Increased Urine Osmolality Research Articles

Metformin, an AMPK activator, stimulates the phosphorylation of aquaporin 2 and urea transporter A1 in inner medullary collecting ducts.

Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations.

RNA binding protein Caprin-2 is a pivotal regulator of the central osmotic defense response.

In response to an osmotic challenge, the synthesis of the antidiuretic hormone arginine vasopressin (AVP) increases in the hypothalamus, and this is accompanied by extension of the 3' poly(A) tail of the AVP mRNA, and the up-regulation of the expression of RNA binding protein Caprin-2. Here we show that Caprin-2 binds to AVP mRNAs, and that lentiviral mediated shRNA knockdown of Caprin-2 in the osmotically stimulated hypothalamus shortens the AVP mRNA poly(A) tail at the same time as reducing transcript abundance. In a recapitulated in vitro system, we confirm that Caprin-2 over-expression enhances AVP mRNA abundance and poly(A) tail length. Importantly, we show that Caprin-2 knockdown in the hypothalamus decreases urine output and fluid intake, and increases urine osmolality, urine sodium concentration, and plasma AVP levels. Thus Caprin-2 controls physiological mechanisms that are essential for the body's response to osmotic stress.

Abstract P018: Periodic Water Intake Exacerbates Hypertension, Decreases Renal Function and Increases Renal Inflammation in Spontaneously Hypertensive Rats

Water is essential for life. The kidneys are major regulators of fluid balance but also appear susceptible to adverse effects of periodic water intake since epidemiological studies have linked low water intake to chronic kidney disease. However, the impact of periodic water intake on renal health has largely been ignored. This study investigated the chronic effects of periodic drinking on blood pressure, kidney function, and immune cell infiltration in rats with pre-existing hypertension. Mean arterial pressure (MAP) was measured continuously in spontaneously hypertensive rats (SHR) that were only given access to water for 2 hours per day versus control rats given free access to water over a 4-week period (n=>8 per group). Glomerular filtration rate (GFR) was measured via transcutaneous FITC-sinistrin clearance at baseline and at the end of the 4-week protocol. Renal immune cell infiltration and cytokine production was assessed by flow cytometry. Basal MAP and renal function were similar between the control and water-restricted (WR) groups. Water restriction led to a significant increase in MAP from baseline (8.8±4.5mmHg versus 0.6±5.5mmHg in WR and control SHR, respectively; P<0.05). Moreover, GFR decreased from baseline by 29±6% in the WR SHR (P<0.001). In comparison, GFR did not change significantly over time in the control SHR (-5.3±2.6% from baseline; P>0.05). The increase in MAP and reduction in renal function in the WR-SHR was associated with periodic increases in urine osmolality (69±26% versus baseline, P<0.05), indicative of cycles of dehydration and replenishment. Renal immune cell infiltration was similar between the groups. However, cytokine analyses revealed a phenotypic shift towards a pro-inflammatory Th1 phenotype in kidney T cell infiltrate from WR versus control SHR. This was indicated by a greater proportion of T cells producing IFN-γ in SHR subjected to water restriction than control SHR (9.5±3.3% versus 2.9±0.9%, respectively; P<0.05). In conclusion, recurrent dehydration associated with periodic drinking exacerbates hypertension, renal dysfunction and inflammation in male SHR. This highlights the importance of regular water intake for the maintenance of kidney health, particularly in populations with existing disease.

Mesenchymal Stromal Cells Improve Renovascular Function in Polycystic Kidney Disease.

Polycystic kidney disease (PKD) is a common cause of end-stage renal failure, for which there is no accepted treatment. Progenitor and stem cells have been shown to restore renal function in a model of renovascular disease, a disease that shares many features with PKD. The objective of this study was to examine the potential of adult stem cells to restore renal structure and function in PKD. Bone marrow-derived mesenchymal stromal cells (MSCs, 2.5 × 10(5)) were intrarenally infused in 6-week-old PCK rats. At 10 weeks of age, PCK rats had an increase in systolic blood pressure (SBP) versus controls (126.22 ± 2.74 vs. 116.45 ± 3.53 mmHg, p < 0.05) and decreased creatinine clearance (3.76 ± 0.31 vs. 6.10 ± 0.48 µl/min/g, p < 0.01), which were improved in PKD animals that received MSCs (SBP: 114.67 ± 1.34 mmHg, and creatinine clearance: 4.82 ± 0.24 µl/min/g, p = 0.001 and p = 0.003 vs. PKD, respectively). MSCs preserved vascular density and glomeruli diameter, measured using microcomputed tomography. PCK animals had increased urine osmolality (843.9 ± 54.95 vs. 605.6 ± 45.34 mOsm, p < 0.01 vs. control), which was improved after MSC infusion and not different from control (723.75 ± 56.6 mOsm, p = 0.13 vs. control). Furthermore, MSCs reduced fibrosis and preserved the expression of proangiogenic molecules, while cyst size and number were unaltered by MSCs. Delivery of exogenous MSCs improved vascular density and renal function in PCK animals, and the benefit was observed up to 4 weeks after a single infusion. Cell-based therapy constitutes a novel approach in PKD.

Sildenafil for the Treatment of Congenital Nephrogenic Diabetes Insipidus

Background: Congenital nephrogenic diabetes insipidus (NDI) is characterized by massive polyuria and polydipsia due to defects in the vasopressin-sensitive signaling system expression of the acuaporin-2 (AQP2) water channel of the kidney collecting duct principal cells. Current conventional treatment regimen including hydration, diuretics and non-steroidal anti-inflammatory drugs can only partially reduce polyuria. Recent experimental studies have suggested that treatment with sildenafil, a selective phosphodiesterase inhibitor, may enhance cyclic guanosine monophosphate (cGMP)-mediated apical trafficking of AQP2 and may be effective in increasing water reabsorption in patients with congenital NDI. Patient and Methods: A 4-year old boy with X-linked NDI resistant to conventional therapy was treated with sildenafil for 10 days after a 2-day washout period between the 2 treatment regimens. Aliquots of the 24-hour urine collections before and after treatment were analyzed for urine volume, osmolality, cGMP and AQP2 determinations. Blood samples were also obtained for sodium and osmolality measurements. The primary endpoint was 24-hour urine volume after 10 days of sildenafil and conventional treatments. Results: Compared to conventional therapy, treatment with sildenafil resulted in substantial reduction in 24-hour urine volume (1,764 vs. 950 ml) and serum sodium (148 vs. 139) mEq/l, and increased urine osmolality (104 vs. 215 mOsm/l), and AQP2 excretion (5 vs. 26 fmol/mg creatinine). The patient tolerated sildenafil well and experienced no adverse effects. Conclusions: Sildenafil citrate should be considered an alternative agent in the treatment of X-linked NDI resistant to conventional therapy.

Estradiol regulates AQP2 expression in the collecting duct: a novel inhibitory role for estrogen receptor α.

While there is evidence that sex hormones influence multiple systems involved in salt and water homeostasis, the question of whether sex hormones regulate aquaporin-2 (AQP2) and thus water handling by the collecting duct has been largely ignored. Accordingly, the present study investigated AQP2 expression, localization and renal water handling in intact and ovariectomized (OVX) female rats, with and without estradiol or progesterone replacement. OVX resulted in a significant increase in urine osmolality and increase in p256-AQP2 in the renal cortex at 7 days post-OVX, as well as induced body weight changes. Relative to OVX alone, estradiol repletion produced a significant increase in urine output, normalized urinary osmolality and reduced both total AQP2 (protein and mRNA) and p256-AQP2 expression, whereas progesterone repletion had little effect. Direct effects of estradiol on AQP2 mRNA and protein levels were further tested in vitro using the mpkCCD principal cell line. Estradiol treatment of mpkCCD cells reduced AQP2 at both the mRNA and protein level in the absence of deamino-8-d-AVP (dDAVP) and significantly blunted the dDAVP-induced increase in AQP2 at the protein level only. We determined that mpkCCD and native mouse collecting ducts express both estrogen receptor (ER)α and ERβ and that female mice lacking ERα displayed significant increases in AQP2 protein compared with wild-type littermates, implicating ERα in mediating the inhibitory effect of estradiol on AQP2 expression. These findings suggest that changes in estradiol levels, such as during menopause or following reproductive surgeries, may contribute to dysregulation of water homeostasis in women.

AMPK Stimulates Urea Permeability and Urine Osmolality

Vasopressin is a key regulator of urine concentration through its actions on water and urea transporters, but what happens when vasopressin signaling breaks down‾ We investigated adenosine monophosphate activated kinase (AMPK) as a possible alternate, vasopressin‐independent regulatory pathway for urea and water transport. Mice lacking a vasopressin receptor (V2R‐KO) are polyuric. Injection of metformin to stimulate AMPK significantly increased urine osmolality from 196 (control) to 399 mOsM at 1h, and 586 mOsM at 5h post treatment in the V2R‐KO mice. We further investigated the role of AMPK using isolated perfused rat inner medullary collecting ducts (IMCDs). We showed that metformin and AICAR, AMPK activators, significantly increased urea permeability (control:metformin 16.4 ± 2.3 to 22.3 ± 3.5 x10‐5 cm/s; control:AICAR 19.3 ± 2.4 to 23.3 ± 3.0 x10‐5 cm/s). Hypertonicity stimulates urea transport though PKC. To determine if AMPK is additive to hypertonic stimulation, 400µM metformin was added to the tubule bath either before or after subjecting the IMCDs to hypertonic stimulation. Increasing the osmolality of perfusate and bath from 290 to 690 mOsM increased urea permeability from 18.9 ± 1.4 to 48.4 ± 3.1 x10‐5 cm/s. Addition of metformin further increased urea permeability from 48.4 ± 3.1 to 57.0 ± 4.1 x10‐5 cm/s. Initial stimulation with metformin raised urea permeability from 23.6 ± 1.9 to 32.2 ± 2.5 x10‐5 cm/s. Subsequent increase of the osmolality from 290 to 690 mOsM in the presence of metformin significantly increased urea permeability from 32.2 ± 2.5 to 68.0 ± 7.9 x10‐5 cm/s. We conclude that AMPK signaling may provide a means to increase urea permeability in addition to, or in place of, PKA and PKC. AMPK activation may be useful to increase urine concentrating ability when vasopressin signaling breaks down.

High-throughput chemical screening identifies AG-490 as a stimulator of aquaporin 2 membrane expression and urine concentration.

A reduction or loss of plasma membrane aquaporin 2 (AQP2) in kidney principal cells due to defective vasopressin (VP) signaling through the VP receptor causes excessive urine production, i.e., diabetes insipidus. The amount of AQP2 on the plasma membrane is regulated by a balance of exocytosis and endocytosis and is the rate limiting step for water reabsorption in the collecting duct. We describe here a systematic approach using high-throughput screening (HTS) followed by in vitro and in vivo assays to discover novel compounds that enhance vasopressin-independent AQP2 membrane expression. We performed initial chemical library screening with a high-throughput exocytosis fluorescence assay using LLC-PK1 cells expressing soluble secreted yellow fluorescent protein and AQP2. Thirty-six candidate exocytosis enhancers were identified. These compounds were then rescreened in AQP2-expressing cells to determine their ability to increase AQP2 membrane accumulation. Effective drugs were then applied to kidney slices in vitro. Three compounds, AG-490, β-lapachone, and HA14-1 increased AQP2 membrane accumulation in LLC-PK1 cells, and both AG-490 and β-lapachone were also effective in MDCK cells and principal cells in rat kidney slices. Finally, one compound, AG-490 (an EGF receptor and JAK-2 kinase inhibitor), decreased urine volume and increased urine osmolality significantly in the first 2-4 h after a single injection into VP-deficient Brattleboro rats. In conclusion, we have developed a systematic procedure for identifying new compounds that modulate AQP2 trafficking using initial HTS followed by in vitro assays in cells and kidney slices, and concluding with in vivo testing in an animal model.

Acetazolamide: an improved treatment for lithium‐induced NDI? (LB726)

Lithium causes nephrogenic diabetes insipidus (Li‐NDI) and hydrochlorothiazide (HCTZ) forms, together with amiloride, the mainstay treatment for Li‐NDI patients. Earlier, we found that HCTZ, primarily known for inhibiting the NaCl‐co‐transporter (NCC), also reduces Li‐NDI in NCC knockout mice, coinciding with alkalinized urine, suggesting that inhibition of carbonic anhydrase (CA) by HCTZ plays a role. To test whether CA inhibition is a useful alternative therapy in Li‐NDI, the effect of acetazolamide (ACZ) was tested.Polarized mouse cortical collecting duct (mpkCCD) cells and mice were used to study the effect of ACZ on Li‐NDI.Treatment of mpkCCD cells with ACZ attenuated AQP2 downregulation by lithium. ACZ did, in contrast to amiloride, not affect transcellular voltage. Treatment of Li‐NDI mice with ACZ revealed a significant antidiuresis and increased urine osmolality, which was indifferent from Li‐NDI mice treated with HCTZ/amiloride. However, unlike HCTZ/amiloride, ACZ treatment did not result in hyponatremia, hyperkalemia, hypercalcaemia and metabolic acidosis. Moreover, ACZ rescued AQP2 expression over the entire length of the collecting duct, whereas HCTZ/amiloride only increased AQP2 levels in the renal papilla.In conclusion, our data reveal that ACZ attenuates lithium‐induced downregulation of AQP2 and NDI development. Moreover, as ACZ caused fewer side effects than the conventional treatment, ACZ may represent a better therapeutic agent than HCTZ/amiloride to treat Li‐NDI.Grant Funding Source: Supported by a VICI grant, a RUNMC grant and a grant from the Society of Experimental Laboratory Medicine.

Codeine-Induced Syndrome of Inappropriate Antidiuretic Hormone: Case Report

The syndrome of inappropriate antidiuretic hormone was first described in 1957 by Schwartz, and is characterised by hyponatraemia, inappropriately increased urine osmolality and urine sodium, and decreased serum osmolality in a euvolemic patient without edema. A patient with the syndrome of inappropriate antidiuretic hormone should have normal cardiac, renal, adrenal, hepatic, and thyroid functions and should not take any diuretics. We present a case of the syndrome of inappropriate antidiuretic hormone caused by codeine and associated with reduced urine volume, increased urine sodium, and decreased serum sodium concentration. The syndrome of inappropriate antidiuretic hormone is a disease that can lead to morbidity and even mortality. Clinicians should measure serum electrolytes intermittently in order to avoid missing the diagnosis of the syndrome of inappropriate antidiuretic hormone in patients using opioid.

Central Diabetes insipidus in a Nigerian child : A case report

Background : Central diabetes insipidus (CDI) is rare in children. About 30 - 50% of cases are idiopathic. Early and accurate diagnosis are crucial for safe and effective treatment. This is the first report of Central diabetes insipidus in a child in Nigeria. Case report: We report a case of central diabetes insipidus in a female toddler who presented at the University of Port Harcourt Teaching Hospital with polydipsia and polyuria with a urine specific gravity of 1.000 and normal blood sugar. The diagnosis of CDI was confirmed by her inability to concentrate urine after a water deprivation test followed by an increase in urine osmolality from 59mOsm/kg to 158mOsm/kg and serum osmolality from 286mOsm/kg to 321Osm/kg following intravenous administration of desmopressin. The patient responded well to oral desmopressin. Conclusion : Central diabetes insipidus occurs in Nigerian children and responds to oral desmopressin. We recommended high index of suspicion in children with polyuria and polydipsia. Key words : Central diabetes insipidus, polyuria, polydipsia, desmopressin, Nigerian child

Fluvastatin Increases AQP2 Urine Excretion in a Dyslipidemic Patient with Nephrogenic Diabetes Insipidus: An In Vivo and In Vitro Study

Objective: Among the pleiotropic effects of statins, we have previously reported that fluvastatin increases the amount of plasma membrane-expressed AQP2 in renal collecting duct cells both in vitro and in vivo, independently of vasopressin. This effect may be of potential clinical significance for the treatment of patients affected by nephrogenic diabetes insipidus forms caused by inactivating mutations of the vasopressin type 2 receptor. Here we report the effect of fluvastatin on AQP2 plasma membrane abundance on an adult male XNDI patient treated with statins. Methods: An adult male NDI patient, carrying an inactivating mutation of the V2R, under conventional treatment to reduce polyuria, was also treated with fluvastatin because of high levels of blood cholesterol. AQP2 plasma membrane expression in the kidney was monitored by measuring urinary excreted AQP2 (u-AQP2), before starting fluvastatin treatment and during a three months follow-up period. The effect of fluvastatin was also tested in vitro in mouse kidney cortical collecting duct MCD4 cells. Results: u-AQP2 increased in a time- and dose-dependent manner after treatment with 40 and 80 mg/day of fluvastatin for 90 days. However, at this drug dosage, increased uAQP2 was not accompanied by reduction of diuresis and increase of urine osmolality. The effect of fluvastatin on AQP2 excretion was confirmed in vitro in cultured renal cells. Conclusions: We first demonstrate that the use of fluvastatin increased AQP2 plasma membrane expression in an NDI dyslipidemic patient. This observation was confirmed by in vitro studies using mouse cultured renal cells treated with fluvastatin. Although a clinical relevant effect of fluvastatin on total diuresis and urine osmolality was not observed at the used dosages, these results suggest further investigation on the possible role of HMG CoA reductase inhibitors to improve the efficacy of the current NDI therapy.

Bradykinin Signal Pathways Modulate Neurohypophyseal Hormones Secretion

Afferent renal nerves stimulate vasopressin (VP) secretion by activation of VPergic neurons of supraoptic (SON) and paraventricular nuclei (PVN). Therefore, intrarenal infusion of bradykinin (BK), which excites afferent renal nerves, increases VP release. However, BK is also a potent intrarenal vasodilatator hence BK may modulate VP secretion not only by stimulating its release via afferent renal nerves, but also inhibiting its renal effects as intrarenal vasodilator via a paracrine control. Furthermore, BK impaires the mechanism of phosphorilation that induces the traslocation of aquaporin 2 containing vesicles to the apical plasma membrane thus inhibiting the osmotic water permeability of the collecting duct cells induced by VP via its V2 receptor, a Gs protein-coupled receptor. In addition, BKB2 receptor knockout mice exhibit decreased urine volume and increased urine osmolality following water deprivation. Oxytocin (OT) increases urine output and sodium excretion in rats. Icatibant, a BKB2 antagonist, suppresses these effects showing that BK mediates diuresis and natriuresis induced by OT. Infusion of OT down-regulates myometrial OT and BK receptors indicating the existence of a common final pathway of OT and BK in the contractile responsiveness of uterine myometrial cells. The frequency of uterine contractions induced by OT and BK are stimulated by nitric oxide via prostaglandins release.

A missense mutation in the arginine‐vasopressin neurophysin‐II gene causes autosomal dominant neurohypophyseal diabetes insipidus in a Chinese family

Familial neurohypophyseal diabetes insipidus, an autosomal dominant disorder, is mostly caused by mutations in the genes that encode AVP or its intracellular binding protein, neurophysin-II. The mutations lead to aberrant preprohormone processing and progressive destruction of AVP-secreting cells, which gradually manifests a progressive polyuria and polydipsia during early childhood, and a disorder of water homeostasis. We characterized the clinical and biochemical features, and sequenced the AVP neurophysin-II(AVP-NPII) gene of the affected individuals with autosomal dominant neurohypophyseal diabetes insipidus(ADNDI)to determine whether this disease was genetically determined. We obtained the histories of eight affected and four unaffected family individuals. The diagnosis of ADNDI was established using a water deprivation test and exogenous AVP administration. For molecular analysis, genomic DNA was extracted and the AVP-NPII gene was amplified using polymerase chain reaction and sequenced. The eight affected individuals showed different spectra of age of onsets (7-15 years) and urine volumes (132-253 ml/kg/24 h). All affected individuals responded to vasopressin administration, with a resolution of symptoms and an increase in urine osmolality by more than 50%. The characteristic hyperintense signal in the posterior pituitary on T1-weighted magnetic resonance imaging was absent in six family members and present in one. Sequencing analysis revealed a missense heterozygous mutation 1516G > T (Gly17Val) in exon 2 of the AVP-NPII gene among the ADNDI individuals. We identified a missense mutation in the AVP-NPII gene and the same mutation showed different spectra of age of onsets and urine volumes in a new Chinese family with ADNDI. The mutation may provide a molecular basis for understanding the characteristics of NPII and add to our knowledge of the pathogenesis of ADNDI, which would allow the presymptomatic diagnosis of asymptomatic subjects.

TRANSGENIC MICE EXPRESSING UT‐A1, BUT LACKING UT‐A3, HAVE INTACT URINE CONCENTRATING ABILITY

Mice lacking the inner medullary collecting duct (IMCD) urea transporters, UT‐A1 and UT‐A3, are unable to concentrate their urine. It is not known whether this defect results from the absence of UT‐A1, UT‐A3, or both. Studies suggest a functional UTA1/ A3 heterodimer. To investigate, we generated a UT‐A1 gene that could not be spliced to produce UT‐A3, behind its original UT‐A promoter. We introduced this transgene into UT‐A1/A3 knock‐out (KO) mice to yield a mouse expressing UT‐A1 but not UT‐A3 (UT‐A1‐only mice). Western blot demonstrated UT‐A1 protein in inner medulla (IM) of UT‐A1‐only and wild‐type (WT) mice, but not in UT‐A1/A3 KO mice. UT‐A1 protein was not detected in brain, heart, colon, lung, liver, spleen, vas deferens, or testes of UT‐A1‐only mice. Immunohistology showed UT‐A1 staining only in the IM of the UT‐A1‐only mice. Basal urine osmolalities were similar in WT (2844 mOsm) and UT‐A1‐only mice (2508 mOsm); both were higher than UT‐A1/A3 KO (920 mOsm)(p&lt;.05). Overnight water restriction increased urine osmolality similarly in WT (4469 mOsm) and UT‐A1‐only (3656 mOsm) mice; both were higher than UT‐A1/A3 KO (1365 mOsm)(p&lt;.05). We conclude that mice expressing UT‐A1, but not UT‐A3, are able to concentrate their urine to similar levels as WT mice. This suggests UT‐A3 may not be essential for urine concentration and that the functional urea channel in the IMCD is not a UT‐A1/UT‐A3 heterodimer. Support:NIDDK

A Case With Recovery of Response to Tolvaptan Associated With Remission of Acute Kidney Injury and Increased Urine Osmolality

Tolvaptan (TLV), a vasopressin type 2 receptor antagonist, has been demonstrated to be effective in patients with decompensated heart failure (HF) refractory to incremental doses of diuretics, but the responsiveness has not always been predictable. We have recently proposed that urine osmolality (U-OSM) is a valuable parameter for the prediction of responses to TLV, because U-OSM reflects the activity of the collecting ducts, where TLV plays its unique role. Acute kidney injury (AKI) is often associated with severe tubular dysfunction, including the collecting ducts, and in such cases a response to TLV may not be expected. We here experienced a patient with HF and AKI in whom TLV was not effective during AKI. We also observed recovery of responsiveness to TLV along with remission of AKI as well as increased U-OSM later on. We believe that this is the first report on the reversibility of the TLV response in relation to U-OSM.

Urine concentration in the diabetic mouse requires both urea and water transporters

The regulation of the inner medullary collecting duct (IMCD) urea transporters (UT-A1, UT-A3) and aquaporin-2 (AQP2) and their interactions in diabetic animals is unknown. We investigated whether the urine concentrating defect in diabetic animals was a function of AQP2, the UT-As, or both transporters. UT-A1/UT-A3 knockout (UT-A1/A3 KO) mice produce dilute urine. We gave wild-type (WT) and UT-A1/A3 KO mice vasopressin via minipump for 7 days. In WT mice, vasopressin increased urine osmolality from 3,000 to 4,550 mosmol/kgH(2)O. In contrast, urine osmolality was low (800 mosmol/kgH(2)O) in the UT-A1/A3 KOs and remained low following vasopressin. Surprisingly, AQP2 protein abundance increased in UT-A1/A3 KO (114%) and WT (92%) mice. To define the role of UT-A1 and UT-A3 in the diabetic responses, WT and UT-A1/A3 KO mice were injected with streptozotocin (STZ). UT-A1/A3 KO mice showed only 40% survival at 7 days post-STZ injection compared with 70% in WT. AQP2 did not increase in the diabetic UT-A1/A3 KO mice compared with a 133% increase in WT diabetic mice. Biotinylation studies in rat IMCDs showed that membrane accumulation of UT-A1 increased by 68% in response to vasopressin in control rats but was unchanged by vasopressin in diabetic rat IMCDs. We conclude that, even with increased AQP2, UT-A1/UT-A3 is essential to optimal urine concentration. Furthermore, UT-A1 may be maximally membrane associated in diabetic rat inner medulla, making additional stimulation by vasopressin ineffective.

Osmoregulatory and metabolic costs of salt excretion in the Rufous-collared sparrow Zonotrichia capensis

Recent experiments on shorebirds have demonstrated that maintaining an active osmoregulatory machinery is energetically expensive. This may, in part, explain diet and habitat selection in birds with salt glands. However little is known about the osmoregulatory costs in birds lacking functional salt glands. In these birds, osmotic work is done almost exclusively by the kidneys. We investigated the osmoregulatory cost in a bird species lacking functional salt glands, the passerine Zonotrichia capensis. After 20days of acclimation to fresh water (FW) and salt water (200mM NaCl, SW), SW birds tended to be heavier than FW birds. However, this difference was not statistically significant. Total basal metabolic rate was higher in SW birds as compared with FW birds. Renal and heart masses were also higher in the SW group. We also found greater medullary development and an increase in urine osmolality in the SW group. In spite of Z. capensis' ability to tolerate a moderate salt load in the laboratory, we hypothesize that increased cost of maintenance produced by salt consumption may significantly affect energy budget, dietary, and habitat choices in the field.

Vasopressin, Copeptin, and Renal Concentrating Capacity in Patients with Autosomal Dominant Polycystic Kidney Disease without Renal Impairment

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent hereditary renal disease, characterized by cyst formation in the kidneys leading to end stage kidney failure. It is clinically acknowledged that ADPKD patients have impaired urine concentrating capacity, but the mechanism behind this observation is unknown. Fifteen ADPKD patients (estimated GFR ≥60 ml/min per 1.73 m(2)) and 15 age- and sex-matched healthy controls underwent a standard prolonged water deprivation test in which urine and plasma osmolality, vasopressin, and copeptin were measured. The effect of a synthetic vasopressin analog (desmopressin) injected at the moment of maximal urine concentrating capacity was also studied. After 14 hours of water deprivation, ADPKD patients tended to have higher plasma osmolality (P=0.07) and significantly higher vasopressin and copeptin levels (both P<0.05), whereas urine osmolality was similar in ADPKD patients and controls (710 versus 742 mOsmol/kg; P=0.61). Maximal urine concentrating capacity was lower in ADPKD patients (758 versus 915 mOsmol/kg in controls; P<0.001). At maximal urine concentrating capacity, plasma osmolality, vasopressin, and copeptin levels were significantly higher in ADPKD patients. The median increase in urine osmolality after desmopressin administration in ADPKD patients was less than in healthy controls. Already early in their disease, ADPKD patients have impaired maximal urine concentrating capacity brought out upon dehydration, with no evidence of impaired hypothalamic response. To maintain fluid balance, vasopressin concentration increases, which is hypothesized to play a role in ADPKD disease progression.

AVP infusion increases the expression of urea transporters and ER stress pathway genes in medullae of female mice with urinary concentrating defect

Abnormal proliferation in collecting duct cells plays a crucial role in polycystic kidney disease (PKD) and in lithium‐induced renal dysfunction. Females with PKD demonstrate lower proliferation rates than males, resulting in a less severe phenotype. We previously reported that vasopressin induced an increase in renal cell proliferation in male AQP1KO mice in addition to ER stress gene activation. In this study we infused AVP for 14 days into female AQP1KO mice. AVP induced a small but significant increase in urine osmolality (155 mosmol/kgH20 vs control). 14 days AVP infusion significantly increased the expression of UTA1 and UTA3 mRNA 2.97 ± 0.29 and 3.34 ± 0.57, respectively, vs control. AQP2 and AQP3 mRNA also increased 3.52 ± 0.28 and 2.59 ± 0.23, vs control. AVP infusion significantly increased the mRNA expression of early growth response gene Egr‐1 2.49 ± 0.49 and transcription factors ATF3 and ATF4 mRNA; 4.91 ± 0.70, and 3.36 ± 0.25, vs control. AVP infusion significantly increased the expression of ER stress factor GADD153 mRNA 2.53 ± 0.41 vs control. AVP did not increase the mRNA expression of PCNA in female kidneys; PCNA is a marker of proliferation. These data suggest that vasopressin regulation of urea transporters is intact in female AQP1KO mice. Further studies will determine if vasopressin induced proliferative pathways are blunted in female AQP1KO mice.Support: DK073611 (HLB)