Our translational research program combines investigation into the basic science rationale (i.e. genetics, molecular biology) behind disease processes with an understanding of the clinical issues faced by patients and providers.
This program represents a fusion between our clinical outcomes program, in which we collect information about a patient's current functional status, and our laboratory effort.
The most common cause of incontinence we see in pediatrics is due to dysfunctional voiding. Our DOVE team is using a new modified questionnaire to develop a better profile of the patient with voiding dysfunction. This enables us to quantify the degree of incontinence and serves as the key measure in clinical trials of new management strategies.
Our urology research laboratory uses a male mouse model of partial bladder outlet obstruction we developed in an effort to better understand how the bladder responds to an increase in outlet resistance. With the imposition of a partial outlet resistance, the bladder may adapt or compensate to its new demands and be able to empty with a higher pressure, but low post-void residual urine. However, with time, the bladder may decompensate, and this state is characterized by increased voiding pressures and a huge increase in post-void residual urine.
Dr. Zedric’s research study was awarded second prize in the basic science research competition during the October 2011 meeting of the American Academy of Pediatrics in Boston.
In this study Stephen Zderic, MD, and his lab team, Joanna Sliwoski and Stephan Butler, set out to understand the relationship between the degree of hypertrophy induced by partial bladder outlet obstruction (pBOO) and the appearance of the following transcription factors: Nuclear Factor of Activated T-cells (NFAT) and Hypoxia Inducible Factor (HIF1-α) as well as the mRNA expression of the slow myosin heavy chain isoform. We hypothesized these transcription factors would increase in a linear relationship with increasing bladder mass and that the slow MHC A isoform would increase in response to HIF1- α.
We found that nuclear accumulation of NFAT and HIF1-α mRNA expression increased in a near linear relationship with the rise in bladder mass following pBOO. In contrast, the nuclear importation of HIF1-α did not follow a linear relationship with rising bladder mass but rather rose sharply once bladder mass exceeded 80 mg implying that bladder wall hypoxia set in after this critical degree of hypertrophy was reached. This in turn correlated with the appearance of the slow myosin heavy chain A isoform which operates best under hypoxic conditions due to its low oxygen consumption; the trade off is a loss of power.
We suspect that the promoter for HIF-1α contains an NFATc1 responsive binding element: a CHIP analysis is currently in progress to test this assertion. This data suggests that as bladder mass exceeds 80 mg, a critical tipping point is reached as hypoxia sets in sending the bladder into a decompensated mode as the rise in the slow myosin A isoform results in a loss of power.
The following projects pertaining to bladder function are currently underway in our lab and are supported by funding from the National Institutes of Health (NIH) as well as our endowed chairs:
We have evidence that with partial outlet obstruction, there is damage to the calcium handling ability of the smooth muscle cell of the bladder. In time, a slow and steady rise in basal cell calcium levels leads to activation of calcineurin, which leads to nuclear importation of the transcription factor NFAT (Nuclear Factor of T-cells). This has been demonstrated using a mouse containing an NFAT-luciferase reporter gene inserted within its genome (a transgenic mouse).
Our current work is directed towards understanding which genes are activated by the 3 NFAT isoforms that are expressed in bladder using Chromatin ImmunoPrecipitation (CHIP) promoter microarrays, as well as by studying the effects of partial outlet obstruction in mice in which these isoforms have been genetically deleted.
We have also shown this pathway is activated within the urothelium. Studies are underway to understand how calcineurin expression within this layer may drive the changes seen in the underlying lamina propria which is the key site for DNA synthesis following outlet obstruction.
This project is of clinical relevance because cyclosporine A is a known inhibitor of calcineurin. In recently completed trials in our mouse model, we have shown that with the administration of cyclosporine A, there is less bladder wall hypertrophy, improved whole organ function and diminished deposition of extracellular matrix elements. It is unlikely that NFAT is the sole transcription factor driving bladder wall hypertrophy; rather, it acts in concert with a number of other as-yet-to-be defined transcription factors to elicit these changes. Two such candidates for which we have gathered evidence are Myocyte Enhancing Factor 2 (Mef-2) and Hypoxia Inducible Factor (HIF1α).
Clement MR, Delaney DP, Austin JC, Sliwoski J, Hii GC, Canning DA, DiSanto ME, Chacko SK, Zderic SA. Activation of the calcineurin pathway is associated with detrusor decompensation – a potential therapeutic target. J Urol. 2006 Sep;176(3):1225-9. Read the abstract»
Austin JC, Chacko SK, DiSanto M, Canning DA, Zderic SA. A male murine model of partial bladder outlet obstruction reveals changes in detrusor morphology, contractility and Myosin isoform expression. J Urol. 2004 Oct;172(4 Pt 1):1524-8. Read the abstract»
Chang, AY, Sliwoski, J, Butler, S, Hearn, G, Lassmann, J, Chacko, SK, Canning, DA, Zderic SA: Calcineurin mediates bladder wall hypertrophy secondary to partial outlet obstruction. AM J Physiol-Renal, 2011, submitted following revision
Following partial outlet obstruction, we have shown changes occur within the cells calcium sequestration system in a manner highly analogous to that seen in the heart following aortic stenosis. We undertook a study of partial bladder outlet obstruction in a mouse in which the SERCA-2 gene was deleted, and expected to find a greater degree of hypertrophy would develop. Much to our surprise, the mice with the gene deletion developed less hypertrophy than their normal littermates. The mice with the genetic deletion of SERCA-2 demonstrated less hypertrophy, improved whole organ function and an improved histologic picture. They also showed evidence of minimal DNA synthesis within the smooth muscle cells and substantial DNA synthesis within the interstitial cell populations of the bladder wall. Understanding the compensatory mechanisms following genetic deletion of SERCA-2 will provide valuable clues to improving bladder function in the face of an ongoing hypertrophic stimulus as seen in patients with neurogenic bladder of the fetus with a posterior urethral valve.
Lassmann J, Sliwoski J, Chang A, Canning DA, Zderic SA. Deletion of one SERCA2 allele confers protection against bladder wall hypertrophy in a murine model of partial bladder outlet obstruction. Am J Physiol Regul Integr Comp Physiol. 2008 Jan;294(1):R58-65. Epub 2007 Oct 31. Read full article»
This publication was featured on the cover of the January 2008 issue of the American Journal of Physiology.
Mice establish a social hierarchy, especially when housed with non-littermates. We have observed that in these situations, the younger and smaller mouse will develop a large distended bladder, and their voiding patterns change dramatically. In addition to undergoing hypertrophy, these mouse bladders also show signs of activation of the calcineurin NFAT pathway and shifts in their expression of myosin heavy chain isoforms. In vivo urodynamic studies done in a freely moving mouse, free of any anesthetics, have shown that social stress leads to an increase in the volume at which micturition is triggered, but does not change the voiding pressure. This data suggests that the social stress results in a suppression of the voiding reflex. All of these changes occur in the absence of any mechanical manipulation, and are the sole result of social stress.
We have also recently shown these changes once established are not reversed once the social stress is removed suggesting that once the voiding reflex is altered, the change is fixed. A key question is whether this is due to shifts in brain stem and spinal neural circuitry or whether memory plays a role.
In effect, this model serves as an example of how the mind has responded to an imposed stress and has lead to an alteration in the function of a pelvic visceral organ with changes in the expression of a transcription factor and mRNA coding for a major filament protein. This exciting new model represents a way in which we can begin to study the mind-body interaction and has clinical translational relevance to the large population of patients we see in the DOVE center.
Chang A, Butler S, Sliwoski J, Valentino R, Canning D, Zderic S. Social stress in mice induces voiding dysfunction and bladder wall remodeling. Am J Physiol Renal Physiol. 2009 Oct;297(4):F1101-8. Epub 2009 Jul 8. Read the abstract»
Butler, S, Luz, S, Sliwoski, J, Valentino, RJ, Canning, DA, Carr, MC, Zderic SA. Social stress induced voiding dysfunction: A permanent phenotypic shift? AM J Physiol-Renal. 2011, manuscript for submission.
Although it has been known for more than a century that boys with undescended testes (UDT) are at risk for low sperm counts and infertility, the reason for this has never been fully understood. The average age at which young boys undergo surgery to correct this problem is about 6 to 12 months, yet the age when one may reliably assess semen quality is 18 years.
We have undertaken a major research effort to follow boys from birth through 18 years to learn more about the long-term problems of infertility and testicular cancer. Over the past two decades, we have enrolled 140 patients who have agreed to periodic semen analysis as well as genetic testing (described below).
Using modern molecular techniques, scientists are able to amplify key segments of human DNA and determine their sequence. We are currently searching for single base-pair alterations that correlate with infertility in the population of patients with consistently low sperm counts.
Another novel approach to the study of infertility in these patients involves the use of microarray methods to search for changes in gene expression within the sperm of patients with cryptorchidism. Using this approach, it has been possible to identify decreases in the expression of the tpx-1 gene that correlate highly with low sperm counts. These surprising findings would not have come to light without the power of modern microarray methods, which allow for the simultaneous detection of shifts in the expression of more than 20,000 genes at one time.
Three areas of cryptorchidism research are currently active, each of which overlap with the others:
Some recent publications from this work include:
Varicocele is an abnormal enlargement of the veins in the scrotum draining the testicles. A urology patient database at The Children's Hospital of Philadelphia tracks the varicocele size, symptoms and testicular volumes over time with follow-up semen analyses as young adults.
Our research has found:
The Urology Division is part of a multispecialty collaboration between clinicians and basic scientists to achieve a goal of identifying and expanding spermatogonial stem cells (SSCs) from testicular tissue in at-risk patients. We offer testicular cryopreservation to prepubertal boys who are at risk for infertility from cancer therapy and explore the laboratory science required for both in vitro expansion of SSC and methods for freezing testicular tissue for future use.
An exciting new line of research focuses on factors associated with genital wound healing. Hypospadias is the most common congenital genital defect affecting 1 in 300 boys. Complications following hypospadias repair are common, especially when the original defect is severe. The most significant complications include the formation of fistulae and strictures. An added area of concern is post-operative cosmesis and scar formation.
Other than severity of presenting condition, few factors have been found to influence the rate of complications or scar formation. Clinically it has been observed that operations conducted when the child is less than 6 months of age seem to have fewer complications than those done when the child is 1 year of age. Basic science research has established that fetal wounds, which produce low levels of inflammatory cytokines, heal without a scar whereas adult wounds, which produce high cytokine levels, do scar. Thus age and cytokine level are implicated in the surgical outcome.
The goal of this project is to unite these two fields of study and determine whether wound healing following hypospadias repair is related to the child’s age at time of repair, the levels of inflammatory cytokines in the child’s genital skin, or both.