A bell-shaped pattern of urinary aquaporin-2-bearing extracellular vesicle release in an experimental model of nephronophthisis.

The DBA/2-FG pcy (pcy) mouse is a model of human nephronophthisis, a recessive cystic kidney disease. Renal expression of aquaporin-2 (AQP2), a water channel protein, has been shown to be altered in pcy mice. However, the relationship between the renal expression and its release in urinary extracellular vesicles (uEV-AQP2), which account for most urinary AQP2, in pcy mice has remained largely unknown. In this study, we examined age-related alterations of this relationship in pcy mice. In comparison with control mice, pcy mice after the age of 14 weeks showed defective urinary concentration ability with an increase in urinary volume. Interestingly, the release of uEV-AQP2 increased progressively up to the age of 16 weeks, but at 21 weeks the release did not significantly differ from that in control mice (i.e., a bell-shaped pattern was evident). Similar results were obtained for uEV marker proteins, including tumor susceptibility gene 101 (TSG101) protein and apoptosis-linked gene 2-interacting protein X (Alix). Immunoblot analysis revealed that renal AQP2 expression increased progressively from 11 weeks, and immunohistochemistry showed that this increase was possibly due to an increase in the number of AQP2-positive cells. Analysis of mRNAs for seven types of AQP expressed in the kidney supported this notion. These data suggest that the level of uEV-AQP2 does not simply mirror the renal expression of AQP2 and that the altered release of uEV-AQP2 in pcy mice depends on the numbers of both renal AQP2-positive cells and EVs released into the urine.

Characterization of urinary exosomal release of aquaporin-1 and -2 after renal ischemia-reperfusion in rats.

Acute kidney injury (AKI) is an important risk factor for the development of chronic kidney disease (CKD), and an alteration in renal water handling has been observed during the transition of AKI to CKD. Urinary exosomal release of aquaporin-1 (AQP1) and AQP2, important proteins for renal water handling, has recently been reported to predict their levels of renal expression. Therefore, we examined the patterns of urinary exosomal release of AQP1 and AQP2, and the exosomal marker proteins tumor susceptibility 101 protein (TSG101) and ALG-2 interacting protein X (Alix), in the acute and chronic phases following induction of AKI by renal bilateral ischemia/reperfusion (I/R) in rats. Blood tests and histological examinations indicated that AKI occurred before at 7 days after renal I/R ( day 7) and that renal fibrosis developed progressively thereafter. Immunoblotting demonstrated significant decreases in the urinary exosomal release of AQP1 and AQP2 during severe AKI. Urinary exosomal release of Alix and TSG101 was significantly increased on day 7. These data were also confirmed in rats with unilateral renal I/R causing more serious AKI. Urinary exosomal release of either the Ser-256- or Ser-269-phosphorylated form of AQP2, both of which are involved in apical trafficking of AQP2, was positively correlated with that of total AQP2. These results suggest that urinary exosomal release of AQP1 and AQP2 is reduced in I/R-induced AKI, whereas that of Alix and TSG101 is increased in the initial phase of renal fibrosis. Furthermore, apical trafficking of AQP2 appears to be related to urinary exosomal release of AQP2.

Aquaporins in Urinary Extracellular Vesicles (Exosomes).

Since the successful characterization of urinary extracellular vesicles (uEVs) by Knepper's group in 2004, these vesicles have been a focus of intense basic and translational research worldwide, with the aim of developing novel biomarkers and therapeutics for renal disease. Along with these studies, there is growing evidence that aquaporins (AQPs), water channel proteins, in uEVs have the potential to be diagnostically useful. In this review, we highlight current knowledge of AQPs in uEVs from their discovery to clinical application.

Acetazolamide enhances the release of urinary exosomal aquaporin-1.

BACKGROUND: Renal aquaporin-1 (AQP1), a water channel protein, is known to be secreted into urine, conveyed by nano-sized extracellular vesicles called exosomes. A previous study has demonstrated that acetazolamide (AZ), a diuretic that inhibits carbonic anhydrases, alters the expression level of AQP1 in cultured cells. Here we investigated whether AZ alters the release of urinary exosomal AQP1 in vivo. METHODS: The effect of AZ on urinary exosomal AQP1 secretion was examined in rats and compared with furosemide (another diuretic), NaHCO3 (an alkalizing agent) and NH4Cl (an acidifying agent). Urine, blood and kidney samples were obtained 2 h after each treatment. Urinary exosomes were isolated by a differential centrifugation technique and urinary exosomal proteins were analyzed by immunoblotting. RESULTS: The release of exosomal AQP1 into urine was markedly increased after treatment with AZ, accompanied by alkaluria and metabolic acidosis. Immunohistochemistry clearly demonstrated that AZ increased the apical membrane expression of AQP1 in the proximal tubules. AZ did not affect the release of exosomal marker proteins (tumor susceptibility gene 101 protein and apoptosis-linked gene 2 interacting protein X). Treatment with furosemide did not change, whereas NaHCO3 and NH4Cl decreased the exosomal release of AQP1. CONCLUSION: The present findings indicate that AZ increases the release of exosomal AQP1 into urine in association with enhanced apical membrane expression of AQP1.

The protective effect of radicicol against renal ischemia--reperfusion injury in mice.

Overexpression of heat shock protein 70 kDa (HSP70) is known to confer cellular protection against ischemia-reperfusion (I/R) injury. Radicicol, a HSP90 inhibitor, has been reported to induce the expression of HSP70 protein. Here we studied whether radicicol attenuated renal I/R injury in vivo. Treatment of mice with radicicol ameliorated renal I/R injury and increased renal HSP70 mRNA and protein. Administration of radicicol with quercetin, an inhibitor of HSP70 induction, eliminated the renoprotective effect of radicicol. Our results suggest that the up-regulation of renal HSP70 protein by radicicol leads to a novel drug therapy against renal I/R injury.

Decreased abundance of urinary exosomal aquaporin-1 in renal ischemia-reperfusion injury.

Urinary exosomes, secreted into urine from renal epithelial cells, are known to contain many types of renal functional membrane proteins. Here, we studied whether renal ischemia-reperfusion (I/R) affects urinary exosomal aquaporin-1 (AQP1) excretion in rats subjected to renal I/R and patients who underwent renal transplantation. Immunoblotting studies demonstrated reduction of the urinary exosomal AQP1 level even at 6 h after renal I/R, and the level continued to be low over 96 h after I/R. Renal AQP1 mRNA and protein analyses revealed that the decreased excretion of urinary exosomal AQP1 is associated with renal AQP1 protein retention in the early phase and with a decreased expression level of renal AQP1 in the later phase of renal I/R injury. Decreased abundance of urinary exosomal AQP1 in a recipient patient was also observed at 48 h after renal allograft transplantation. No significant decrease in urinary exosomal AQP1 was observed in a rat model of nephropathy or in patients with proteinuria. Our studies suggest that the renal AQP1 expression level is possibly controlled by its urinary exosomal excretion and indicate that urinary exosomal AQP1 is a novel urinary biomarker for renal I/R injury.

A protective role of unfolded protein response in mouse ischemic acute kidney injury.

Although renal ischemia-reperfusion is known to activate the unfolded protein response, the renal site and role of activation of this response following the insult in vivo remains largely unknown. Here we studied the renal spatio-temporal expression pattern of glucose-regulated protein (GRP) 78, a central regulator of the unfolded protein response network, following renal ischemia-reperfusion and the effects of the specific chemical unfolded protein response inducers, tunicamycin and thapsigargin, on renal ischemia-reperfusion injury in mice. Renal ischemia-reperfusion resulted in expression of the spliced form of the X-box binding protein-1 (XBP-1s) transcript, an unfolded protein response target, at 1 and 2 h after the insult. This response was followed by an increase in the GRP78 transcript and protein. The increased amount of GRP78 protein after ischemia-reperfusion was largely localized in proximal tubule cells. Pretreatment with tunicamycin or thapsigargin significantly ameliorated renal dysfunction and injury after ischemia-reperfusion. Taken together with these results, the unfolded protein response was activated following renal ischemia-reperfusion at sites that are susceptible to ischemia-reperfusion injury, and this activation had a protective effect against renal ischemia-reperfusion injury in vivo. Molecules involved in the unfolded protein response may offer new opportunities for pharmacological intervention against renal ischemia-reperfusion injury, which is an important cause of acute kidney injury.

Inhibitory effect of tyrphostin 47 on Shiga toxin-induced cell death.

The inhibitory effects of tyrosine kinase inhibitors including tyrphostins 25, 47 and 51 on Shiga toxin 1-induced cell death and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation were examined in Vero cells. Tyrphostin 47 significantly inhibited Shiga toxin 1-induced cell death and p38 MAPK phosphorylation. In contrast, tyrphostins 25 and 51 had no significant effect on the Shiga toxin 1-induced responses. These data indicate that Shiga toxin 1-induced cell injury occurs through a pathway sensitive to tyrphostin 47, and the target molecule for tyrphostin 47 opens up new opportunities for pharmacological intervention against Shiga toxin-producing Escherichia coli infectious diseases.