Hiroko Sonoda

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.

Aquaporin 11 insufficiency modulates kidney susceptibility to oxidative stress.

Aquaporin 11 (AQP11) is a newly described member of the protein family of transport channels. AQP11 associates with the endoplasmic reticulum (ER) and is highly expressed in proximal tubular epithelial cells in the kidney. Previously, we identified and characterized a recessive mutation of the highly conserved Cys227 to Ser227 in mouse AQP11 that caused proximal tubule (PT) injury and kidney failure in mutant mice. The current study revealed induction of ER stress, unfolded protein response, and apoptosis as molecular mechanisms of this PT injury. Cys227Ser mutation interfered with maintenance of AQP11 oligomeric structure. AQP11 is abundantly expressed in the S1 PT segment, a site of major renal glucose flux, and Aqp11 mutant mice developed PT-specific mitochondrial injury. Glucose increased AQP11 protein expression in wild-type kidney and upregulation of AQP11 expression by glucose in vitro was prevented by phlorizin, an inhibitor of sodium-dependent glucose transport across PT. Total AQP11 levels in heterozygotes were higher than in wild-type mice but were not further increased in response to glucose. In Aqp11 insufficient PT cells, glucose potentiated increases in reactive oxygen species (ROS) production. ROS production was also elevated in Aqp11 mutation carriers. Phenotypically normal mice heterozygous for the Aqp11 mutation repeatedly treated with glucose showed increased blood urea nitrogen levels that were prevented by the antioxidant sulforaphane or by phlorizin. Our results indicate an important role for AQP11 to prevent glucose-induced oxidative stress in proximal tubules.

Excretion of urinary exosomal AQP2 in rats is regulated by vasopressin and urinary pH

Urinary exosomes are small vesicles secreted into urine from all renal epithelial cell types and known to contain proteins that are involved in renal secretion and reabsorption. Among these proteins, urinary exosomal aquaporin-2 (AQP2) has been suggested to be useful for diagnosis of renal disease. However, the mechanisms underlying the excretion of urinary exosomal AQP2 are largely unknown. In this study, we examined the mechanisms of urinary exosomal AQP2 excretion in vivo, using diuretics including furosemide (FS), an inhibitor of the sodium-potassium-chloride symporter; acetazolamide (ACTZ), an inhibitor of carbonic anhydrase; OPC-31260 (OPC), a vasopressin type 2 receptor antagonist; and NaHCO3, a urinary alkalizing agent. Samples of urine from rats were collected for 2 h just after treatment with each diuretic, and urinary exosomes were isolated by ultracentrifugation. Urinary exosomal AQP2 excretion was dramatically increased by treatment with FS accompanied by urine acidification or with ACTZ accompanied by urine alkalization. Immunohistochemistry showed that apical localization of AQP2 was clearly evident and the plasma vasopressin level was increased after each treatment. Although treatment with OPC alone had no significant effect, coadministration of OPC completely inhibited the FS-induced and partially reduced the ACTZ-induced responses, respectively. Treatment with NaHCO3 increased the excretion of urinary exosomal AQP2 accompanied by urine alkalization. This increased response was partially inhibited by coadministration of OPC. These data suggest that an increased plasma level of vasopressin promoted the excretion of urinary exosomal AQP2 and that urine alkalinization also increased it independently of vasopressin.

Activation of renal angiotensin type 1 receptor contributes to the pathogenesis of progressive renal injury in a rat model of chronic cardiorenal syndrome

Although chronic cardiac dysfunction is known to progressively exacerbate renal injury, a condition known as type 2 cardiorenal syndrome (CRS), the mechanism responsible is largely unknown. The present study was undertaken to clarify the mechanism of renal injury in rats with both unilateral nephrectomy (NX) and surgically induced myocardial infarction (MI), corresponding to a model of type 2 CRS. Compared with a control group, rats with both MI and NX (MI+NX) exhibited progressive proteinuria during the experimental period (34 wk after MI surgery), whereas proteinuria was not observed in rats with MI alone and was moderate in rats with NX alone. The proteinuria in rats with MI+NX was associated with renal lesions such as glomerulosclerosis and infiltration of mononuclear cells and upregulation of the renal proinflammatory and -fibrotic cytokine and angiotensin II type 1a receptor (AT1aR) genes. In contrast, plasma renin activity was lowered in rats with MI+NX. Immunohistochemistry revealed that the increased AT1R protein was present mainly in renal interstitial mononuclear cells. Olmesartan medoxomil, an AT1R blocker, markedly reduced the proteinuria and infiltration of mononuclear cells, whereas spironolactone, a mineralocorticoid receptor blocker, did not. The present findings demonstrate the pathogenetic role of renal interstitial AT1R signaling in a model of type 2 CRS, providing evidence that AT1R blockade can be a useful therapeutic option for this syndrome.

Urinary excretion pattern of exosomal aquaporin-2 in rats that received gentamicin

Urinary exosomes are nano-sized vesicles secreted into urine from all types of renal epithelial cells and are known to contain possible biomarker proteins for renal diseases. Gentamicin has been reported to decrease the level of renal aquaporin (AQP)2, which is known to be mainly expressed in renal collecting ducts and excreted into the urine via exosomes. In the present study, we investigated whether urinary exosomal AQP2 could serve as a potential biomarker for gentamicin-induced nephrotoxicity, especially collecting duct cell dysfunction. Gentamicin was given to rats intraperitoneally once every day starting on day 0. Gentamicin significantly increased the plasma creatinine concentration from day 5 and beyond. Also, gentamicin induced polyuria and a defective urine concentration mechanism on day 7, suggesting gentamicin-induced collecting duct cell dysfunction. Immunoblot analysis showed that gentamicin significantly increased urinary exosomal AQP2 excretion on day 1 but decreased it on day 7 compared with the control group. Similarly, increased excretion of exosomal tumor susceptibility gene 101 protein, frequently used as an exosome marker protein, was observed on day 1. However, gentamicin did not significantly affect the urinary excretion of exosomal tumor susceptibility gene 101 on day 7. Gentamicin slightly decreased renal AQP2 expression on day 2 and markedly decreased it on day 8. These data strongly suggest that the use of urinary exosomal AQP2 as a biomarker may allow detection of gentamicin-induced collecting duct cell dysfunction. Furthermore, urinary exosomal AQP2 might also be useful for the early detection of gentamicin-induced renal injury in addition to collecting duct injury.

The role of Cysteine 227 in subcellular localization, water permeability, and multimerization of aquaporin-11.

Aquaporin‐11 (AQP11) is the latest member of the mammalian water channel protein family to be described. Recent in vivo studies have shown that mutation at Cys227 causes renal failure. However the importance of Cys227 for the molecular function of AQP11 is largely unknown. In this study, we examined the subcellular localization, water permeability, and multimerization of AQP11 with a mutation at Cys227. Interestingly, cells expressing the mutants had significantly higher osmotic water permeability. In contrast, the mutation lowered the cell surface expression and multimerization levels. Our observations suggest that Cys227 is crucial for the proper molecular function of AQP11.

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.

Evaluation of Olmesartan Medoxomil in the Rat Monocrotaline Model of Pulmonary Hypertension

It is suggested that angiotensin II is involved in the pathogenesis of pulmonary hypertension and subsequent right ventricular hypertrophy; therefore, an angiotensin AT1 receptor antagonist could be beneficial for the treatment of this disease. We tested the effect of the new AT1 receptor antagonist olmesartan medoxomil on monocrotaline-induced pulmonary hypertension in rats. At 3 weeks after a single subcutaneous injection of monocrotaline (50 mg/kg), the lung/body weight ratio, the right ventricle/(left ventricle plus septum) weight ratio [RV/(LV+S)], and right ventricular systolic pressure were increased, indicating establishment of pulmonary hypertension and right ventricular hypertrophy. Oral administration of olmesartan medoxomil (2 or 5 mg/kg/day for 3 weeks) restored RV/(LV+S) and right ventricular systolic pressure, and a higher dose (5 mg/kg/day) improved the lung/body weight ratio. Pulmonary arteries isolated from monocrotaline-treated rats exhibited an increase in basal tone in the resting state, indicating that they had intrinsic tone. Three weeks of treatment with olmesartan decreased this intrinsic tone. These data suggest that long-term treatment with olmesartan has beneficial effects on monocrotaline-induced pulmonary hypertension and subsequent right ventricular hypertrophy.

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.