Wilna Oosthuyzen

Quantification of human urinary exosomes by nanoparticle tracking analysis

•  Exosomes are vesicles that are released from the kidney into the urine. They contain RNA and protein from the cell of origin and can track changes in renal physiology non‐invasively. •  Current methods for the identification and quantification of urinary exosomes are time consuming and only semi‐quantitative. •  In this study, we applied nanoparticle tracking analysis to human urine and identified particles with a range of sizes, including a subpopulation of characteristic exosomal size that labelled positively with antibodies to exosome proteins. •  Nanoparticle tracking analysis was able to track an increase in exosomal aquaporin 2 concentration following desmopressin treatment of a kidney cell line, a rodent model and a patient with central diabetes insipidus. •  With appropriate sample storage, nanoparticle tracking analysis has potential as a tool for the rapid characterization and quantification of exosomes in human urine. This new method can be used to develop urinary extracellular vesicles further as a non‐invasive tool for investigating human renal physiology.

Diurnal Variation in Blood Pressure and Arterial Stiffness in Chronic Kidney DiseaseNovelty and Significance: The Role of Endothelin-1

Abstract—Hypertension and arterial stiffness are important independent cardiovascular risk factors in chronic kidney disease (CKD) to which endothelin-1 (ET-1) contributes. Loss of nocturnal blood pressure (BP) dipping is associated with CKD progression, but there are no data on 24-hour arterial stiffness variation. We examined the 24-hour variation of BP, arterial stiffness, and the ET system in healthy volunteers and patients with CKD and the effects on these of ET receptor type A receptor antagonism (sitaxentan). There were nocturnal dips in systolic BP and diastolic BP and pulse wave velocity, our measure of arterial stiffness, in 15 controls (systolic BP, −3.2±4.8%, P<0.05; diastolic BP, −6.4±6.2%, P=0.001; pulse wave velocity, −5.8±5.2%, P<0.01) but not in 15 patients with CKD. In CKD, plasma ET-1 increased by 1.2±1.4 pg/mL from midday to midnight compared with healthy volunteers (P<0.05). Urinary ET-1 did not change. In a randomized, double-blind, 3-way crossover study in 27 patients with CKD, 6-week treatment with placebo and nifedipine did not affect nocturnal dips in systolic BP or diastolic BP between baseline and week 6, whereas dipping was increased after 6-week sitaxentan treatment (baseline versus week 6, systolic BP: −7.0±6.2 versus −11.0±7.8 mm Hg, P<0.05; diastolic BP: −6.0±3.6 versus −8.3±5.1 mm Hg, P<0.05). There was no nocturnal dip in pulse pressure at baseline in the 3 phases of the study, whereas sitaxentan was linked to the development of a nocturnal dip in pulse pressure. In CKD, activation of the ET system seems to contribute not only to raised BP but also the loss of BP dipping. The clinical significance of these findings should be explored in future clinical trials. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01770847 and NCT00810732.

Glucocorticoids Induce Nondipping Blood Pressure by Activating the Thiazide-Sensitive Cotransporter

Blood pressure (BP) normally dips during sleep, and nondipping increases cardiovascular risk. Hydrochlorothiazide restores the dipping BP profile in nondipping patients, suggesting that the NaCl cotransporter, NCC, is an important determinant of daily BP variation. NCC activity in cells is regulated by the circadian transcription factor per1. In vivo, circadian genes are entrained via the hypothalamic–pituitary–adrenal axis. Here, we test whether abnormalities in the day:night variation of circulating glucocorticoid influence NCC activity and BP control. C57BL6/J mice were culled at the peak (1:00 AM) and trough (1:00 PM) of BP. We found no day:night variation in NCC mRNA or protein but NCC phosphorylation on threonine53 (pNCC), required for NCC activation, was higher when mice were awake, as was excretion of NCC in urinary exosomes. Peak NCC activity correlated with peak expression of per2 and bmal1 (clock genes) and sgk1 and tsc22d3 (glucocorticoid-responsive kinases). Adrenalectomy reduced NCC abundance and blunted the daily variation in pNCC levels without affecting variation in clock gene transcription. Chronic corticosterone infusion increased bmal1, per1, sgk1, and tsc22d3 expression during the inactive phase. Inactive phase pNCC was also elevated by corticosterone, and a nondipping BP profile was induced. Hydrochlorothiazide restored rhythmicity of BP in corticosterone-treated mice without affecting BP in controls. Glucocorticoids influence the day:night variation in NCC activity via kinases that control phosphorylation. Abnormal glucocorticoid rhythms impair NCC and induce nondipping. Night-time dosing of thiazides may be particularly beneficial in patients with modest glucocorticoid excess.Blood pressure (BP) normally dips during sleep, and nondipping increases cardiovascular risk. Hydrochlorothiazide restores the dipping BP profile in nondipping patients, suggesting that the NaCl cotransporter, NCC, is an important determinant of daily BP variation. NCC activity in cells is regulated by the circadian transcription factor per1. In vivo, circadian genes are entrained via the hypothalamic–pituitary–adrenal axis. Here, we test whether abnormalities in the day:night variation of circulating glucocorticoid influence NCC activity and BP control. C57BL6/J mice were culled at the peak (1:00 AM) and trough (1:00 PM) of BP. We found no day:night variation in NCC mRNA or protein but NCC phosphorylation on threonine53 (pNCC), required for NCC activation, was higher when mice were awake, as was excretion of NCC in urinary exosomes. Peak NCC activity correlated with peak expression of per2 and bmal1 (clock genes) and sgk1 and tsc22d3 (glucocorticoid-responsive kinases). Adrenalectomy reduced NCC abundance and blunted the daily variation in pNCC levels without affecting variation in clock gene transcription. Chronic corticosterone infusion increased bmal1, per1, sgk1, and tsc22d3 expression during the inactive phase. Inactive phase pNCC was also elevated by corticosterone, and a nondipping BP profile was induced. Hydrochlorothiazide restored rhythmicity of BP in corticosterone-treated mice without affecting BP in controls. Glucocorticoids influence the day:night variation in NCC activity via kinases that control phosphorylation. Abnormal glucocorticoid rhythms impair NCC and induce nondipping. Night-time dosing of thiazides may be particularly beneficial in patients with modest glucocorticoid excess.

Diurnal Variation in Blood Pressure and Arterial Stiffness in Chronic Kidney Disease: The Role of Endothelin-1

Abstract—Hypertension and arterial stiffness are important independent cardiovascular risk factors in chronic kidney disease (CKD) to which endothelin-1 (ET-1) contributes. Loss of nocturnal blood pressure (BP) dipping is associated with CKD progression, but there are no data on 24-hour arterial stiffness variation. We examined the 24-hour variation of BP, arterial stiffness, and the ET system in healthy volunteers and patients with CKD and the effects on these of ET receptor type A receptor antagonism (sitaxentan). There were nocturnal dips in systolic BP and diastolic BP and pulse wave velocity, our measure of arterial stiffness, in 15 controls (systolic BP, −3.2±4.8%, P<0.05; diastolic BP, −6.4±6.2%, P=0.001; pulse wave velocity, −5.8±5.2%, P<0.01) but not in 15 patients with CKD. In CKD, plasma ET-1 increased by 1.2±1.4 pg/mL from midday to midnight compared with healthy volunteers (P<0.05). Urinary ET-1 did not change. In a randomized, double-blind, 3-way crossover study in 27 patients with CKD, 6-week treatment with placebo and nifedipine did not affect nocturnal dips in systolic BP or diastolic BP between baseline and week 6, whereas dipping was increased after 6-week sitaxentan treatment (baseline versus week 6, systolic BP: −7.0±6.2 versus −11.0±7.8 mm Hg, P<0.05; diastolic BP: −6.0±3.6 versus −8.3±5.1 mm Hg, P<0.05). There was no nocturnal dip in pulse pressure at baseline in the 3 phases of the study, whereas sitaxentan was linked to the development of a nocturnal dip in pulse pressure. In CKD, activation of the ET system seems to contribute not only to raised BP but also the loss of BP dipping. The clinical significance of these findings should be explored in future clinical trials. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01770847 and NCT00810732.

Vasopressin Regulates Extracellular Vesicle Uptake by Kidney Collecting Duct Cells

Extracellular vesicles (ECVs) facilitate intercellular communication along the nephron, with the potential to change the function of the recipient cell. However, it is not known whether this is a regulated process analogous to other signaling systems. We investigated the potential hormonal regulation of ECV transfer and report that desmopressin, a vasopressin analogue, stimulated the uptake of fluorescently loaded ECVs into a kidney collecting duct cell line (mCCDC11) and into primary cells. Exposure of mCCDC11 cells to ECVs isolated from cells overexpressing microRNA-503 led to downregulated expression of microRNA-503 target genes, but only in the presence of desmopressin. Mechanistically, ECV entry into mCCDC11 cells required cAMP production, was reduced by inhibiting dynamin, and was selective for ECVs from kidney tubular cells. In vivo, we measured the urinary excretion and tissue uptake of fluorescently loaded ECVs delivered systemically to mice before and after administration of the vasopressin V2 receptor antagonist tolvaptan. In control-treated mice, we recovered 2.5% of administered ECVs in the urine; tolvaptan increased recovery five-fold and reduced ECV deposition in kidney tissue. Furthermore, in a patient with central diabetes insipidus, desmopressin reduced the excretion of ECVs derived from glomerular and proximal tubular cells. These data are consistent with vasopressin-regulated uptake of ECVs in vivo We conclude that ECV uptake is a specific and regulated process. Physiologically, ECVs are a new mechanism of intercellular communication; therapeutically, ECVs may be a vehicle by which RNA therapy could be targeted to specific cells for the treatment of kidney disease.

Urinary extracellular vesicle protein profiling and endogenous lithium clearance support excessive renal sodium wasting and water reabsorption in thiazide induced hyponatremia

Introduction Thiazide diuretics are among the most widely used antihypertensive medications worldwide. Thiazide-induced hyponatremia (TIH) is 1 of their most clinically significant adverse effects. A priori TIH must result from excessive saliuresis and/or water reabsorption. We hypothesized that pathways regulating the thiazide-sensitive sodium-chloride cotransporter NCC and the water channel aquaporin-2 (AQP2) may be involved. Our aim was to assess whether patients with TIH would show evidence of altered NCC and AQP2 expression in urinary extracellular vesicles (UEVs), and also whether abnormalities of renal sodium reabsorption would be evident using endogenous lithium clearance (ELC). Methods Blood and urine samples were donated by patients admitted to hospital with acute symptomatic TIH, after recovery to normonatremia, and also from normonatremic controls on and off thiazides. Urinary extracellular vesicles were isolated and target proteins evaluated by western blotting and by nanoparticle tracking analysis. Endogenous lithium clearance was assessed by inductively coupled plasma mass spectrometry. Results Analysis of UEVs by western blotting showed that patients with acute TIH displayed reduced total NCC and increased phospho-NCC and AQP2 relative to appropriate control groups; smaller differences in NCC and AQP2 expression persisted after recovery from TIH. These findings were confirmed by nanoparticle tracking analysis. Renal ELC was lower in acute TIH compared to that in controls and convalescent case patients. Conclusion Reduced NCC expression and increased AQP2 expression would be expected to result in saliuresis and water reabsorption in TIH patients. This study raises the possibility that UEV analysis may be of diagnostic utility in less clear-cut cases of thiazide-associated hyponatremia, and may help to identify patients at risk for TIH before thiazide initiation.

Diurnal Variation in Blood Pressure and Arterial Stiffness in Chronic Kidney Disease

Abstract—Hypertension and arterial stiffness are important independent cardiovascular risk factors in chronic kidney disease (CKD) to which endothelin-1 (ET-1) contributes. Loss of nocturnal blood pressure (BP) dipping is associated with CKD progression, but there are no data on 24-hour arterial stiffness variation. We examined the 24-hour variation of BP, arterial stiffness, and the ET system in healthy volunteers and patients with CKD and the effects on these of ET receptor type A receptor antagonism (sitaxentan). There were nocturnal dips in systolic BP and diastolic BP and pulse wave velocity, our measure of arterial stiffness, in 15 controls (systolic BP, −3.2±4.8%, P<0.05; diastolic BP, −6.4±6.2%, P=0.001; pulse wave velocity, −5.8±5.2%, P<0.01) but not in 15 patients with CKD. In CKD, plasma ET-1 increased by 1.2±1.4 pg/mL from midday to midnight compared with healthy volunteers (P<0.05). Urinary ET-1 did not change. In a randomized, double-blind, 3-way crossover study in 27 patients with CKD, 6-week treatment with placebo and nifedipine did not affect nocturnal dips in systolic BP or diastolic BP between baseline and week 6, whereas dipping was increased after 6-week sitaxentan treatment (baseline versus week 6, systolic BP: −7.0±6.2 versus −11.0±7.8 mm Hg, P<0.05; diastolic BP: −6.0±3.6 versus −8.3±5.1 mm Hg, P<0.05). There was no nocturnal dip in pulse pressure at baseline in the 3 phases of the study, whereas sitaxentan was linked to the development of a nocturnal dip in pulse pressure. In CKD, activation of the ET system seems to contribute not only to raised BP but also the loss of BP dipping. The clinical significance of these findings should be explored in future clinical trials. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01770847 and NCT00810732.

Diurnal Variation in Blood Pressure and Arterial Stiffness in Chronic Kidney DiseaseNovelty and Significance

Abstract—Hypertension and arterial stiffness are important independent cardiovascular risk factors in chronic kidney disease (CKD) to which endothelin-1 (ET-1) contributes. Loss of nocturnal blood pressure (BP) dipping is associated with CKD progression, but there are no data on 24-hour arterial stiffness variation. We examined the 24-hour variation of BP, arterial stiffness, and the ET system in healthy volunteers and patients with CKD and the effects on these of ET receptor type A receptor antagonism (sitaxentan). There were nocturnal dips in systolic BP and diastolic BP and pulse wave velocity, our measure of arterial stiffness, in 15 controls (systolic BP, −3.2±4.8%, P<0.05; diastolic BP, −6.4±6.2%, P=0.001; pulse wave velocity, −5.8±5.2%, P<0.01) but not in 15 patients with CKD. In CKD, plasma ET-1 increased by 1.2±1.4 pg/mL from midday to midnight compared with healthy volunteers (P<0.05). Urinary ET-1 did not change. In a randomized, double-blind, 3-way crossover study in 27 patients with CKD, 6-week treatment with placebo and nifedipine did not affect nocturnal dips in systolic BP or diastolic BP between baseline and week 6, whereas dipping was increased after 6-week sitaxentan treatment (baseline versus week 6, systolic BP: −7.0±6.2 versus −11.0±7.8 mm Hg, P<0.05; diastolic BP: −6.0±3.6 versus −8.3±5.1 mm Hg, P<0.05). There was no nocturnal dip in pulse pressure at baseline in the 3 phases of the study, whereas sitaxentan was linked to the development of a nocturnal dip in pulse pressure. In CKD, activation of the ET system seems to contribute not only to raised BP but also the loss of BP dipping. The clinical significance of these findings should be explored in future clinical trials. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01770847 and NCT00810732.