Hypothesis / aims of study
Urinary tract infections (UTI) occur about 10 times more frequently with increased severity in patients with diabetes mellitus. However, the mechanisms of increased UTI susceptibility in these patients are not well understood. Because of the growing incidence of diabetes (nearly half of the adult population of the United States is affected), understanding these mechanisms may improve clinical outcomes and reduce a burden on the healthcare system. Previous studies have demonstrated insulin signaling to be involved in antibacterial defences in the kidney [1]. However, hyperglycemia was shown to be involved in urothelial barrier dysfunction in diabetic rodents [2]. We have evaluated the role of urothelial insulin receptors (IR) in maintaining urothelial barrier in mouse bladders.
Study design, materials and methods
Mice with IR deleted in superficial and intermediate urothelial cells as well as mice with IR deleted in basal urothelial cells were generated in one of author’s laboratory. These mice with their respective littermate controls were anesthetized with isoflurane (5% induction, 2% maintenance), bladders excised and placed immediately into Ringer’s solution (111.2 mM NaCl, 25 mM NaHCO3, 4.8 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4 and 11.1 mM glucose, 95% O2 / 5% CO2, 37 °C). The organ was cut open along the ventral axis and mounted on a ring insert with a 0.09 cm2 opening. The ring with the tissue was then mounted between two halves of an Ussing chamber filled with 95% O2 / 5% CO2 bubbled Ringer’s solution at 37 °C. After tissue stabilization, current was regularly passed through Ag–AgCl electrodes and voltage deflection across the tissue measured. TER was calculated as maximal voltage change divided by current amplitude and multiplied by area (in cm2). Capacitance was determined from the Resistor-Capacitor (RC) time constant (τ, time required to charge the tissue) and resistance. To measure water and urea permeabilities, [3H] water (1 µCi/ml) and [14C] urea (0.25 µCi/ml) were added to the urothelial side and duplicate samples taken every 15 min from both hemichambers for 1-1.5 hours. Samples were placed into scintillation fluid and diffusive coefficients calculated as:
PD = Φ ⁄ A ∙ ∆C,
where Φ – tracer flux (increase in basolateral side (counts/sec), A – area of the apical membrane (cm2, from capacitance, 1 µF ≈ 1 cm2), ΔC – difference in concentrations of isotope across the membrane (counts/cm3).
Results
All knockout mice had normal phenotypes, bladder histopathology, serum insulin concentrations, and no evidence of hyperglycemia or glucosuria (not shown). Bladders from the mice with IR-deficient superficial and intermediate urothelial cells demonstrated a significant decrease in TER accompanied by an increase in permeabilities to water and urea compared to littermate controls. In contrast, bladders from mice with IR deletion in basal urothelial cells showed no difference in these values when compared to their littermate controls. (n = 4 – 6 mice/genotype, unpaired Student t-test, P < 0.05).
Interpretation of results
Our results indicate that deletion of IR in superficial and intermediate, but not in basal urothelial cells, independently from hyperglycemia or glucosuria, compromises the bladder urothelial barrier function that may lead to increased permeation of bladder wall to urine pathogens and solutes.