Hypothesis / aims of study
Although bolstered by intuition and confirmation bias, the dogma of "water-tight" bladder lining is undermined by the tritiated water absorption rate of 0.3-1mL/min from human bladder(ref.1) and saline absorption rate of 0.001mL/min from rat bladder (ref.2). This bladder surface area dependent, 300-fold decline in water absorption rate is facilitated by aquaporin channel subtypes expressed on intermediate cell layer of urothelium. However, asymmetric unit membrane of umbrella cells is renowned for restricting transcellular diffusion of urine constituents and the marked absence of aquaporin channels on luminal surface raises the question: what is the physical mechanism of water movement from bladder lumen to intermediate urothelium cells? Since tritiated water absorption rate (ref.1) increases with distended bladder wall expanding gap of tight junctions gap, a plausible mechanism for water movement preceding the facilitated diffusion of water by aquaporins into systemic circulation is the passive, paracellular diffusion of free and bound water through tight junctions.
The premise of paracellular diffusion is consistent with the decline in bladder volume recorded by periodic ultrasound measurement during sleep of healthy adults which mimics an average 500mL decline in 24hour urine volume with volitional extension of awake voiding interval from 3h to 5h (ref.3) for a week by healthy adults. However, instead of diffusion, water movement into human bladder was conjectured to be osmosis (ref.3), which formed our null hypothesis: water moves from urine to aquaporin channels of intermediate urothelium by osmosis or alternatively by paracellular passive diffusion. Since the net movement of free water from low osmolality of glomerular filtrate in the loop of Henle to the high osmolality of renal medulla is emblematic of osmosis, we relied on a well-established relationship between spin-lattice (T1) and spin-spin relaxation time (T2) constants of water protons with the osmolality to benchmark the osmolality gradient between urine and urothelium to the gradient between renal medulla and cortex for a conceptual evaluation of null hypothesis.
Study design, materials and methods
Four months old female B6D2F1 mice (n=3) were anesthetized by 1-2% isoflurane and abdomen was secured to platform for reducing motion artifacts during MRI at 7 Tesla by 30-cm AVIII spectrometer using an 86 mm quadrature RF volume coil with a 4-channel receive surface array. T2-weighted coronal scans were acquired by rapid acquisition with resolution enhancement (RARE) sequence with following parameters: repetition time (TR)/echo time (TE) = 3000/40 ms, field of view (FOV) of 40 mm2, acquisition matrix = 128 × 128, slice thickness of 0.8 mm for 15 slices, 2 signal averages, and a RARE factor = 8. Coronal T1 maps were acquired using a variable TR sequence: 400, 842, 1,410, 2,208, 3,554 and 10,000 ms, echo time (TE) = 7 ms, 9 contiguous 0.8 mm slices, RARE factor = 2, 2 signal averages, 28 mm2 FOV and matrix = 218 × 218. T1 maps were processed using a 3-parameter single exponential function and unpaired Student's t test assessed the difference between urothelium and urine T1 for correlation with published osmolality values.
Interpretation of results
Since true osmosis is antithetical to free water movement from significantly higher osmolality of urine to lower osmolality of urothelium, T1 mapping of urine and urothelium affirms the alternative hypothesis of passive paracellular diffusion of free (ref.1) and bound water (ref.2) as spheres of Stokes-Einstein radius (>1.37Angstrom) through the tortuous gap of tight junctions assembled at mammalian umbrella cell borders. The compliance of paracellular diffusion with Stokes diffusion principle-inverse size dependence-is self-evident from three times faster diffusion rate of three times smaller hydrated sodium (1.3 Angstrom) than dextrose (~3.28Angstrom), determining the three times higher systemic uptake of saline than dextrose (ref.2). As the radius of water molecule is three times smaller than polar dyes (Fluorescein and Gadolinium chelate), the fluorescence and image contrast of umbrella cell borders visually affirms the paracellular diffusion of water whereas dark apical surface of umbrella cells attests the restricted transcellular diffusion of water. Furthermore, free water reabsorption is inevitable to cause a feed-forward rise in the osmolality of residual urine, which elevates the concentration gradient for Fickian diffusion of water bound to Na+/K+ or urea from lumen. Importantly, local buildup of diffused agents triggers a reflexive, homeostatic acceleration of urothelial blood flow as measured during potassium sensitivity test (PST) and corroborated by the rapid systemic distribution of instilled drugs (DMSO, Formalin and Lidocaine). While the concentration gradient generated by higher osmolality of residual urine provides the pushing force, the clearance of diffused agent from urothelial blood provides the pulling force in same direction for sustaining the concentration gradient which is also facilitated by a forty-fold upregulation of aquaporins in the intermediate cell layer of urothelium (ref.2) to accelerate the Fickian diffusion of free as well as bound water into systemic circulation at the rate of 1mL/min(ref.1). Thus, the reduction in 24h urine volume with mere extension of voiding interval by 2h (ref.3) exhibits a homeostatic mechanism of reflexive acceleration of urothelial blood flow to augment Fickian diffusion of water that delays the awakening of healthy adults while they sleep and prevents the buildup of K+ from irritating sensory nerve endings of urothelium that occurs in PST of cystitis patients but not in healthy adults. While rich vasculature saves dense innervation of urothelium from irritation, sparse innervation tolerates counter-current multiplier mechanism erected by slow blood flow of vasa recta capillaries for osmotic movement of water into renal medulla (Fig.2).