Diabetic Bladder Dysfunction Is Associated with Bladder Inflammation Triggered Through Hyperglycemia, Not Polyuria
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Journal name: Research and Reports in Urology Article Designation: Original Research Year: 2018 Volume: 10 Research and Reports in Urology Dovepress Running head verso: Inouye et al Running head recto: Hyperglycemia causes diabetic bladder dysfunction open access to scientific and medical research DOI: http://dx.doi.org/10.2147/RRU.S177633 Open Access Full Text Article ORIGINAL RESEARCH Diabetic bladder dysfunction is associated with bladder inflammation triggered through hyperglycemia, not polyuria Brian M Inouye1 Purpose: Diabetes is a grave and progressive condition characterized by debilitating compli- Francis M Hughes Jr1,2 cations. Diabetic bladder dysfunction (DBD) is a very common complication with no specific Huixia Jin1 treatments currently available. Unlike other tissues affected by this disease, the bladder is Robin Lütolf3 subjected to two independent insults; 1) polyuria, created by the osmotic effects of glucose Kunal C Potnis1 in the urine, and 2) hyperglycemia itself. Based on our understanding of inflammation as a major contributor to the underlying organ damage in several other diabetic complications, its Jonathan C Routh1,4 presence in the bladder during DBD and the contribution of polyuria and hyperglycemia to its Douglas C Rouse5 development were assessed. Wen-Chi Foo6 Methods: Awake, restrained cystometry was performed on wild type C57BL/6 mice and 1,2,4 J Todd Purves diabetic (Akita) mice on a C57BL/6 background at 15 weeks of age. A subgroup of the Akita 1Department of Surgery, Division mice were treated with phlorizin, an inhibitor of sodium-glucose linked transporter types 1 and of Urology, Duke University 2 that prevents glucose reabsorption in the kidney. All groups were assessed for serum glucose, Medical Center, Durham, NC, USA; 2Department of Bioengineering, 4-hour voiding totals, and inflammation in the bladder (Evans blue assay). Clemson University, Clemson, SC, Results: Akita mice develop cystometrically-defined DBD by 15 weeks of age, as evidenced USA; 3Department of Health Science by an increase in urinary frequency, a decrease in voiding volume, and an increase in post- and Technology, ETH Zurich, Zürich 8092, Switzerland; 4Department of voiding residual volume. Phlorizin effectively normalized serum glucose in these animals while Pediatrics, Duke University Medical increasing the urine output. Inflammation in the bladder was present in the diabetic animals at Center, Durham, NC, USA; 5Division of Laboratory Animal Medicine, Duke this time point, but not detectable in animals receiving phlorizin. University Medical Center, Durham, Conclusion: Inflammation in the bladder of diabetic mice correlates with the development of 6 NC, USA; Department of Pathology, DBD and is triggered by hyperglycemia, not polyuria. Duke University Medical Center, Durham, NC, USA Keywords: diabetes, inflammation, urinary bladder, urodynamics, cystitis, immunity, innate Introduction The WHO has estimated that, in 2014, there were 422 million people worldwide living with diabetes.1 Among those afflicted, somewhere between 25% and 90% had diabetic uropathy, a complication more common than neuropathy or nephropathy,2,3 and one that has been recognized since 1935.4 The wide range of this estimate is most likely due to the absence of a validated and standardized method for diagnosis and the 5,6 Correspondence: Francis M Hughes Jr selected referral of patients. Diabetic uropathy consists of diabetic bladder dysfunc- Department of Surgery, Division of tion (DBD), a more inclusive term than the previously en vogue diabetic cystopathy,7 Urology, Duke University Medical sexual or erectile dysfunction, and recurrent urinary tract infection. In recent years, Center, PO Box 3831, Durham, NC 27710, USA DBD has come to be recognized as a progressive set of symptoms, characterized in Tel +1 843 709 2128 the early stages by increased frequency, urgency, and even urge incontinence.8 These Fax +1 919 681 5507 Email [email protected] changes are also characteristic of the irritative symptoms associated with overactive submit your manuscript | www.dovepress.com Research and Reports in Urology 2018:10 219–225 219 Dovepress © 2018 Inouye et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work http://dx.doi.org/10.2147/RRU.S177633 you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Inouye et al Dovepress bladder (OAB).6 As diabetes develops, chronic DBD becomes Methods characterized by an insensate, decompensated bladder that Animals results in overflow incontinence and high post-void residual The Akita mouse strain contains a spontaneous mutation 6,8 volumes. However, it must be stated that, clinically, not in the insulin 2 (Ins2) gene, which causes it to fold incor- all patients will progress smoothly through these stages or rectly, leading to an inactive protein as well as producing present at the beginning of their disease. toxicity in pancreatic β cells.23 Mice heterozygous for the In the early stages of diabetes, hyperglycemia overwhelms Ins2 mutation are viable and fertile and begin developing the capacity for renal reabsorption of glucose, leading to diabetes around the time of weaning (4 weeks). Akita mice glucosuria and osmotic polyuria. Initially the bladder com- on a C57BL/6 background (C57BL/6-Ins2Akita/J, stock pensates, leading to increased urinary frequency. Contempo- number: 003548), and the wild type C57BL/6J strain (stock raneously in the bladder, as in all insulin-independent tissues, number: 000664) were purchased from Jackson labs and hyperglycemia drives enhanced oxidative phosphorylation bred in the mouse breeding core at Duke University Medi- that causes significant oxidative stress. Oxidative stress is cal Center. Wild type and heterozygote (ie, diabetic) female an imbalance between the production of reactive oxygen mice were provided to the lab upon weening (≈ 4 weeks of species (free radicals) and antioxidant defenses, resulting in age). Mice were housed socially, as required by the Guide excess radicals that react and damage all components of the for the Care and Use of Laboratory Animals published by cell including proteins, lipids, and DNA. Increased oxida- the US Public Health Service. Animals were housed 2–5 tive stress has been implicated in a vast number of diseases per cage, depending on how they were transferred from the 9 10 11 including heart disease, cancer, and, critically, diabetes, breeding core. Individual cages were not necessarily com- 6,12 and DBD in particular. It is this oxidative stress that trig- posed of one genotype or experimental group or targeted for 13–15 gers an inflammatory response in many target tissues. one endpoint. The exception to social housing were with the While for many years there has been general evidence animals implanted with a suprapubic tube for urodynamics. that inflammation plays an important role in the develop- These animals were housed individually to prevent them 16,17 ment of several diabetic complications, there has been from chewing one another’s tubes. Within 3 days of arrival little evidence that it is occurring in the bladder. Suggestive one group of mice were anesthetized (80 mg/kg ketamine/5 18 indications were provided when Wang and Kuo recently mg/kg xylazine, ip), treated prophylactically with carpro- found that mast cells in the urothelium and suburothelial fen (5 mg/kg, sc) and enrofloxacin (0.5 mL/kg of a 2.27% layer were increased in diabetic patients. In this study, we solution), and implanted with osmotic mini-pumps (Alzet, have examined the presence of inflammation in the bladder Cupertino, CA; cat# 2004; release rate 0.25 µL/h for 4 weeks) of the Akita diabetic mice at a time where they demonstrate containing phlorizin (40% w/v in propylene glycol).24 The DBD symptoms. To assess the contribution of polyuria effective dose was 120 mg/kg/day. Bupivacaine (0.25%) was and hyperglycemia to the activation of inflammation, we applied topically to the sutures after the procedure. Pumps have treated a group of these diabetic mice with phlorizin. were replaced every 4 weeks. Prior to any surgery mice were Phlorizin is a dihydrochalcone (a bicyclic flavonoid) that given preemptive analgesic (carprofen, 5 mg/kg). All animals functions as a competitive inhibitor of the sodium-glucose were maintained on a normal light:dark cycle (12 hours on, linked transporter types 1 and 2 (SGLT1 and SGLT2) in the 12 hours off) until 15 weeks of age, when they were used kidney and gut. Treatment with phlorizin prevents glucose for various studies. Animal protocols were reviewed and 19,20 reabsorption and results in increased glucose in the urine. approved by the Institutional Animal Care and Use Commit- Thus, phlorizin results in significantly lower blood glucose tee at Duke University Medical Center and conformed to the coupled with an enhanced polyuria (due to osmotic effects of Guide for the Care and Use of Laboratory Animals published glucosuria) allowing for discrimination between the effects by the US Public Health Service. of polyuria