Reduced Urine Osmolality Research Articles

Renal Expression of Adhesion GPCR Gpr116 (ADGRF5) Plays a Role in Urinary Concentration in Mice

G‐protein coupled receptors (GPCR) are a large and diverse family of integral membrane proteins that recognize a tremendous assortment of extracellular molecules including neurotransmitters, hormones, light and odors. They are a common target of pharmaceutical drug development, and uncovering the function of novel GPCRs in the kidney represents a wealth of untapped therapeutic potential. We previously performed an mRNA screen for novel GPCRs in the kidney to identify promising yet overlooked renal GPCRs. This screen revealed that Gpr116, an adhesion‐class GPCR, is highly expressed in the kidney. To understand the role of Gpr116 in renal physiology, we have taken a multidisciplinary approach. Using transfected HEK293 cells expressing cloned Gpr116, we have validated a monoclonal antibody for Gpr116. In murine kidney, this antibody indicates localization of Gpr116 to intercalated cells in the collecting duct of mouse kidneys. This staining is absent in kidneys from targeted knockout (KO) of Gpr116 in renal tubules (Gpr116flox/flox, ksp‐Cre). Additionally, kidney‐specific KO animals have significantly reduced urine osmolality (1552±71 mOsm/kg, N=11) compared to wild‐type (WT) (2097±76, N=10, p<0.0001) and heterozygous (2423±86, N=6, p<0.0001) littermates. Lastly, preliminary data of urine osmolality following 12‐hour water restriction (dark‐cycle) suggests KO animals are capable of concentrating urine, though only to about 75% that of WT littermates (3492±150 vs. 4361±525; N=3, N=4, respectively). Overall, these results suggest a role for Gpr116 in enhancing the ability to concentrate urine. This study establishes a physiologic role of the previously understudied Gpr116 in the murine kidney and demonstrates the scientific potential of future investigations into novel GPCRs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Absence of the Na‐Glucose Cotransporter SGLT1 Ameliorates Kidney Recovery in a Murine Model of Acute Kidney Injury

The renal Na‐glucose cotransporter SGLT1 reabsorbs ~3% of the filtered glucose in the late proximal tubule, a hypoxia‐sensitive tubular segment that enters the outer medulla. Pharmacological blockade of SGLT2, which in the early proximal tubule reabsorbs most of the filtered glucose, was found protective in a mouse model of acute kidney injury (AKI) induced by ischemia‐reperfusion (IR). Since little is known about the role of SGLT1 in this context, we determined whether lack of SGLT1 affects kidney injury or recovery following IR.SGLT1‐knockout (KO) and WT mice (C57BL/6J males) underwent IR or sham surgery. IR was induced by 20 minutes of bilateral renal artery clamping, while body temperature was maintained at 36–37□. Sham operation was similar to IR surgery without artery clamping. All groups (WT Sham, WT IR, SGLT1‐KO Sham and SGLT1‐KO IR, n=7–8/group) were fed a low‐glucose high‐protein diet to prevent diarrhea in SGLT1‐KO. Urine and blood were collected at several time points during recovery. GFR was measured 14 days after IR by plasma elimination kinetics of FITC‐sinistrin in conscious mice. Kidneys were harvested on day 16.On day 1 after IR, plasma creatinine (by LC‐MS‐MS) increased to similar levels in SGLT1‐KO and WT (0.554±0.08 vs. 0.470±0.1 mg/dL, p=0.351), indicating similar initial kidney impairment. In accordance, IR reduced urine osmolality and increased plasma osmolality to similar levels in SGLT1‐KO and WT on day 1, possibly as a consequence of impaired urine concentration or renal retention of waste products. This was associated with a greater increase in fractional glucose excretion in SGLT1‐KO (1 to 19%) vs WT (0.1 to 7%) (p=0.003) indicating a prominent role of SGLT1 in glucose reabsorption at this time point. On day 14, GFR was significantly lower in WT IR vs SGLT1‐KO IR (6.56±0.6 vs. 9.13±0.8 μL/min/g BW, p<0.05), associated with higher plasma creatinine levels on harvest day (0.130±0.016 vs. 0.093±0.009 mg/dL, p<0.05). Moreover, plasma osmolality remained elevated in WT IR but was normalized in SGLT1‐KO IR on day 16 (345±11 vs. 316±4 mosm/kg, p=0.006). On day 16, plasma urea levels in sham groups were higher in SGLT1 KO vs WT (18.0±0.3 vs. 13.2±0.9 mmol/L, p=0.028), possibly reflecting a proposed role of SGLT1 in renal urea secretion, unmasked by high protein diet. Plasma urea was strongly increased in WT IR vs sham (24.6±2.2 vs. 13.2±0.9 mmol/L, p<0.001), whereas levels were preserved in SGLT1‐KO IR vs sham. RT‐qPCR analyses on whole kidney tissue revealed that Na‐2Cl‐K cotransporter NKCC2 mRNA expression was decreased in WT IR but preserved in SGLT1‐KO IR, whereas renal mRNA expression of markers of injury, fibrosis and inflammation (KIM‐1, type 1 collagen, MCP‐1) was lower in SGLT1‐KO IR vs WT IR.Lack of SGLT1 improved kidney recovery in a mouse model of IR‐induced AKI. The results may reflect a deleterious role of SGLT1 activity in the late proximal tubule for outer medullary integrity in the aftermath of IR‐induced AKI. This may impair tubular function (reduced NKCC2) with consequences on GFR, kidney integrity, and body homeostasis, and have therapeutical implications.Support or Funding InformationNIH R01DK106102, R01DK112042, P30DK079337, and the Department of Veterans AffairsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of adenylyl cyclase 6 in the development of lithium-induced nephrogenic diabetes insipidus.

Psychiatric patients treated with lithium (Li+) may develop nephrogenic diabetes insipidus (NDI). Although the etiology of Li+-induced NDI (Li-NDI) is poorly understood, it occurs partially due to reduced aquaporin-2 (AQP2) expression in the kidney collecting ducts. A mechanism postulated for this is that Li+ inhibits adenylyl cyclase (AC) activity, leading to decreased cAMP, reduced AQP2 abundance, and less membrane targeting. We hypothesized that Li-NDI would not develop in mice lacking AC6. Whole-body AC6 knockout (AC6-/-) mice and potentially novel connecting tubule/principal cell-specific AC6 knockout (AC6loxloxCre) mice had approximately 50% lower urine osmolality and doubled water intake under baseline conditions compared with controls. Dietary Li+ administration increased water intake and reduced urine osmolality in control, AC6-/-, and AC6loxloxCre mice. Consistent with AC6-/- mice, medullary AQP2 and pS256-AQP2 abundances were lower in AC6loxloxCre mice compared with controls under standard conditions, and levels were further reduced after Li+ administration. AC6loxloxCre and control mice had a similar increase in the numbers of proliferating cell nuclear antigen-positive cells in response to Li+. However, AC6loxloxCre mice had a higher number of H+-ATPase B1 subunit-positive cells under standard conditions and after Li+ administration. Collectively, AC6 has a minor role in Li-NDI development but may be important for determining the intercalated cell-to-principal cell ratio.

The role of amiloride in managing patients with lithium‐induced nephrogenic diabetes insipidus

AbstractBackgroundDespite lithium being a highly effective drug, it is commonly associated with inducing nephrogenic diabetes insipidus (NDI) in a high number of patients. In this case report we describe the successful use of amiloride as an effective means to manage and minimise NDI occurring in patients while they are on lithium therapy.Clinical detailsThe patient had been taking lithium for management of her bipolar disorder for approximately 5 years. While on this medication the patient suffered from acute kidney impairment, hypernatraemia, reduced urine osmolality, increased plasma osmolality, polyuria, polydipsia and severe dehydration. These clinical results were consistent with an adverse reaction known as NDI acquired from long‐term lithium use and fluid restriction.OutcomesLithium was ceased on day two of admission. Intravenous fluid replacements were provided until the patient's renal function and sodium levels returned to normal. Once the patient was symptomatically stable, lithium was reintroduced at initiating dosage levels concomitantly with amiloride with aims of reversing the concentrating defect and preventing this side effect from occurring again. The patient was discharged 15 days later.ConclusionIn most cases NDI is reversible with the cessation of lithium. However, a subset of the mental health population is resistant to other forms of therapy, leaving lithium as their only option to successfully manage their mood disorder. For patients who suffer from mental health disorders, the literature has found that amiloride can be an effective means to restore a patient's impaired urine concentrating ability.

Spilanthol from Acmella Oleracea Lowers the Intracellular Levels of cAMP Impairing NKCC2 Phosphorylation and Water Channel AQP2 Membrane Expression in Mouse Kidney.

Acmella oleracea is well recognized in Brazilian traditional medicine as diuretic, although few scientific data have been published to support this effect. Aim of this study was to determine the molecular effect of Acmella oleracea extract and its main alkylamide spilanthol on two major processes involved in the urine concentrating mechanism: Na-K-2Cl symporter (NKCC2) activity in the thick ascending limb and water channel aquaporin 2 accumulation at the apical plasma membrane of collecting duct cells. Phosphorylation of NKCC2 was evaluated as index of its activation by Western blotting. Rate of aquaporin 2 apical expression was analyzed by confocal laser microscopy. Spilanthol-induced intracellular signalling events were dissected by video-imaging experiments. Exposure to spilanthol reduced the basal phosphorylation level of NKCC2 both in freshly isolated mouse kidney slices and in NKCC2-expresing HEK293 cells. In addition, exposure to spilanthol strongly reduced both desmopressin and low Cl−-dependent increase in NKCC2 phosphorylation in mouse kidney slices and NKCC2-expressing HEK293 cells, respectively. Similarly, spilanthol reduced both desmopressin- and forskolin-stimulated aquaporin 2 accumulation at the apical plasma membrane of collecting duct in mouse kidney slice and MCD4 cells, respectively. Of note, when orally administered, spilanthol induced a significant increase in both urine output and salt urinary excretion associated with a markedly reduced urine osmolality compared with control mice. Finally, at cellular level, spilanthol rapidly reduced or reversed basal and agonist-increased cAMP levels through a mechanism involving increases in intracellular [Ca2+]. In conclusion, spilanthol-induced inhibition of cAMP production negatively modulates urine-concentrating mechanisms thus holding great promise for its use as diuretic.

Systemic PPARγ deletion causes severe disturbance in fluid homeostasis in mice.

The pharmacological action of peroxisome proliferator-activated receptor (PPAR)γ in promoting sodium and water retention is well documented as highlighted by the major side-effect of body weight gain and edema associated with thiazolidinedione use. However, a possible physiological role of PPARγ in regulation of fluid metabolism has not been reported by previous studies. Here we analyzed fluid metabolism in inducible whole-body PPARγ knockout mice. The null mice developed severe polydipsia and polyuria, reduced urine osmolality, and modest hyperphagia. The phenomenon persisted during 3 days of pair feeding and pair drinking, accompanied by progressive weight loss. After 24 h water deprivation, the null mice had a lower urine osmolality, a higher urine volume, a greater weight loss, and a greater rise in hematocrit than the floxed control. Urinary vasopressin (AVP) excretion was not different between the genotypes under basal condition or after WD. The response of urine osmolality to acute and chronic 1-desamino-8-D-arginine vasopressin treatment was attenuated in the null mice, but the total abundance or phosphorylation of aquaporin 2 (AQP2) in the kidney or AVP-induced cAMP production in inner medullary collecting duct suspensions was unaffected. Overall, PPARγ participates in physiological control of fluid homeostasis through an unknown mechanism involving cAMP/AQP2-independent enhancement of AVP response.

Salt-sparing diuretic action of a water-soluble urea analog inhibitor of urea transporters UT-A and UT-B in rats

Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics (‘urearetics’) with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10–20 μM. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2–3 mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500 mg/kg DMTU, peak plasma concentration was 9 mM with t1/2 of about 10 hours, and a urine concentration of 20–40 mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of Syndrome of Inappropriate Antidiuretic Hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition.

Inhibition of mTOR activity in diabetes mellitus reduces proteinuria but not renal accumulation of hyaluronan

Objectives. Accumulation of extracellular matrix (ECM) components is an early sign of diabetic nephropathy. Also the glycosaminoglycan hyaluronan (HA) is elevated in the renal interstitium during experimental diabetes. The mammalian target of rapamycin (mTOR) pathway participates in the signaling of hyperglycemia-induced ECM accumulation in the kidney, but this has not yet been investigated for HA. We hypothesized that interstitial HA accumulation during diabetes may involve mTOR activation.Methods. Diabetic rats (6 weeks post-streptozotocin (STZ)) were treated with rapamycin to inhibit mTOR or vehicle for 2 additional weeks. Kidney function (glomerular filtration rate, renal blood flow, urine output) and regional renal HA content were thereafter analyzed. The ability of the animals to respond to desmopressin was also tested.Results. Diabetic animals displayed hyperglycemia, proteinuria, hyperfiltration, renal hypertrophy, increased diuresis with reduced urine osmolality, and reduced weight gain. Cortical and outer medullary HA was elevated in diabetic rats. Urine hyaluronidase activity was almost doubled in diabetic rats compared with controls. The ability to respond to desmopressin was absent in diabetic rats. Renal blood flow and arterial blood pressure were unaffected by the diabetic state. In diabetic rats treated with rapamycin the proteinuria was reduced by 32%, while all other parameters were unaffected.Conclusion. Regional renal accumulation of the ECM component HA is not sensitive to mTOR inhibition by rapamycin, while proteinuria is reduced in established STZ-induced diabetes. Whether the diabetes-induced renal accumulation of HA occurs through different pathways than other ECM components, or is irreversible after being established, remains to be shown.

Glycogen synthase kinase 3α regulates urine concentrating mechanism in mice.

In mammals, glycogen synthase kinase (GSK)3 comprises GSK3α and GSK3β isoforms. GSK3β has been shown to play a role in the ability of kidneys to concentrate urine by regulating vasopressin-mediated water permeability of collecting ducts, whereas the role of GSK3α has yet to be discerned. To investigate the role of GSK3α in urine concentration, we compared GSK3α knockout (GSK3αKO) mice with wild-type (WT) littermates. Under normal conditions, GSK3αKO mice had higher water intake and urine output. GSK3αKO mice also showed reduced urine osmolality and aquaporin-2 levels but higher urinary vasopressin. When water deprived, they failed to concentrate their urine to the same level as WT littermates. The addition of 1-desamino-8-d-arginine vasopressin to isolated inner medullary collecting ducts increased the cAMP response in WT mice, but this response was reduced in GSK3αKO mice, suggesting reduced responsiveness to vasopressin. Gene silencing of GSK3α in mpkCCD cells also reduced forskolin-induced aquaporin-2 expression. When treated with LiCl, an isoform nonselective inhibitor of GSK3 and known inducer of polyuria, WT mice developed significant polyuria within 6 days. However, in GSK3αKO mice, the polyuric response was markedly reduced. This study demonstrates, for the first time, that GSK3α could play a crucial role in renal urine concentration and suggest that GSK3α might be one of the initial targets of Li(+) in LiCl-induced nephrogenic diabetes insipidus.

Efficacy of tolvaptan in patients with refractory ascites in a clinical setting.

To elucidate the efficacies of tolvaptan (TLV) as a treatment for refractory ascites compared with conventional treatment. We retrospectively enrolled 120 refractory ascites patients between January 1, 2009 and September 31, 2014. Sixty patients were treated with oral TLV at a starting dose of 3.75 mg/d in addition to sodium restriction (> 7 g/d), albumin infusion (10-20 g/wk), and standard diuretic therapy (20-60 mg/d furosemide and 25-50 mg/d spironolactone) and 60 patients with large volume paracentesis in addition to sodium restriction (less than 7 g/d), albumin infusion (10-20 g/wk), and standard diuretic therapy (20-120 mg/d furosemide and 25-150 mg/d spironolactone). Patient demographics and laboratory data, including liver function, were not matched due to the small number of patients. Continuous variables were analyzed by unpaired t-test or paired t-test. Fisher's exact test was applied in cases comparing two nominal variables. We analyzed factors affecting clinical outcomes using receiver operating characteristic curves and multivariate regression analysis. We also used multivariate Cox's proportional hazard regression analysis to elucidate the risk factors that contributed to the increased incidence of ascites. TLV was effective in 38 (63.3%) patients. The best cut-off values for urine output and reduced urine osmolality as measures of refractory ascites improvement were > 1800 mL within the first 24 h and > 30%, respectively. Multivariate regression analysis indicated that > 25% reduced urine osmolality [odds ratio (OR) = 20.7; P < 0.01] and positive hepatitis C viral antibodies (OR = 5.93; P = 0.05) were positively correlated with an improvement of refractory ascites, while the total bilirubin level per 1.0 mg/dL (OR = 0.57; P = 0.02) was negatively correlated with improvement. In comparing the TLV group and controls, only the serum sodium level was significantly lower in the TLV group (133 mEq/L vs 136 mEq/L; P = 0.02). However, there were no significant differences in the other parameters between the two groups. The cumulative incidence rate was significantly higher in the control group with a median incidence time of 30 d in the TLV group and 20 d in the control group (P = 0.01). Cox hazard proportional multivariate analysis indicated that the use of TLV (OR = 0.58; P < 0.01), uncontrolled liver neoplasms (OR = 1.92; P < 0.01), total bilirubin level per 1.0 mg/dL (OR = 1.10; P < 0.01), and higher sodium level per 1.0 mEq/L (OR = 0.94; P < 0.01) were independent factors that contributed to incidence. Administration of TLV results in better control of refractory ascites and reduced the incidence of additional invasive procedures or hospitalization compared with conventional ascites treatments.

O-071 Salt-sparing Diuretic Action Of A Urea Analogue Inhibitor Of Urea Transporters Ut-a And Ut-b In Rats

Background and aims Urea is end metabolite of protein metabolism and is crucial for generation of hypertonic renal medulla. Urea transport to medullary interstitium is facilitated by urea transporters (UT-A and UT-B). UT inhibitors have potential use as a novel class of salt-sparing diuretics. Methods UT inhibitor effect of urea analogue dimethylthiourea (DMTU) was investigated and characterised in cell-based assays. Kidney function tests were investigated in both 1-day and 7-days DMTU-treated (500 mg/kg ip initially, then 125 mg/kg ip every 12h) rats. The effect of DMTU on maximum urine concentrating function was investigated in low (6%) and normal (20%) protein-fed rats. Results DMTU non-competitively inhibited UT-A and UT-B with IC 50 of ~3 mM. Following 500 mg/kg ip injection, plasma DMTU concentration was initially 10 mM (plasma elimination t 1/2 ~10 h) and urine DMTU concentration was >20 mM for 12 h. DMTU-treated rats showed reversible, sustained reduction in urine osmolality (>60%) and 3-fold increased daily urine output. DMTU increased renal electrolyte-free water excretion without altering solute excretion. DMTU impaired maximum urinary concentrating function only in normal protein-fed rats. Methylurea, a non-UT inhibitor urea analogue, had no effect on either urine volume or osmolality. DMTU-treated rats had greater diuresis and much reduced urinary salt loss compared to that of furosemide-treated rats. Conclusions These results establish a rat model of sustained UT inhibition and demonstrate remarkable diuretic efficacy of UT inhibition. Prominent effect of UT inhibitors on net renal water excretion implies a novel therapeutic strategy for treatment of oedema in hypervolemic diseases.

Diuresis and reduced urinary osmolality in rats produced by small-molecule UT-A-selective urea transport inhibitors.

Urea transport (UT) proteins of the UT-A class are expressed in epithelial cells in kidney tubules, where they are required for the formation of a concentrated urine by countercurrent multiplication. Here, using a recently developed high-throughput assay to identify UT-A inhibitors, a screen of 50,000 synthetic small molecules identified UT-A inhibitors of aryl-thiazole, γ-sultambenzosulfonamide, aminocarbonitrile butene, and 4-isoxazolamide chemical classes. Structure-activity analysis identified compounds that inhibited UT-A selectively by a noncompetitive mechanism with IC50 down to ∼1 μM. Molecular modeling identified putative inhibitor binding sites on rat UT-A. To test compound efficacy in rats, formulations and administration procedures were established to give therapeutic inhibitor concentrations in blood and urine. We found that intravenous administration of an indole thiazole or a γ-sultambenzosulfonamide at 20 mg/kg increased urine output by 3-5-fold and reduced urine osmolality by ∼2-fold compared to vehicle control rats, even under conditions of maximum antidiuresis produced by 1-deamino-8-D-arginine vasopressin (DDAVP). The diuresis was reversible and showed urea > salt excretion. The results provide proof of concept for the diuretic action of UT-A-selective inhibitors. UT-A inhibitors are first in their class salt-sparing diuretics with potential clinical indications in volume-overload edemas and high-vasopressin-associated hyponatremias.

Potentiation of the Effect of Thiazide Derivatives by Carbonic Anhydrase Inhibitors: Molecular Mechanisms and Potential Clinical Implications

BackgroundCarbonic anhydrase inhibitors (CAI) are mild diuretics, hence not widely used in fluid overloaded states. They are however the treatment of choice for certain non-kidney conditions. Thiazides, specific inhibitors of Na-Cl cotransport (NCC), are mild agents and the most widely used diuretics in the world for control of mild hypertension.HypothesisIn addition to inhibiting the salt reabsorption in the proximal tubule, CAIs down-regulate pendrin, therefore leaving NCC as the major salt absorbing transporter in the distal nephron, and hence allowing for massive diuresis by the inhibitors of NCC in the setting of increased delivery of salt from the proximal tubule.Experimental Protocols and ResultsDaily treatment of rats with acetazolamide (ACTZ), a known CAI, for 10 days caused mild diuresis whereas daily treatment with hydrochlorothiazide (HCTZ) for 4 days caused hardly any diuresis. However, treatment of rats that were pretreated with ACTZ for 6 days with a combination of ACTZ plus HCTZ for 4 additional days increased the urine output by greater than 2 fold (p<0.001, n = 5) compared to ACTZ-treated animals. Sodium excretion increased by 80% in the ACTZ plus HCTZ group and animals developed significant volume depletion, metabolic alkalosis and pre-renal failure. Molecular studies demonstrated ∼75% reduction in pendrin expression by ACTZ. The increased urine output in ACTZ/HCTZ treated rats was associated with a significant reduction in urine osmolality and reduced membrane localization of AQP-2 (aquaporin2).ConclusionsThese results indicate that ACTZ down-regulates pendrin expression and leaves NCC as the major salt absorbing transporter in the distal nephron in the setting of increased delivery of salt from the proximal tubule. Despite being considered mild agents individually, we propose that the combination of ACTZ and HCTZ is a powerful diuretic regimen.

Nephrotoxizität und Langzeitbehandlung mit Lithium

With particular focus on clinical, pathophysiologic and epidemiologic aspects this systematic review article presents the available data on nephrotoxic effects of a long-term treatment with lithium. Lithium may lead to tubular dysfunction (LITD = nephrogenic diabetes insipidus, hyperchloremic metabolic acidosis, increased natriuresis) and lithium-induced nephropathy (LIN) with reduced glomerular filtration rate (GFR). The histopathologic finding of LIN is chronic tubulo-interstitial nephritis. LITD frequently presents with polydipsia/-uria and reduced urine osmolality, while LIN features a wide clinical spectrum ranging from clinically asymptomatic presentations with reduced GFR to end-stage renal failure. LIN seems to feature slow progression and is significantly less frequent than LITD. Regular monitoring of renal function is indispensable for patients treated with lithium. Patients with reduced GFR under treatment with lithium should always be presented to a nephrologist. Currently, there are no guidelines for the handling of patients with LIN and ongoing treatment with lithium. Thus, regarding continuation of lithium-treatment an individual benefit/risk assessment is necessary.

Early alteration of urinary exosomal aquaporin 1 and transforming growth factor β1 after release of unilateral pelviureteral junction obstruction

Background/PurposeDown-regulation of aquaporin 1 (AQP1) and up-regulation of transforming growth factor β1 (TGF-β1) in the renal parenchyma have been demonstrated in children who underwent pyeloplasty for pelviureteral junction obstruction. However, no information about urinary exosomal AQP1 and TGF-β1 during postobstructive polyuria in children with congenital unilateral hydronephrosis is available. The aim of the present study is to evaluate the urine concentration of exosomal AQP1 and TGF-β1 on the first and the second day after surgery in children who underwent pyeloplasty. MethodsTwenty-two patients (age, 36.2 ± 17.1 months) with unilateral pelviureteral junction obstruction were examined in the study. For the first 2 days after the operation, the urine was collected separately from pyelostomy draining only from the postobstructed kidney and from the bladder catheter draining mostly from the contralateral kidney, which was used as an internal control. Urinary output, urinary osmolality, sodium, β2-microglobulin (β2-MG), and creatinine, as well as urinary exosomal AQP1 and TGF-β1 excretion, were tested in each sample. ResultsAfter pyeloplasty, a significantly decreased urinary excretion of exosomal AQP1 (∼64%) was found in the postobstructed kidney. The patients developed polyuria (807 ± 216 mL/24 h vs 484 ± 144 mL/24 h at day 1, 1021 ± 348 mL/24 h vs 603 ± 228 mL/24 h at day 2; P < .01) and reduced urine osmolality (115 ± 44 mOsm/kg vs 282 ± 61 mOsm/kg at day 1, 139 ± 39 vs 303 ± 46 mOsm/kg at day 2; P < .01) that persisted for 48 hours. In parallel, urinary TGF-β1 and β2-MG (normalized for creatinine) from the postobstructed kidney were significantly higher compared with the contralateral kidney. The urine output and urinary sodium concentration from the postobstructed kidney elevated significantly on the second day after the release of obstruction compared with those on the first day. The contralateral kidney also showed same trends. ConclusionsThe down-regulation of urinary exosomal AQP1 in the postobstructed kidney may account for the polyuria, hypotonic urine, and elevated urinary β2-MG. The urinary TGF-β1 level locally increased in the postobstructed kidney may be involved in renal AQP1 down-regulation.

Triazolothienopyrimidine Inhibitors of Urea Transporter UT-B Reduce Urine Concentration

Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB(inh)-14, fully and reversibly inhibited urea transport with IC(50) values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB(inh)-14 competed with urea binding at an intracellular site on the UT-B protein. UTB(inh)-14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB(inh)-14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H(2)O lower in UTB(inh)-14-treated mice than vehicle-treated mice. UTB(inh)-14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB(inh)-14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.

Mice with targeted disruption of the acyl-CoA binding protein display attenuated urine concentrating ability and diminished renal aquaporin-3 abundance

The acyl-CoA binding protein (ACBP) is a small intracellular protein that specifically binds and transports medium to long-chain acyl-CoA esters. Previous studies have shown that ACBP is ubiquitously expressed but found at particularly high levels in lipogenic cell types as well as in many epithelial cells. Here we show that ACBP is widely expressed in human and mouse kidney epithelium, with the highest expression in the proximal convoluted tubules. To elucidate the role of ACBP in the renal epithelium, mice with targeted disruption of the ACBP gene (ACBP(-/-)) were used to study water and NaCl balance as well as urine concentrating ability in metabolic cages. Food intake and urinary excretion of Na(+) and K(+) did not differ between ACBP(-/-) and (+/+) mice. Interestingly, however, water intake and diuresis were significantly higher at baseline in ACBP(-/-) mice compared with that of (+/+) mice. Subsequent to 20-h water deprivation, ACBP(-/-) mice exhibited increased diuresis, reduced urine osmolality, elevated hematocrit, and higher relative weight loss compared with (+/+) mice. There were no significant differences in plasma concentrations of renin, corticosterone, and aldosterone between mice of the two genotypes. After water deprivation, renal medullary interstitial fluid osmolality and concentrations of Na(+), K(+), and urea did not differ between genotypes and cAMP excretion was similar. Renal aquaporin-1 (AQP1), -2, and -4 protein abundances did not differ between water-deprived (+/+) and ACBP(-/-) mice; however, ACBP(-/-) mice displayed increased apical targeting of pS256-AQP2. AQP3 abundance was lower in ACBP(-/-) mice than in (+/+) control animals. Thus we conclude that ACBP is necessary for intact urine concentrating ability. Our data suggest that the deficiency in urine concentrating ability in the ACBP(-/-) may be caused by reduced AQP3, leading to impaired efflux over the basolateral membrane of the collecting duct.

Renal distal tubule proliferation and increased aquaporin 2 level but decreased urine osmolality in db/db mouse: Treatment with chromium picolinate

Hallmark features of type 2 diabetes mellitus include glucosuria and polyuria. Further, renal aquaporin 2 is pivotal to regulation of fluid excretion and urine osmolality. Accordingly, we tested the hypothesis that the db/db mouse displays increased glucosuria and fluid excretion but reduced urine osmolality in association with decreased renal aquaporin 2 level. In addition, we examined the effect of chromium picolinate (Cr(pic)3) which is purported to improve glycemic control. The db/db mice excreted more urine in association with marked glucose excretion but lower urine osmolality than db/m control group. Light microscopic examination of renal tissue revealed proliferation of tubular structures in db/db compared to the db/m mice, a feature validated with Ki67 immunostaining. Further, these tubules showed generally similar immunostaining intensity and pattern for aquaporin 2 indicating that proliferated tubules are of distal origin. On the other hand, renal aquaporin 2 protein level was significantly higher in the db/db than db/m group. Treatment of db/db mice with Cr(pic)3 reduced plasma glucose and hemoglobin A1c (~15–17%, p<0.05) and Ki67 positive cells but other parameters were similar to their untreated counterparts. Collectively, these findings suggest that proliferation of renal distal tubules and increased aquaporin 2 level likely represent an adaptive mechanism to regulate fluid excretion to prevent dehydration in the setting of marked glucosuria in the db/db mouse, features not affected by Cr(pic)3 treatment. These observations are of relevance to increasing interest in developing therapeutic agents that facilitate renal glucose elimination.

αENaC-Mediated Lithium Absorption Promotes Nephrogenic Diabetes Insipidus

Lithium-induced nephrogenic diabetes insipidus (NDI) is accompanied by polyuria, downregulation of aquaporin 2 (AQP2), and cellular remodeling of the collecting duct (CD). The amiloride-sensitive epithelial sodium channel (ENaC) is a likely candidate for lithium entry. Here, we subjected transgenic mice lacking αENaC specifically in the CD (knockout [KO] mice) and littermate controls to chronic lithium treatment. In contrast to control mice, KO mice did not markedly increase their water intake. Furthermore, KO mice did not demonstrate the polyuria and reduction in urine osmolality induced by lithium treatment in the control mice. Lithium treatment reduced AQP2 protein levels in the cortex/outer medulla and inner medulla (IM) of control mice but only partially reduced AQP2 levels in the IM of KO mice. Furthermore, lithium induced expression of H(+)-ATPase in the IM of control mice but not KO mice. In conclusion, the absence of functional ENaC in the CD protects mice from lithium-induced NDI. These data support the hypothesis that ENaC-mediated lithium entry into the CD principal cells contributes to the pathogenesis of lithium-induced NDI.

Furosemide impairs nasal mucociliary clearance in humans

Furosemide, a potent diuretic, affects ion and water movement across the respiratory epithelium. However, the effects of furosemide, as clinically used, on mucociliary clearance, a critical respiratory defense mechanism, are still lacking in humans. Fourteen young healthy subjects were assigned to three random interventions, spaced one-week apart: no intervention (control), oral furosemide (40mg), and furosemide+oral volume replacement (F+R). Nasal mucociliary clearance was assessed by saccharine test (STT), and mucus properties were in vitro evaluated by means of contact angle and transportability by sneeze. Urine output and osmolality were also evaluated. Urine output increased and reduced urine osmolality in furosemide and F+R compared to the control condition. STT remained stable in the control group. In contrast, STT increased significantly (40%) after furosemide and F+R. There were no changes in vitro mucus properties in all groups. In conclusion, furosemide prolongs STT in healthy young subjects. This effect is not prevented by fluid replacement, suggesting a direct effect of furosemide on the respiratory epithelium.