Sarcoglycan Subcomplex Expression in Refluxing Ureteral Endings

Sarcoglycan Subcomplex Expression in Refluxing Ureteral Endings Salvatore Arena,* Angelo Favaloro, Giuseppina Cutroneo, Angela Consolo, Francesco Arena, Giuseppe Anastasi and Vincenzo Di Benedetto From the Department of Pediatric Surgery, Unit of Pediatric Surgery, University of Catania, Catania (SA, VDB), and Departments of Biomorphology and Biotechnologies (AF, GC, AC, GA), and Medical and Surgical Pediatric Sciences, Unit of Pediatric Surgery (FA), University of Messina, Messina, Italy

Purpose: Functional and structural lesions of ureteral endings seem to alter the active valve mechanism of the ureterovesical junction, causing vesicoureteral reflux. The interaction of the dystroglycan complex with components of the extracellular matrix may have an important role in force transmission and sarcolemma protection, and the sarcoglycan complex is an essential component of the muscle membrane located dystroglycan complex. We performed immunofluorescence and molecular analysis on the expression of sarcoglycan complex subunits. Materials and Methods: A total of 21 specimens of refluxing ureteral endings were obtained during ureteral reimplantation. Six ureteral ends obtained during organ explantation were used as controls. Immunohistochemical analysis and reverse transcriptase polymerase chain reaction evaluation were performed for ␣, ␤, ␥, ␦ and ␧-sarcoglycan complex. Results: The Spearman test revealed a significant positive correlation between ␣-sarcoglycan complex immunofluorescence intensity and grade of vesicoureteral reflux, while a negative correlation was recorded between ␧-sarcoglycan complex immunofluorescence intensity and grade of vesicoureteral reflux. Conclusions: Semiquantitative analysis demonstrated a significant grade related impairment of ␧-sarcoglycan complex coupled with an increased expression of ␣-sarcoglycan complex. This observation suggests that the structural deficiency of the trigonal ureterovesical junction could cause a passive stretching of refluxing urine on the ureter, deranging the multimodular tensegrity architecture of the sarcoglycan subcomplex, or that the sarcoglycan complex could have a key role in the physiopathology of vesicoureteral reflux. In fact, the defect in any of the sarcoglycan complexes results in degeneration of membrane integrity and muscle fiber. An altered configuration of the sarcoglycan complex could explain the structural and functional changes in refluxing ureteral endings. Our observations underline the assumption that primary vesicoureteral reflux might be regarded as a sarcoglycanopathy with marked quantitative deficiency of ␧-sarcoglycan complex and over expression of ␣-sarcoglycan complex. Key Words: sarcoglycans; vesico-ureteral reflux; muscle, smooth

rimary VUR is thought to occur as a consequence of a congenital structural deficiency of the trigonal UVJ due to an abnormal maturation.1 Historically, passive compression of the roof of the intravesical ureter against the underlying detrusor was considered an efficient antireflux protection, and the length of the intravesical ureter relative to its diameter was judged the crucial point perpetuating the “passive” reflux defense mechanism.2,3 According to this theory, a lateral intravesical ostium, and the brevity of ureteral transmural and submucosal course were believed to be the main cause of VUR,4 and the spontaneous resolution of VUR was considered to be due to bladder growth, during which submucosal tunnel elongation occurs.5 It has recently been shown that a lower ratio than expected of intravesical ureteral length-to-ureteral diameter occurs in human newborns, and it has been suggested that an intrinsic pathophysiological lesion may have an important role in impairing the so-called active antireflux mech-

P

Submitted for publication September 17, 2007. * Correspondence: Unit of Pediatric Surgery, University of Catania, Vittorio Emanuele Hospital, Via Plebiscito, n. 628-95124 Catania, Italy (telephone: 0039-095-743-6501; FAX: 0039-095-7435324; e-mail: [email protected]).

0022-5347/08/1795-1980/0 THE JOURNAL OF UROLOGY® Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION

anism.1,5,6 Functional and structural lesions of ureteral endings seem to alter the active valve mechanism of the UVJ, causing VUR.1,3 Dysplasia, atrophy and architectural derangement of smooth muscle bundles appear essential for the deficient active valve mechanisms in refluxing ureteral endings and, in particular, the reduction of the total smooth muscle fascicles or defective configuration, creating insufficient or uncoordinated ureteral peristalsis accompanied by a grade related loss of c-kit positive interstitial ICCs at the UVJ.1,5,7 On the other hand, it has also been supposed that an impairment of overall microperfusion in RUs, leading to tissue ischemia, and diminished ureteral perfusion are likely to induce and support smooth muscle dysfunction and apoptosis, so that functional and structural alterations may further deteriorate the active valve mechanism of the ureterovesical junction, causing VUR.6 Previous findings in skeletal and smooth muscles supported the idea that one function of DGC is to maintain membrane integrity during cycles of contraction and relaxation.8,9 The interaction of DGC with components of the extracellular matrix may have an important role in force transmission and sarcolemma protection.8,9 The SG complex is an essential component of the muscle membrane located DGC.10 The defect of even one SG, essential for muscle

1980

Vol. 179, 1980-1986, May 2008 Printed in U.S.A. DOI:10.1016/j.juro.2008.01.059

SARCOGLYCANS AND VESICOURETERAL REFLUX viability and maintaining sarcolemmal stability, reportedly causes various forms of muscular dystrophy and disruption of the complex.11 Loss of the SG complex clearly has an adverse effect on the survival and maintenance of proper function in myocytes,10 leading to necrosis.12 Previous studies have revealed that in smooth muscle ␧-SG replaces ␣-SG, forming a novel complex that consists of ␧, ␤, ␥ and ␦-SG.13 In comparison, immunofluorescence analysis of SGs in smooth muscle fibers of ureters has recently demonstrated the contemporary presence of ␣, ␤, ␥, ␦ and ␧-sarcoglycan.14 To our knowledge a hypothetical role of SGs has not been elucidated in the pathogenesis of altered active antireflux mechanism of RUs. On this basis, we performed immunofluorescence and molecular analysis on the expression of SG subunits, comparing samples of normal and refluxing human ureteral endings. MATERIALS AND METHODS A total of 9 boys and 5 girls (mean age 15.8 ⫾ 4.0 months) with primary VUR undergoing transvesical antireflux surgery were included in the study, after informed parental consent was obtained. The indication for open antireflux surgery was poor parental compliance with treatment or reflux nephropathy. Four boys and 3 girls had bilateral VUR, yielding a total of 21 RUs. No child had a history of neurogenic bladder, neuromuscular pathology or voiding dysfunction. Reflux was recorded as low grade in 6 RUs (grade II in 3 and grade III in 3) and high grade in 15 (grade IV in 9 and grade V in 6). Antireflux surgery was used in patients with grade II reflux in the context of a contralateral procedure for high grade VUR. The 21 specimens of the distal intravesical refluxing ureter were obtained during ureteral refluxing reimplantation. Six archival ureteral ends of matched age (20.2 ⫾ 8.1 months), obtained during organ explantation with no evidence of urological disease and no history of VUR or UTI, were investigated as a comparison. To identify the distal part of the ureter, colored sutures were inserted into the distal ureteral ends. The resected ureteral segments were fixed in 3% paraformaldehyde in 0.2 M phosphate buffer at pH 7.4 for 2 hours. After 9 rinses in 0.2 M phosphate buffer and PBS the biopsies were infiltrated with saccharose at 12% and 18% to obtain a gradual substitution of saline solution with gluconate solution, and to avoid the disruption of cellular membranes during the successive phase. Finally, the specimens were frozen in liquid nitrogen. For immunohistochemistry 20 ␮m thick sections were cut on a cryostat, and collected on glass coated with 0.5% gelatin and 0.005% chromium potassium sulfate. To block nonspecific sites and to render the membranes permeable, the sections were preincubated with 1% bovine serum albumin and 0.3% Triton® X-100 in PBS at room temperature for 15 minutes. Finally, the sections were incubated with primary antibodies for 2 hours. The primary antibodies used consisted of mouse monoclonal anti-␣-sarcoglycan diluted at 1:100, mouse monoclonal anti-␤-sarcoglycan diluted at 1:200, mouse monoclonal anti-␥-sarcoglycan diluted at 1:100, mouse monoclonal anti-␦-sarcoglycan diluted at 1:50 and mouse monoclonal anti-␧-sarcoglycan diluted at 1:100. In all reactions trimeric intracellular cation conjugated IgG anti-mouse in goat was used as the first fluorochrome,

1981

diluted at 1:100 and applied for 1 hour, after incubation with the primary antibody. For double localization reactions, after many rinses with PBS and incubation with a biotinylated IgG in goat to obtain saturation of residual-free binding sites, sections were incubated with a second antibody conjugated with fluorescein-5-isothiocyanate conjugated secondary IgG as the second fluorochrome, diluted at 1:100. Slides were finally washed in PBS and sealed with mounting medium. Sections were then observed and photographed using a photon laser scanning confocal microscope equipped with argon laser (458, 488 ␭) and 2 helium-neon lasers (543 and 633 ␭). All images were digitalized at a resolution of 8 bits into an array of 2,048 ⫻ 2,048 pixels. Optical sections of fluorescence specimens were obtained using helium-neon laser (543 nm) and argon laser (458 nm) at a 1-minute, 2-second scanning speed with up to 8 averages. We obtained 1.50 ␮m thick sections using a pinhole of 250. For each reaction at least 100 fibers were observed to obtain a statistical analysis. Contrast and brightness were established by examining the most brightly labeled pixel and choosing the setting that allowed clear visualization of structural details, while keeping the highest pixel intensities near 200. The same settings were used for all of the images obtained from the other samples that had been processed in parallel. The function called “display profile” allowed us to show the intensity profile across the image along a freely selectable line. Digital images were cropped, and figure montages were prepared using Adobe Photoshop® 10.0. We collected tissue samples of human control and refluxing ureters. We evaluated the expression of ␣, ␤, ␥, ␦ and ␧-SG in each of them by RT PCR. Total RNA Isolation A 50 to 100 mg section of each tissue sample was homogenized using a power homogenizer. Total RNA was isolated by procedures based on the monophasic solution of phenol and guanidine isothiocyanate on single step RNA isolation. Reverse Transcriptase Polymerase Chain Reaction Analysis The RT PCR procedure was carried out using the 2-step protocol in a thermal cycler. In the first step an initial RT reaction was carried out in 20 ␮l volume containing 3 ␮g total RNA, 10 U RNase inhibitor, 10 mM dithiothreitol tetraacetate, 15 U MultiScribe® Reverse Transcriptase and oligo(dT) 16, 1.25 ␮M under the thermal cycler conditions of holding 10 minutes at 25C and 12 minutes at 42C. In the second step a further independent PCR was carried out in 50 ␮l volume containing 5 ␮l cDNA of the first step (RT) as template, 2.5 U AmpliTaq Gold® DNA polymerase, with 0.2 ␮M each primer designed by us of mRNA sequences (table 1). The DNA amplification was performed conventionally. Each sample together with an internal control was subjected to 30 cycles of amplification (exponential phase of amplification) consisting of 30 seconds of denaturation, 30 seconds of annealing and 40 seconds of extension. The final extension step at 72C was extended to 7 minutes. The annealing temperature was optimized for each primer set. For each component of the SG complex human D-glyceraldehyde-3-phosphate dehydrogenase cDNA was used as an internal control.

1982

SARCOGLYCANS AND VESICOURETERAL REFLUX TABLE 1. Oligonucleotide primer sequences used for genotype

Primers

Forward

Reverse

Length (bp)

Exons

Nucleotides

National Center for Biotechnology Information Accession No.

SGCA SGCB SGCG SGCD SGCE SGCZ GAPDH

5=-ACTTCTGTCTTGCTACGAC-3= 5=-GTGAGAAGGCTGTTGAGA-3= 5=-CCTGTCTGTGGCCGGTGTGA-3= 5=-AGTGGTAGTAGGAGCTGA-3= 5=-ACATTCTTGCTGACAGTGT-3= 5=-ATGCAGAGACAATCAAGCT-3= 5=-AACCTGCCAAATATGATGAC-3=

5=-TCACCTGTGTGCACATTG-3= 5=-ATAGTCTGTGCTGAATAAG-3= 5=-GCGTTTACTTCCCATCCACGCTGC-3= 5=-TCGCAGCATCTAACTTAAT-3= 5=-TGGATATATCGAAGCCAT-3= 5=-TGCTACTGGACTGACAAG-3= 5=-ACTGAGTGTGGCAGGGACTC-3=

423 485 168 251 221 165 340

6–9 1–3 8 6–9 1–3 7 8–9

695–1117 89–573 943–1110 987–1237 162–382 1415–1579 854–1192

NM-000023 NM-000232 NM-000231 NM-000337 NM-003919 NM-139167 NM-002046

The sequence of sarcoglycans was later confirmed by nucleotide sequencing analysis. Nucleotide Sequencing Analysis Amplified DNA was purified using a commercially available kit (GFX™ PCR DNA and Gel Band Purification Kit). The fragments extracted were directly sequenced with the primers used for the RT PCR assay and labeled with the ABI Prism® BigDye Terminator Cycle Sequencing Kit, according to the manufacturer instructions, on a 377 ABI Prism Sequencer Analyzer. ABI Sequencing Analysis 3.4.1 was used to process the raw sequence data, and ABI Sequencing Navigator was used to align sequenced data. Student’s t test was used to compare fluorescent intensity values (obtained via display profile mode) of each SG subunit (␣, ␤, ␥, ␦ and ␧) in each group (control, low grade and high grade). The Spearman p test was used to correlate the values of fluorescent intensity of each immunohistochemical reaction and vesicoureteral reflux grade. Quantitative values are expressed as the mean (standard error), and statistical significance was accepted at p ⬍0.05.

RESULTS Using confocal laser scanning microscopy, we studied the immunostaining patterns of ␣, ␤, ␥, ␦ and ␧-SG in control and refluxing ureteral ends. A common feature of all SGs was their sarcolemmal expression. In control ureters indirect immunofluorescence revealed a normal staining pattern of ␣, ␤, ␥, ␦ and ␧-SG. Immunofluorescence analysis demonstrated that the value of fluorescence intensity was 42.48 (SD 4.34) for ␣-SG, 76.25 (9.82) for ␤-SG, 72.97 (35.12) for ␥-SG, 69.68 (7.27) for ␦-SG and 86.15 (8.96) for ␧-SG (fig. 1). Student’s t test values are summarized in table 2. In low grade RUs immunofluorescence intensity values were 78.80 (SD 3.02), 65.72 (5.74), 67.75 (6.51), 61.68 (6.10) and 45.30 (1.13), respectively, for ␣, ␤, ␥, ␦ and ␧-SG (fig. 2). Student’s t test values are summarized in table 2. In high grade RUs immunofluorescence intensity was evaluated as 137.20 (SD 4.65), 67.97 (3.93), 75.24 (4.44), 70.66 (4.11) and 28.81 (1.86), respectively, for ␣, ␤, ␥, ␦ and ␧-SG (fig. 3). Student’s t test data values are summarized in table 2. All values are included in table 3 for easier comparison.

FIG. 1. Compound panel of immunohistochemical findings in control ureteral endings

SARCOGLYCANS AND VESICOURETERAL REFLUX

1983

TABLE 2. Statistical comparison of fluorescent intensities of each SG subunit

Control group: ␣ ␤ ␥ ␦ Low grade reflux group: ␣ ␤ ␥ ␦ High grade reflux group: ␣ ␤ ␥ ␦

␤

␥

␦

␧

⬍0.05 — — —

⬍0.05 0.592 (NS) — —

⬍0.001 0.459 (NS) 0.157 (NS) —

⬍0.001 0.842 (NS) 0.404 (NS) 0.539 (NS)

⬍0.05 — — —

⬍0.05 0.815 (NS) — —

⬍0.001 0.473 (NS) 0.367 (NS) —

⬍0.001 ⬍0.001 ⬍0.001 ⬍0.05

⬍0.001 — — —

⬍0.001 0.039 (NS) — —

⬍0.001 0.517 (NS) 0.157 (NS) —

⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001

p Values were obtained using Student’s t test.

Using RT PCR and nucleotide sequencing analysis with specific primers, we confirmed the presence of ␣ and ␧-SG, as well as ␤, ␥ and ␦-SG (data not shown) in control and refluxing ureteral ends (fig. 4). The Spearman test revealed a significant positive correlation between ␣-SG immunofluorescence intensity and VUR grade (p ⬍0.001, r ⫽ 0.6748, 95% CI 0.5987 to 0.7388), while a negative correlation between ␧-SG immunofluorescence intensity and VUR grade (p ⬍0.001, r ⫽ ⫺0.4969; 95% CI ⫺0.5873 to ⫺0.3942) was recorded. DISCUSSION It has recently been confirmed that RUs share a grade dependent disorganization of smooth muscle fibers and an altered smooth muscle cell structure, with subsequent dys-

function of the ostial valve and an inadequately active antireflux mechanism.1,15 In our study manometry demonstrated a significant decrease in peak isovolumetric pressure and basal pressure in RUs, despite the control values and a highly significant negative correlation of these values with the VUR grade. Moreover, in high grade VUR (IV to V) manometry showed severe impairment of basal pressure and ureteral arrhythmia in 73% of RUs, and a “silent” pressure profile pattern in 27%.1 In the former the manometric record featured irregular (bicuspid) and intermittent peristaltic waves. The silent ureter might represent an advanced stage of ureteral arrhythmia, suggesting a more damaged ureter that resembles a ureteral arrhythmia state, and it could be argued that in the silent pattern of VUR superficial contractions can occur, with no changes of pres-

FIG. 2. Compound panel of immunohistochemical findings in low grade refluxing ureteral endings. Moderate enhancement of ␣-SG is evident, coupled with impairment of ␧-SG.

1984

SARCOGLYCANS AND VESICOURETERAL REFLUX

FIG. 3. Compound panel of immunohistochemical findings in high grade refluxing ureteral endings. Marked increase in ␣-SG is accompanied by loss of ␧-SG.

sure in the urine column.1 The variable or inconsistent pressures of the peristaltic waves and the irregular wave rhythm are likely to result in disturbed urine transport along the distal part of the RUs. Sarcoglycans seem to be functionally and pathologically important in maintaining sarcolemmal stability, so that mutations in SG genes lead to muscular dystrophy in humans and mice.16 In control ureters immunofluorescence analysis revealed a normal staining pattern of ␤, ␥, ␦ and ␧-SG, while ␣-SG staining was reduced but clearly detectable, suggesting a pentameric or hexameric organization of this subcomplex.14 To our knowledge the immunofluorescence of SG in refluxing ureteral ends has never been investigated. Our immunohistochemical data and relative semiquantitative analysis provided by the software function “display profile” applied to each SG subunit, and our molecular data obtained by RT PCR demonstrated a significant grade related deficiency of ␧-SG and increased expression of ␣-SG. We believe this varied behavior of SG subunits could be explained in 2 ways. First, the SG subcomplex could have a

TABLE 3. Fluorescence intensity values of all tested sarcoglycans in controls, and low and high grade VUR cases

␣ ␤ ␥ ␦ ␧

Control Mean ⫾ SD

Low Grade VUR Mean ⫾ SD

High Grade VUR Mean ⫾ SD

42.48 ⫾ 4.34 76.25 ⫾ 9.82 72.97 ⫾ 35.12 72.97 ⫾ 35.12 69.68 ⫾ 7.27

78.80 ⫾ 3.02 65.72 ⫾ 5.74 67.75 ⫾ 6.51 67.75 ⫾ 6.51 61.68 ⫾ 6.10

137.20 ⫾ 4.65 67.97 ⫾ 3.93 75.24 ⫾ 4.44 75.24 ⫾ 4.44 28.81 ⫾ 1.86

p Values were obtained using limited sampling model function “display profile.”

key role in the physiopathology of VUR. In fact, mouse ␣ and ␧-SG are 43% identical at the amino acid level and, thus, the intracellular domain of ␧-SG (98AAs) is larger than that of ␣-SG (76AAs).17 Thus, the 2 SGs are similar but not identical, and could have distinct roles in the context of the plasma membrane. Previous biochemical studies have shown that the SG complex reinforces the dystrophin-dystroglycan molecular linkage between the extracellular matrix and cytoskeletal actin,18 preventing the dystrophic feature.19 A defect in any of the SGs results in specific loss of the SG subcomplex, destabilization of ␣-dystroglycan, loss of membrane integrity and fiber degeneration.19 Furthermore, this subcomplex is critical for conferring stability to the muscle membrane, thus, protecting the sarcolemma from stress that develops during muscle fiber contraction.19 The other explanation for the variation in SG subunit expression could be a structural deficiency of the trigonal UVJ, which could provoke a passive stretching of refluxing urine on the ureter with derangement of the SG subcomplex. This effect can also be explained by reduced amplitude of contraction and relaxation of the smooth muscle fibers of the ureter caused by structural deficiency and altered ureteral peristalsis.1 This condition could provoke a loss of mechanical stress transmitted over cell surface receptors that physically couple the cytoskeleton to the extracellular matrix or to other cells. Therefore, mechanical signals could be integrated with other environmental signals and transduced into a biochemical response through force dependent changes in scaffold geometry or molecular mechanisms by multimodular tensegrity architecture.20 Resultant changes in the topology of these networks could alter cellular biochemistry directly. Consequently, these mechanical changes

SARCOGLYCANS AND VESICOURETERAL REFLUX

1985

RUs might be the consequence of the disruption of smooth muscle cells, progenitors of ICCs, or secondary to mechanical stress, affecting the expression of developmental genes, during episodes of VUR.1 Conversely, ␧-SG is reported to be highly expressed in vascular smooth muscle, and it is known that abnormalities in smooth muscle SGC lead to vascular disturbance because endothelial cells release agents that could regulate the function of underlying vasculature smooth muscle, thereby controlling vascular tone by modulating the local concentration of vasoactive substances.12,22 Loss of ␧-SG in RUs might explain the overall microperfusion, ongoing functional and structural alterations of vesicoureteral junction, and deterioration of the active antireflux mechanism.6 CONCLUSIONS

FIG. 4. Two percent agarose gel electropherogram of RT PCR products amplified using human RNA as template, primer pairs of ␣ and ␧-SG, and primer pairs of GAPDH (internal control). bp, base pair. C⫹, positive correlation. C⫺, negative correlation.

could cause modifications of chemical signals, although variations of the structural pathway of the SG subcomplex and ␣-SG could replace or compensate the loss of ␧-SG. Our results are consistent with previously reported data.1,3,5–7 In fact, an altered configuration of the SG complex, in particular the deficiency of ␧-SG and over expression of ␣-SG, could explain the structural and functional changes in RUs, displaying smooth muscle cell apoptosis, smooth muscular atrophy, increased interstitial fibrosis, hypoperistaltic or ureteroarrhythmic manometric pattern and, at least, the impaired overall microperfusion, diminishing the active valve mechanism of the ureterovesical junction. A changed membrane permeability of mitochondrial membranes in smooth muscle cells of RUs has recently been described, and this event is considered an important step in the apoptotic or necrotic cell death cascade.15 An altered expression of SGs, causing a structural instability to the muscle plasma membrane, increases permeability of muscle fibers to intravenous Evans blue dye,20,21 and might promote damage from stress that develops during smooth muscle fiber contraction in RUs, loss of membrane integrity, smooth muscle cell apoptosis and fiber degeneration. Moreover, loss or mutation of ␧-SG is reported to cause the myoclonic dystonia syndrome in humans, and manometric findings in the vesicoureteral junction of RUs, including hypoperistalsis and ureteroarrhythmia or “silent” pattern, are in accordance with local and grade related impairment of ␧-SG, with subsequent altered motility in distal RUs.1,22 It is noteworthy that the depletion of c-kit positive ICCs in

It is unknown why impairment of ␧-SG and increased expression of ␣-SG occur in RUs. However, delayed expression during development of ␧-SG and subsequent late maturation of ureteral ends are consistent with a possible spontaneous postnatal resolution of VUR. Our observations underscore the assumption that primary VUR might be regarded as a sarcoglycanopathy with marked quantitative deficiency of ␧-SG and over expression of ␣-SG. These data open a new area in our understanding of the real protein organization and behavior of the SG subcomplex during smooth muscular pathology. Nevertheless, additional physiological data are necessary to determine if the replacement of ␧-SG by ␣-SG is primary or secondary with respect to VUR. ACKNOWLEDGMENTS Dr. Antony Bridgewood, University of Catania, revised and corrected the language in the article.

Abbreviations and Acronyms DGC ICCs NS PBS PCR RT RUs SG UVJ VUR

⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽

dystrophin-glycoprotein complex interstitial cells of Cajal not significant phosphate buffered saline polymerase chain reaction reverse transcriptase refluxing ureteral endings sarcoglycan ureterovesical junction vesicoureteral reflux

REFERENCES 1.

Arena S, Fazzari C, Arena F, Scuderi MG, Romeo C, Nicotina PA et al: Altered “active” antireflux mechanism in primary vesico-ureteric reflux: a morphological and manometric study. BJU Int 2007; 100: 407. 2. Paquin AJ Jr: Ureterovesical anastomosis: the description and evaluation of a technique. J Urol 1959; 82: 573. 3. Schwentner C, Oswald J, Lunacek A, Fritsch H, Deibl M, Bartsch G et al: Loss of interstitial cells of Cajal and gap junction protein connexin 43 at the vesicoureteral junction in children with vesicoureteral reflux. J Urol 2005; 174: 1981. 4. Tanagho EA, Guthrie TH and Lyon RP: The intravesical ureter in primary reflux. J Urol 1969; 101: 824.

1986 5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

SARCOGLYCANS AND VESICOURETERAL REFLUX

Oswald J, Brenner E, Deibl M, Fritsch H, Bartsch G and Radmayr C: Longitudinal and thickness measurement of the normal distal and intravesical ureter in human fetuses. J Urol 2003; 169: 1501. Schwentner C, Oswald J, Lunacek A, Schlenck B, Berger AP, Deibl M et al: Structural changes of the intravesical ureter in children with vesicoureteral reflux— does ischemia have a role? J Urol 2006; 176: 2212. Oswald J, Schwentner C, Brenner E, Deibl M, Fritsch H, Bartsch G et al: Extracellular matrix degradation and reduced nerve supply in refluxing ureteral endings. J Urol 2004; 172: 1099. Weller B, Karpati G and Carpenter S: Dystrophin-deficient mdx muscle fibers are preferentially vulnerable to necrosis induced by experimental lengthening contractions. J Neurol Sci 1990; 100: 9. Petrof BJ, Shrager JB, Stedman HH, Kelly AM and Sweeney HL: Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci U S A 1993; 90: 3710. Wheeler MT and McNally EM: Sarcoglycans in vascular smooth and striated muscle. Trends Cardiovasc Med 2003; 13: 238. Nigro V, de Sa Moreira E, Piluso G, Vainzof M, Belsito A, Politano L et al: Autosomal recessive limb-girdle muscular dystrophy, LGMD2F, is caused by a mutation in the deltasarcoglycan gene. Nat Genet 1996; 14: 195. Barresi R, Moore SA, Stolle CA, Mendell JR and Campbell KP: Expression of gamma-sarcoglycan in smooth muscle and its interaction with the smooth muscle sarcoglycan-sarcospan complex. J Biol Chem 2000; 275: 38554. Straub V, Ettinger AJ, Durbeej M, Venzke DP, Cutshall S, Sanes JR et al: ␧-Sarcoglycan replaces ␣-sarcoglycan in smooth muscle to form a unique dystrophin-glycoprotein complex. J Biol Chem 1999; 274: 27989. Anastasi G, Cutroneo G, Sidoti A, Rinaldi C, Bruschetta D, Rizzo G et al: Sarcoglycan subcomplex expression in normal human smooth muscle. J Histochem Cytochem 2007; 55: 831. Sofikerim M, Sargon M, Oruc O, Dogan HS and Tekgul S: An electron microscopic examination of the intravesical ureter in children with primary vesico-ureteric reflux. BJU Int 2007; 99: 1127. Wheeler MT, Zarnegar S and McNally EM: Zeta-sarcoglycan, a novel component of the sarcoglycan complex, is reduced in muscular dystrophy. Hum Mol Genet 2002; 11: 2147. McNally EM, Ly CT and Kunkel LM: Human epsilon-sarcoglycan is highly related to alpha-sarcoglycan (adhalin), the limb girdle muscular dystrophy 2D gene. FEBS Lett 1998; 422: 27.

18.

Araishi K, Sasaoka T, Imamura M, Noguchi S, Hama H, Wakabayashi E et al: Loss of the sarcoglycan complex and sarcospan leads to muscular dystrophy in beta-sarcoglycandeficient mice. Hum Mol Genet 1999; 8: 1589. 19. Crosbie RH, Lebakken CS, Holt KH, Venzke DP, Straub V, Lee JC et al: Membrane targeting and stabilization of sarcospan is mediated by the sarcoglycan subcomplex. J Cell Biol 1999; 145: 153. 20. Ingber DE: Tensegrity: the architectural basis of cellular mechanotransduction. Annu Rev Physiol 1997; 59: 575. 21. Holt KH, Lim LE, Straub V, Venzke DP, Duclos F, Anderson RD et al: Functional rescue of the sarcoglycan complex in the BIO 14.6 hamster using ␦-sarcoglycan gene transfer. Mol Cell 1998; 1: 841. 22. Imamura M, Mochizuki Y, Engvall E and Takeda S: Epsilonsarcoglycan compensates for lack of alpha-sarcoglycan in a mouse model of limb-girdle muscular dystrophy. Hum Mol Genet 2005; 14: 775.

EDITORIAL COMMENT The authors should be congratulated for bringing up the important issue of sarcoglycan subcomplex expression in primary refluxing ureteral endings with regard to the pathophysiology of this important congenital disease. This study fits in perfectly with recently published series about sarcoglycan expression in skeletal muscle (reference 20 in article). These findings add another stone to the huge mosaic of the pathophysiological background of vesicoureteral reflux. It is now becoming more and more obvious that we need to revisit our understanding of conditions causing this most frequent congenital disease. More data are now available on the microanatomy1 and microenvironment (references 3 and 7 in article) of the ureterovesical junction, a crucial region that determines whether there is reflux (or not) or obstruction (or not). On the other hand, it is still too early to draw definitive conclusions with regard to the underlying molecular defects. Much more basic research is still necessary to rule out these conditions. Christian Radmayr Department of Pediatric Urology Medical University Innsbruck Innsbruck, Austria 1.

Oswald J, Brenner E, Schwentner C, Deibl M, Bartsch G, Fritsch H et al: The intravesical ureter in children with vesicoureteral reflux: a morphological and immunohistochemical characterization. J Urol 2003; 170: 2423.