tubular epithelial cells and was slightly increased in glomeruli (endothelial and capsular epithelial cells), as well as in vessels (endothelial and smooth muscle cells) (Figure 4, D and E). In addition, among patients with high-grade proteinuria, staining for ET-1 was prominent in mesangial cells (Figure 4E).
Figure 4: Immunohistochemical staining. (A and B) Biopsy from a patient with IgA nephropathy and high-grade proteinuria, which served as a negative control sample for ET-1 (A) and ET-RB (B) immunohistochemical staining. (C to E) Representative immunohistochemical staining for ET in biopsy samples from a healthy control subject (C), a patient with IgA glomerulonephritis (GN) and lower-grade proteinuria (D), and a patient with IgA GN and higher-grade proteinuria (E). (F to H) Representative immunohistochemical staining for ET-RB in biopsy samples from a healthy control subject (F), a patient with IgA GN and lower-grade proteinuria (G), and a patient with IgA GN and higher-grade proteinuria (H). Magnifications: ×360 in A, B, and D through H; ×180 in C.
Among patients with lower-grade proteinuria, expression of ET-1, as indicated by the staining score, was lower in glomeruli than in proximal tubules in the majority of cases. Expression of ET-1 in glomeruli and proximal tubular epithelial cells was significantly greater among patients with higher-grade proteinuria than among patients with lower-grade proteinuria or control subjects (Figure 5A). The correlation between individual proteinuria values and staining scores for proximal tubular epithelial cells was statistically significant (r2 = 0.32. P < 0.02).
Figure 5: Staining scores for immunoreactive ET-1 (A) and ET-RB (B) in glomeruli and proximal tubules of patients with IgA GN and lower-grade proteinuria (<2 g/24 h, n = 8) (□) and patients with IgA GN and higher-grade proteinuria (≥2 g/24 h, n = 8) (▪). Staining scores were as follows: 0, no staining; 1, weak staining; 2, intermediate staining; 3, strong staining, * P < 0.05 versus IgA GN with lower-grade proteinuria.
Immunhistochemical Staining for Immunoreactive ET-RB. A negative control assay for ET-RB demonstrated no significant nonspecific staining in a biopsy from a patient with IgA nephropathy and high-grade proteinuria (Figure 4B). In healthy control biopsies, we observed variable, but usually weak, staining for immunoreactive ET-RB in glomeruli (parietal epithelial cells and podocytes) (Figure 4F), in vessels (endothelial and smooth muscle cells), and in some proximal tubular epithelial cells. For patients with IgA nephropathy (even those with lower-grade proteinuria), staining in proximal tubular epithelial cells was increased, compared with control subjects. Furthermore, staining in glomeruli (capsular epithelial cells, podocytes, endothelial cells, and mesangial cells) and in vessels (endothelial and smooth muscle cells) demonstrated a similar pattern, compared with control samples, or was slightly increased (Figure 4, G and H).
In proximal tubular epithelial cells, the staining scores for ET-RB were significantly higher for patients with IgA nephropathy and higher-grade proteinuria than for patients with lower-grade proteinuria or control subjects (Figure 5B). Staining was more widespread, as well as more intense. In glomeruli, immunoreactivity for ET-RB was not different between control subjects and patients with IgA nephropathy (with higher- or lower-grade proteinuria).
Discussion
These studies with two different patient cohorts demonstrated that the renal ET system was activated in human chronic renal disease. This was true for expression on the mRNA level as well as on the protein level. Such activation was related to the degree of proteinuria.
In renal biopsies, the concentrations of ET-1 and ET-RB mRNA determined by quantitative PCR were significantly increased, at least for patients with higher-grade proteinuria. In parallel, ET-RA expression tended to be higher, but this did not reach statistical significance. In a separate cohort of patients with IgA nephropathy and different degrees of proteinuria, increased expression of ET-1 and ET-RB protein was observed and the intensity of staining was again related to proteinuria.
The increase in ET-1 and ET-RB mRNA levels was independent of the type of renal disease; specifically, no difference was observed when inflammatory (IgA nephropathy, lupus nephritis, and mesangioproliferative GN) and noninflammatory (diabetic nephropathy, amyloidosis, and membranous GN) types of renal disease were compared. There was no correlation between age and ET-1 expression, as reported previously for plasma and vascular endothelium of healthy subjects (32,33).
For healthy control subjects, immunohistochemical analyses confirmed faint staining for ET in the renal vasculature, in glomeruli, and in proximal tubular epithelial cells (15). Semi-quantitative evaluation demonstrated marked ET peptide expression in both glomeruli and tubular epithelial cells of patients with higher-grade proteinuria. The observation of increased expression in renal tissue is of interest in view of the report by Ohta et al. (4) that indicated that ET excretion was increased among patients with renal disease. Those authors concluded that urinary ET originated from ET expressed in tubular cells (4). The in vitro studies by Zoja et al. (34) provide further support for this hypothesis; exposure of proximal tubular epithelial cells to albumin and other serum proteins increased tubular ET-1 expression and secretion.
An additional argument for increased activity of the ET system in human renal disease involves the concomitant increases in ET-1 and ET-RB expression. Expression of ET-RA was not significantly increased in whole biopsy samples, but the possibility of increased expression of ET-RA in critical compartments certainly cannot be excluded.
Overall, the concomitant increases in ET-1 and ET-RB expression strongly argue for a potential role of the ET system in renal disease. The increase in ET-1 expression, when examined in isolation, may underestimate the degree of activation of the ET system, because ET-RB is upregulated in parallel with, and presumably in response to, ET-1. Upregulation of ET-RB is surprising, because negative feedback inhibition would be anticipated a priori. For example, in failing hearts, Zolk et al. (35) observed increased ET-1 expression but decreased ET-RB expression. Among our healthy control subjects, ET-RB was faintly expressed in glomeruli, vessels, and proximal tubular epithelial cells, confirming the findings reported by Karet et al. (19). In contrast, for patients with higher-grade proteinuria, staining for immunoreactive ET-RB was prominent in proximal tubular cells but was also observed in glomeruli (Figure 4, B and C). This observation is in good agreement with findings in experimental models of renal disease, e. g., aminonucleoside-induced nephrosis (24) or Thy-1 nephritis (25). It is of interest that, in those models, increased ET-RB expression was noted in the proteinuric phase in parallel with increased ET-1 expression, as observed in the biopsies from our patients with renal disease. It has been argued that exposure of tubular epithelial cells to proteins is the major signal for upregulation of ET-1 (36). Recent findings in lupus nephritis, however, suggest predominant glomerular expression (23); therefore, the mechanisms of activation in different glomerular diseases may be more diverse and complex.
The preferential upregulation of ET-RB (compared with ET-RA) may be of interest in another respect. Iwasaki et al. (37) demonstrated ET-RB-mediated autoinduction of ET-1 in rat mesangial cells, and Ong et al. (20) demonstrated similar autoinduction in human tubular epithelial cells. Both groups demonstrated that autoinduction could be blocked by ET-RB antagonists. The role of ET-RB is still unclear. ET-RB-mediated signals have been implicated in vasodilation, nitric oxide and bradykinin release, and sodium transport (11,38,39). Furthermore, Oksche et al. (40) recently demonstrated that ET-1 is cleared by internalization through ET-RB. The net final result of these diverse local ET-1 actions in tubular epithelial cells is currently unknown. It is most unlikely that the upregulation of ET-1 and ET-RB represents the result of generalized activation of protein-loaded tubular epithelial cells, because analysis of additional renal biopsies from patients with similar degrees of proteinuria (data not shown) confirmed our previous observation (27) that, in contrast to ET-RB, angiotensin II (AngII) receptor type 1 mRNA is downregulated. In summary, the aforementioned results are clearly in accordance with recent proposals that the “nephrotoxic” effects of protein in the urine are mediated, at least to a large extent, via activation of the renal ET system (36).
The primary aim of this study was to assess the effects of proteinuria on the renal ET system. In addition, we observed that activation of the renal ET system was less for a limited number of proteinuric patients who were receiving ACE inhibitors. This finding requires a brief discussion; AngII and the ET system interact. In vitro and in vivo studies demonstrated that AngII induces ET-1 expression (41,42,43). Increased plasma and renal ET-1 peptide concentrations were observed in rats treated with AngII infusions (44). AngII also increased the release of ET-1 in human endothelial cell cultures (45). Decreased ET-1 excretion and decreased expression of ET-1 peptide and prepro-ET-1 mRNA were noted after pharmacologic blockade of the renin-angiotensin system (RAS) by ACE inhibitors and/or AngII receptor type 1 blockers, in several experimental models of renal disease (31,46,47,48,49). The effects of blockade of the RAS may depend on the baseline activity of the RAS, because we observed, at best, modest effects in a low-renin model of chronic renal failure (9). In this study, patients with higher-grade proteinuria who were receiving ACE inhibitors exhibited significantly lower renal ET-1 and ET-RB expression, compared with patients with higher-grade proteinuria who were not receiving ACE inhibitors. A local RAS is present in tubular epithelial cells. Tubular epithelial cells are exposed to very high local AngII concentrations (50). When proteinuria is provoked by daily injections of albumin, the local RAS in proximal tubular epithelial cells is activated (51). This observation is consistent with the idea that an “inflammatory phenotype” of proximal tubular epithelial cells is associated with higher local availability of AngII. Our observation of lower renal expression of ET-1 and ET-RB among patients receiving ACE inhibitors does not allow determination of whether these effects result from local or systemic inhibition of the RAS and whether they are mediated by direct actions on the RAS or indirectly, via reduction of proteinuria. In summary, we conclude that increased expression of ET-RB and ET-1, at the mRNA and protein levels, is observed in renal tubular epithelial cells of patients with severe proteinuria.
Mr. Ingo Lehrke was supported by the Deutsche Forschungsge-meinschaft (Graduiertenkolleg Experimentelle Nieren- und Kreislauf-forschung). We thank Prof. Gerd Staehler and Prof. Manfred Wiesel (Department of Urology, University of Heidelberg) for support in the collection of tumor nephrectomy specimens and Prof. Ivan Zuna (German Cancer Research Center) for help and advice regarding statistical analyses.
Dr. Roland Blantz served as Guest Editor and supervised the review and final disposition of this manuscript.
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