Markers of fibrosis and inflammation in urinary bladder across six rodent models of type 1 and type 2 diabetes

Michel-Reher M1, Matthes J2, Castaneda T3, Elvert R3, Kannt A3, Christen U4, Arioglu-Inan E5, Pautz A1, Michel M1

Research Type

Pure and Applied Science / Translational

Abstract Category

Female Lower Urinary Tract Symptoms (LUTS) / Voiding Dysfunction

Abstract 366
Open Discussion ePosters
Scientific Open Discussion Session 10
Wednesday 27th September 2023
17:25 - 17:30 (ePoster Station 2)
Exhibit Hall
Animal Study Pathophysiology Detrusor Overactivity Underactive Bladder Basic Science
1. Johannes Gutenberg University, 2. University of Cologne, 3. Sanofi Research and Development, 4. Goethe University Frankfurt, 5. Ankara University
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Poster

Abstract

Hypothesis / aims of study
Bladder dysfunction is common in diabetes and can manifest as detrusor over- and underactivity. Conditions such as the overactive bladder syndrome are less efficiently treated in the presence of diabetes. As part of diabetic cystopathy, a major enlargement of the urinary bladder (often a doubling in weight) occurs in all rodent models of type 1 diabetes and many, but not all of type 2 diabetes [1, 3]. An enlargement (hypertrophy and/or hyperplasia) of tissues including prostate, skeletal muscle, heart, and kidney in non-diabetic animals is typically accompanied by fibrosis and (non-infectious) inflammation. This is also the case with bladder enlargement in euglycemic models, e.g., after bladder outlet obstruction. Nonetheless, it has been reported repeatedly that the bladder enlargement in the streptozotocin (STZ) model of type 1 diabetes is not accompanied by fibrosis, if anything the opposite [1]. However, no such information is available for other models of type 1 diabetes or any model of type 2 diabetes. Therefore, we have explored expression of markers of fibrosis (collagens I and III, and TGF-β) and of inflammation (MCP-1) in the urinary bladder of six models type 1 and 2 diabetes including examples that do and do not exhibit bladder enlargement.
Study design, materials and methods
Urinary bladder specimens were obtained from the following rodent models: A) STZ rats (female, type 1 model, enlarged bladder; including a treatment arm with empagliflozin n = 4-6); B) RIP-LCMV mice (both sexes; type 1 model, enlarged bladder; n = 9-11), C) ZSF rats (male, type 2 model, enlarged bladder, n = 4-6), D) insulin receptor substrate 2 (IRS2) knock-out mice (both sexes; type 2 model, no bladder enlargement, n = 8-12), E) ob/ob mice (both sexes, enlarged bladder, n = 7-15), and F) db/db mice (both sexes; including an additional ob/ob arm, enlarged bladder in ob/ob and moderately enlarged bladder in db/db, n = 13-16). Each study had been approved by the locally responsible animal committee.
mRNA isolation and expression analysis was performed by quantitative PCR using the 2(−ΔΔCT) method with normalization for dual GAPDH and β-actin expression (2). Data were excluded from the analysis if melting curves for the target mRNA or the references gene products GADPH and β-actin were inappropriate, or when a value differed by more than 2 log units from the others within the group. 
Except for the STZ rats, all data and samples came from studies primarily performed for other purposes, i.e., only the STZ rats were sacrificed for the purpose of our study. All available bladders from each study were used. Therefore, sample sizes were not based on formal power calculations for bladder endpoints, and the findings should be considered as exploratory, not as hypothesis-testing. Accordingly, no hypothesis-testing and only descriptive statistical analysis was applied. It was performed by unpaired two-tailed t-tests for the 2-armed studies and by ANOVA followed by Sidak’s multiple comparison tests for the 3-armed studies.
Results
In contrast to many non-diabetic models and in confirmation of findings in STZ-induced diabetes, the mRNA expression of collagen I was not increased in any of the six models (Figure 1). Similarly, expression of collagen III was not increased, except in RIP-LMCV mice. Neither was expression of TGF-β. The inflammatory marker MCP-1 also was not increased in any of the six models (Figure 2).
Interpretation of results
Our data are based on studies performed for other purposes, except for that on STZ rats. However, all bladder analyses had been prespecified. This led to different sample sizes between groups, i.e., ranging from 4-6 in STZ and ZSF rats, to more than 10 in the mouse studies. While the studies with the smaller sample sizes may have had too little statistical power, the general picture was consistent across all six studies, i.e., that the enlarged diabetic bladders did not exhibit an increased expression of collagens I and III (with one exception) and TGF-β or the inflammatory marker MCP-1.
Concluding message
This is the first report on markers of fibrosis and inflammation in the bladder of animal models of diabetes other than the STZ model of type 1 diabetes and based on five such models including four models of type 2 diabetes. Together with previously reported data in STZ-injected rats and mice, these data show that a lack of fibrosis and inflammation (at least at the level of mRNA expression for the markers studied here) is not limited to a single model of type 1 diabetes but a universal finding in rodent models of diabetic cystopathy. Apparently, the bladder enlargement developing in diabetes has a fundamentally distinct pathophysiology from that occurring in non-diabetic animal models.
Figure 1 Figure 1: mRNA expression of collagen I in bladders of six studies. Each datapoint represents one animal.
Figure 2 Figure 2: mRNA expression of MCP-1 in bladders of six studies. Each datapoint represents one animal.
References
  1. Ellenbroek JH, Arioglu-Inan E, and Michel MC. A systematic review of urinary bladder hypertrophy in experimental diabetes: Part 2. Comparison of animal models and functional consequences. Neurourol Urodyn 37: 2346-2360, 2018.
  2. Erdogan BR, Michel MB, Matthes J, Castañeda TR, Christen U, Arioglu-Inan E, Michel MC, and Pautz A. A comparison of urinary bladder weight in male and female mice across five models of diabetes and obesity. Front Pharmacol 14: 92355, 2023.
  3. Yesilyurt ZE, Matthes J, Hintermann E, Castañeda TR, Elvert R, Beltran-Ornelas JH, Silva-Velasco DL, Xia N, Kannt A, Christen U, Centurión D, Li H, Pautz A, Arioglu-Inan E, and Michel MC. Analysis of 16 studies in nine rodent models does not support the hypothesis that diabetic polyuria is a main reason of urinary bladder enlargement. Front Physiol 13: 923555, 2022.
Disclosures
Funding This work was funded in part by TÜBITAK-SBAG 118S443 and 119S769, Landesoffensive zur Entwicklung wissenschaftlich-ökonomischer Exzellenz (LOEWE; LOEWE Center for Translational Medicine and Pharmacology) of the State of Hessen, Germany, and Deutsche Forschungsgemeinschaft XI 139/2-1 (to NX), LI-1042/5-1 (to HL) and Mi 294/10-1. Some underlying studies were performed and/or funded by Sanofi-Aventis (identified as “Hoechst”) for purposes unrelated to this manuscript. Clinical Trial No Subjects Animal Species rat and mouse Ethics Committee Animal welfare committee of Ankara University, Ethics Committee of the State Ministry of Agriculture, Nutrition and Forestry, State of Hessen, Landesamt für Natur-, Umwelt- und Verbraucherschutz Nordrhein-Westfalen
22/11/2024 17:33:45