Role Of Spinal Cord K~+-Cl~- Cotransporter Isoform 2 In The Visceral Hypersensitivity Of Irritable Bowel Syndrome

Background and aims: Visceral hypersensitivity represents an important hallmark in the pathophysiology of irritable bowel syndrome(IBS), of which the mechanisms remain elusive. K+-Cl- cotransporter isoform 2(KCC2), which is essential for the maintenance of chloride homeostasis, may play an important role in controlling the efficacy of visceral nociceptive transmission in spinal cord by modulating the strength of γ-aminobutyric acid(GABA)/glycine mediated synaptic inhibition. The present study was designed to examine the role of KCC2 in the development of IBS-like visceral hypersensitivity.Methods:(1) Chronic visceral hypersensitivity was induced by exposing male Wistar rats to water avoidance stress(WAS) 1 hour per day for consecutive 10 days. Visceral sensitivity was estimated by measuring the abdominal withdrawal reflex(AWR) and visceromotor response(VMR) in response to colorectal distension(CRD). H&E staining of the colon was performed for histology evaluation.(2) Reverse transcription-polymerase chain reaction(RT-PCR), western blotting and immunofluorescence were used to assess the expression and cellular localization of proteins responsible for chloride homeostasis in lumbosacral and thoracolumbar spinal dorsal horn.(3) Whole-cell patch-clamp recordings were performed on adult spinal cord slices to evaluate Cl- homeostasis and Cl- extrusion capacity of lamina I neurons.(4) Visceral sensitivity was estimated after intrathecal injection of a KCC2 inhibitor R-(+)-butylindazone(DIOA).Results:(1) WAS exposure for 10 days induced visceral hypersensitivity without significant inflammation, manifested as decreased nociceptive thresholds(sham, 35.8± 1.8 mm Hg; WAS, 25.8± 2.4 mm Hg; p<0.01) and increased AWR scores and VMR amplitudes in response to CRD stimulus.(2) Compared with the control group, levels of both total protein(reduced to ~65% of the control level, p=0.01) and the monomeric(reduced to ~74% of the control level, p<0.01) and oligomeric forms(reduced to ~57% of the control level, p<0.01) of KCC2, but not Na+-K+-2Cl- cotransporter isoform 1(NKCC1) which is another protein responsible for the maintenance of chloride homeostasis, were significantly decreased in the lumbosacral spinal dorsal horn of the WAS rats.(3) The downregulation of KCC2 resulted in a depolarizing shifted equilibrium potential of GABAergic inhibitory postsynaptic current(ECl)(sham,-62.4±0.9 m V; WAS,-51±1.7 m V; p<0.01) and impaired Cl- extrusion capacity in lamina I neurons of the lumbosacral spinal cord from WAS rats.(4) Pharmacological blockade of KCC2 activity by intrathecal injection of a KCC2 inhibitor DIOA caused a rapid and reversible sensitization of visceral nociception in sham rats. By twenty minutes after DIOA application(30 μg), the VMR to CRD in sham rats were significantly increased by 30% and 36%, at 40 and 60 mm Hg pressures, respectively(p<0.05 or p<0.01 vs. vehicle). The pro-nociceptive effects of DIOA gradually diminished, and the VMR returned to the vehicle control level at 2 h after DIOA application(p>0.05 vs. vehicle). DIOA had no effect on VMR to CRD when administered to WAS rats(p>0.05 vs. vehicle).Conclusions: Our results suggest that KCC2 downregulation-mediated spinal cord chloride homeostasis disruption may play an important role in chronic stress-induced visceral hypersensitivity.