Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow-to-fast fibre type transition of the slow-twitch soleus muscle (Sol). After 2 weeks HU, the resting ClC-1 chloride conductance (g Cl ) of sarcolemma, which controls muscle excitability, increases in Sol toward a value typical of the fast-twitch EDL muscle. After 3 days of HU, the g Cl increases as well before initiation of fibre type transition. Since ClC-1 channels are acutely silenced by PKC-dependent phosphorylation, we studied the modulation of g Cl by PKC and serine-threonine phosphatase in Sol during HU, using a number of pharmacological tools. We show that a fraction of ClC-1 channels of control Sol are maintained in an inactive state by PKC basal activity, which contributes to the lower g Cl in control Sol compared to EDL. After 14 days of HU, PKC/phosphatase manipulation produces effects on Sol g Cl that corroborate the partial slow-to-fast transition. After 3 days of HU, the early increase of g Cl in Sol is entirely attributable to a reduction of PKC activity and/or activation of phosphatase, maintaining ClC-1 channels in a fully active state. Accordingly, we found that HU reduces expression of PKCα, ε, and θ isoenzymes in Sol and EDL muscles and reduces total PKC activity. Moreover, we show that the rheobase current is increased in Sol muscle fibres as soon as after 3 days of HU, most probably in relation to the increased g Cl . In conclusion, Sol muscle disuse is characterized by a rapid reduction of PKC activity, which reduces muscle excitability and is likely to contribute to disuse-induced muscle impairment.

Aquaporin-2 (AQP2) increases the water permeability of renal collecting ducts in response to vasopressin. Vasopressin stimulation is accompanied by a profound remodeling of actin cytoskeleton whose dynamics are regulated by crosstalk between intracellular and extracellular signals. Here, we report that AQP2 contains a conserved RGD domain in its external C-loop. Co-immunoprecipitation experiments demonstrated that AQP2 binds integrin β1 in renal tissue and in MCD4 cells. To investigate the role of this interaction on AQP2 trafficking, cells were exposed to synthetic RGD-containing peptides, GRGDNP or GRGDSP, able to bind certain integrins. Incubation with these peptides increased the membrane expression of AQP2 in the absence of hormonal stimulation as assessed by confocal analysis and cell surface biotinylation. To identify the signals underlying the effects of peptides on AQP2 trafficking, some possible intracellular messengers were evaluated. Exposure of MCD4 cells to GRGDNP increased intracellular cAMP as assessed by FRET studies while GRGDSP increased intracellular calcium concentration. Taken together, these data propose integrins as new players controlling the cellular localization of AQP2, via two distinct signal transduction pathways dependent on cAMP and calcium respectively.

Diabetic nephropathy (DN) is a microangiopathic complication of diabetes mellitus (DM) affecting one-third of diabetic patients. The large variability in the clinical presentation of renal involvement in patients with DM makes kidney biopsy a prerequisite for a correct diagnosis. However, renal biopsy is an invasive procedure associated with risk of major complications. Numerous studies aimed to identify a noninvasive biomarker of DN but, so far, none of these is considered to be sufficiently specific and sensitive. Water channel aquaporins (AQPs), expressed at the plasma membrane of epithelial tubular cells, are often dysregulated during DN. In this work, we analyzed the urine excretion of AQP5 and AQP2 (uAQP5 and uAQP2), via exosomes, in 35 diabetic patients: 12 normoalbuminuric with normal renal function (DM), 11 with proteinuric nondiabetic nephropathy (NDN), and 12 with histological diagnosis and classification of DN. ELISA and WB analysis independently showed that uAQP5 was significantly increased in DN patients. Interestingly, linear regression analysis showed a positive correlation between uAQP5 and the histological class of DN. The same analysis, focusing on uAQP2, showed comparable results. Taken together, these data suggest a possible use of AQP5 and AQP2 as novel noninvasive biomarkers to help in classifying the clinical stage of DN.

One mechanism proposed for reducing the risk of calcium renal stones is activation of the calcium-sensing receptor (CaR) on the apical membranes of collecting duct principal cells by high luminal calcium. This would reduce the abundance of aquaporin-2 (AQP2) and in turn the rate of water reabsorption. While evidence in cells and in hypercalciuric animal models supports this hypothesis, the relevance of the interplay between the CaR and AQP2 in humans is not clear. This paper reports for the first time a detailed correlation between urinary AQP2 excretion under acute vasopressin action (DDAVP treatment) in hypercalciuric subjects and in parallel analyzes AQP2-CaR crosstalk in a mouse collecting duct cell line (MCD4) expressing endogenous and functional CaR. In normocalciurics, DDAVP administration resulted in a significant increase in AQP2 excretion paralleled by an increase in urinary osmolality indicating a physiological response to DDAVP. In contrast, in hypercalciurics, baseline AQP2 excretion was high and did not significantly increase after DDAVP. Moreover DDAVP treatment was accompanied by a less pronounced increase in urinary osmolality. These data indicate reduced urinary concentrating ability in response to vasopressin in hypercalciurics. Consistent with these results, biotinylation experiments in MCD4 cells revealed that membrane AQP2 expression in unstimulated cells exposed to CaR agonists was higher than in control cells and did not increase significantly in response to short term exposure to forskolin (FK). Interestingly, we found that CaR activation by specific agonists reduced the increase in cAMP and prevented any reduction in Rho activity in response to FK, two crucial pathways for AQP2 translocation. These data support the hypothesis that CaR–AQP2 interplay represents an internal renal defense to mitigate the effects of hypercalciuria on the risk of calcium precipitation during antidiuresis. This mechanism and possibly reduced medulla tonicity may explain the lower concentrating ability observed in hypercalciuric patients.

We screened human kidney-derived multipotent CD133+/CD24+ ARPCs for the possible expression of all 13 aquaporin isoforms cloned in humans. Interestingly, we found that ARPCs expressed both AQP5 mRNA and mature protein. This novel finding prompted us to investigate the presence of AQP5 in situ in kidney. We report here the novel finding that AQP5 is expressed in human, rat and mouse kidney at the apical membrane of type-B intercalated cells. AQP5 is expressed in the renal cortex and completely absent from the medulla. Immunocytochemical analysis using segment- and cell type-specific markers unambiguously indicated that AQP5 is expressed throughout the collecting system at the apical membrane of type-B intercalated cells, where it co-localizes with pendrin. No basolateral AQPs were detected in type-B intercalated cells, suggesting that AQP5 is unlikely to be involved in the net trans-epithelial water reabsorption occurring in the distal tubule. An intriguing hypothesis is that AQP5 may serve an osmosensor for the composition of the fluid coming from the thick ascending limb. Future studies will unravel the physiological role of AQP5 in the kidney.