TMEM16A channel upregulation in arterial smooth muscle cells produces vasoconstriction during diabetes.

The pathological involvement of anion channels in vascular dysfunction that occurs during type 2 diabetes (T2D) is unclear. Here, we tested the hypothesis that TMEM16A, a calcium-activated chloride (Cl-) channel, contributes to modifications in arterial contractility during T2D. Our data indicate that T2D increased TMEM16A mRNA in arterial smooth muscle cells and total and surface TMEM16A protein in resistance-size cerebral and hindlimb arteries of mice. To examine vascular cell types in which TMEM16A protein increased and the functional consequences of TMEM16A upregulation during T2D, we generated tamoxifen-inducible, smooth muscle cell-specific TMEM16A knockout (TMEM16A smKO) mice. T2D increased both TMEM16A protein and Cl- current density in arterial smooth muscle cells of control (TMEM16Afl/fl) mice. In contrast, T2D did not alter arterial TMEM16A protein or Cl- current density in smooth muscle cells of TMEM16A smKO mice. Intravascular pressure stimulated greater vasoconstriction (myogenic tone) in the arteries of T2D TMEM16Afl/fl mice than in the arteries of nondiabetic TMEM16Afl/fl mice. This elevation in myogenic tone in response to T2D was abolished in the arteries of T2D TMEM16A smKO mice. T2D also reduced Akt2 protein and activity in the arteries of T2D mice. siRNA-mediated knockdown of Akt2, but not Akt1, increased arterial TMEM16A protein in nondiabetic mice. In summary, data indicate that T2D is associated with an increase in TMEM16A expression and currents in arterial smooth muscle cells that produces vasoconstriction. Data also suggest that a reduction in Akt2 function drives these pathological alterations during T2D.NEW & NOTEWORTHY We investigated the involvement of TMEM16A channels in vascular dysfunction during type 2 diabetes (T2D). TMEM16A message, protein, and currents were higher in smooth muscle cells of resistance-size arteries during T2D. Pressure stimulated greater vasoconstriction in the arteries of T2D mice that was abolished in the arteries of TMEM16A smKO mice. Akt2 protein and activity were both lower in T2D arteries, and Akt2 knockdown elevated TMEM16A protein. We propose that a decrease in Akt2 function stimulates TMEM16A expression in arterial smooth muscle cells, leading to vasoconstriction during T2D.

TMEM16A deficiency: a potentially fatal neonatal disease resulting from impaired chloride currents.

INTRODUCTION: TMEM16A is a calcium-activated chloride channel expressed in various secretory epithelia. Two siblings presented in early infancy with reduced intestinal peristalsis and recurrent episodes of haemorrhagic diarrhoea. In one of them, the episodes were characterised by hepatic pneumatosis with gas bubbles in the portal vein similar to necrotising enterocolitis of the newborn. METHODS: Exome sequencing identified a homozygous truncating pathogenic variant in ANO1. Expression analysis was performed using reverse transcription PCR, western blot and immunohistochemistry. Electrophysiological and cell biological studies were employed to characterise the effects on ion transport both in patient respiratory epithelial cells and in transfected HEK293 cells. RESULTS: The identified variant led to TMEM16A dysfunction, which resulted in abolished calcium-activated Cl- currents. Secondarily, CFTR function is affected due to the close interplay between both channels without inducing cystic fibrosis (CF). CONCLUSION: TMEM16A deficiency is a potentially fatal disorder caused by abolished calcium-activated Cl- currents in secretory epithelia. Secondary impairment of CFTR function did not cause a CF phenotyp, which may have implications for CF treatment.

Nephron-specific knockout of TMEM16A leads to reduced number of glomeruli and albuminuria.

TMEM16A is a transmembrane protein from a conserved family of calcium-activated proteins that is highly expressed in the kidney. TMEM16A confers calcium-activated chloride channel activity, which is of importance for various cellular functions in secretory epithelia and involved in secretion-dependent renal cyst growth. However, its specific function in renal physiology has remained elusive so far. Therefore, we generated conditional nephron-specific TMEM16A-knockout mice and found that these animals suffered from albuminuria. Kidney histology demonstrated an intact corticomedullary differentiation and absence of cysts. Electron microscopy showed a normal slit diaphragm. However, the total number of glomeruli and total nephron count was decreased in TMEM16A-knockout animals. At the same time, glomerular diameter was increased, presumably as a result of the hyperfiltration in the remaining glomeruli. TUNEL and PCNA stainings showed increased cell death and increased proliferation. Proximal tubular cilia were intact in young animals, but the number of properly ciliated cells was decreased in older, albuminuric animals. Taken together, our data suggest that TMEM16A may be involved in ureteric bud branching and proper nephron endowment. Loss of TMEM16A resulted in reduced nephron number and, subsequently, albuminuria and tubular damage.