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Introduction: G protein-coupled bile acid receptor (TGR5), the first G protein-coupled receptor for bile acids identified, is capable of activating a variety of intracellular signaling pathways after interacting with bile acids. TGR5 plays an important role in multiple physiological processes and is considered to be a potential target for the treatment of various metabolic diseases, including type 2 diabetes. Evidence has emerged that genetic deletion of TGR5 results in an increase in basal urine output, suggesting that it may play a critical role in renal water and salt reabsorption. The present study aims to elucidate the effect and mechanism of TGR5 activation on urine concentration. Methods: Mice were treated with TGR5 agonists (LCA and INT-777) for 3 days. The 24-h urine of mice was collected and analyzed for urine biochemical parameters. The mRNA expressions were detected by real-time PCR, and the protein expressions were detected by western blot. Immunohistochemistry and immunofluorescence were performed to examine the cellular location of proteins. The cultured primary medullary collecting duct cells were pretreated with H89 (a PKA inhibitor) for 1 h, followed by 12-h treatment of LCA and INT-777. Luciferase reporter assays were used to detect the effect of CREB on the gene transcription of AQPs. Gel electrophoretic mobility shift assays were used to analyze DNA–protein interactions. Results: Treatment of mice with the TGR5 agonist LCA and INT-777 markedly reduced urine output and increased urine osmolality, accompanied by a marked increase in AQP2 and AQP3 protein expression and membrane translocation. In cultured primary medullary collecting duct cells, LCA and INT-777 dose-dependently upregulated AQP2 and AQP3 expression in a cAMP/PKA-dependent manner. Mechanistically, both AQP2 and AQP3 gene promoter contains a putative CREB-binding site, which can be bound and activated by CREB as assessed by both gene promoter-driven luciferase and gel shift assays. Conclusion: Collectively, our findings demonstrate that activation of TGR5 can promote urine concentration by upregulation of AQP2 and AQP3 expression in renal collecting ducts. TGR5 may represent an attractive target for the treatment of patients with urine concentration defect.

The aim of this study was to report some of the morphological characteristics of the kidney involved in urine concentration and hence water conservation in the dromedaries. A total of 20 fresh kidneys of 10 apparently healthy camels were used in this study. The architecture of the renal pelvis was revealed by dissection and polyvinyl chloride corrosion casts. Samples were also processed for histology and for enzyme histochemistry. The camel kidney is bean shaped, smooth, multilobar, unipapillary, in which the fusion of renal papillae is complete forming a common renal papilla or crest, which channel urine into a central renal pelvis. It is more or less similar to equine, caprine, ovine and canine kidney. Under certain anatomical requisites the renal pelvis is known to play a role in urine concentration through recycling of urea to increase the medullary osmotic concentration which favors the counter-current mechanism. One of these requisites is an elaborate renal pelvis which is closely associated with the renal medulla. The renal pelvis of the camel has a main crescentic cavity following the long axis and curvature of the kidney. A thick extensive renal crest projects into the cavity of the pelvis. The thick renal crest contains large numbers of long loops of Henle and vasa recta which are important for urine concentration. The renal crest is formed by convergence of the medullary pyramids before it projects into the cavity of the renal pelvis. The crescentic main cavity of the pelvis forms 20–24 three dimensional radiating collateral recesses which contain the medullary pyramids. This close association of the renal pelvis and medulla provide a large surface area for the recycling of urea and hence urine concentration. This large pelvic-medullary interface is lined by simple low cuboidal epithelium which enhances the recycling of urea and water from the pelvic urine into the medulla and directly contributes to urine concentration. The rest of the wall of the renal pelvis and its recesses facing away from the renal crest and medullary pyramids is lined by impermeable transitional epithelium. Another feature is the intense activity of alkaline phosphatase demonstrated in the proximal convoluted tubules which indicates increased membrane transport. It is concluded that the kidney in dromedaries has the anatomical and histochemical requisites for the production of concentrated urine. These requisites enable the kidney to adequately contribute to the ability of the camel to conserve water and withstand the aridity of its habitat.

Abstract Objective The present study analyzed whether renally eliminated antibiotics achieve sufficient urinary concentrations based on their pharmacokinetic/pharmacodynamic principles to effectively eradicate organisms deemed resistant by automated susceptibility testing. Results Lower median minimum inhibitory concentrations against enterobacteriaceae were noted for ceftriaxone, cefepime, and doripenem when comparing Etest® to Vitek®. All Pseudomonas aeruginosa isolates were susceptible to cefepime, ciprofloxacin, and doripenem with both susceptibility methods, but higher median minimum inhibitory concentrations were observed with Etest®. Urine concentrations/time profiles were calculated for standard doses of ceftriaxone, cefepime, doripenem, and ciprofloxacin. The data presented in the current study suggests high urine concentrations of antibiotics may effectively eradicate bacteria which were determined to be resistant per in vitro susceptibility testing.

Miriam Zacchia, Giovanna Capolongo, Luca Rinaldi, Giovambattista Capasso Division of Nephrology, Department of Cardio-Thoracic and Respiratory Sciences, Università della Campania “Luigi Vanvitelli”, Naples, Italy Abstract: The thick ascending limb (TAL) of Henle’s loop is a crucial segment for many tasks of the nephron. Indeed, the TAL is not only a mainstay for reabsorption of sodium (Na+), potassium (K+), and divalent cations such as calcium (Ca2+) and magnesium (Mg2+) from the luminal fluid, but also has an important role in urine concentration, overall acid–base homeostasis, and ammonia cycle. Transcellular Na+ transport along the TAL is a prerequisite for Na+, K+, Ca2+, Mg2+ homeostasis, and water reabsorption, the latter through its contribution in the generation of the cortico-medullar osmotic gradient. The role of this nephron site in acid–base balance, via bicarbonate reabsorption and acid secretion, is sometimes misunderstood by clinicians. This review describes in detail these functions, reporting in addition to the well-known molecular mechanisms, some novel findings from the current literature; moreover, the pathophysiology and the clinical relevance of primary or acquired conditions caused by TAL dysfunction are discussed. Knowing the physiology of the TAL is fundamental for clinicians, for a better understanding and management of rare and common conditions, such as tubulopathies, hypertension, and loop diuretics abuse. Keywords: TAL, sodium handling, potassium handling, acid–base homeostasis, urine concentration

Carbonic anhydrase II (CAII) is expressed along the nephron where it interacts with a number of transport proteins augmenting their activity. Aquaporin-1 (AQP1) interacts with CAII to increase water flux through the water channel. Both CAII and aquaporin-1 are expressed in the thin descending limb (TDL); however, the physiological role of a CAII-AQP1 interaction in this nephron segment is not known. To determine if CAII was required for urinary concentration, we studied water handling in CAII-deficient mice. CAII-deficient mice demonstrate polyuria and polydipsia as well as an alkaline urine and bicarbonaturia, consistent with a type III renal tubular acidosis. Natriuresis and hypercalciuria cause polyuria, however, CAII-deficient mice did not have increased urinary sodium nor calcium excretion. Further examination revealed dilute urine in the CAII-deficient mice. Urinary concentration remained reduced in CAII-deficient mice relative to wild-type animals even after water deprivation. The renal expression and localization by light microscopy of NKCC2 and aquaporin-2 was not altered. However, CAII-deficient mice had increased renal AQP1 expression. CAII associates with and increases water flux through aquaporin-1. Water flux through aquaporin-1 in the TDL of the loop of Henle is essential to the concentration of urine, as this is required to generate a concentrated medullary interstitium. We therefore measured cortical and medullary interstitial concentration in wild-type and CAII-deficient mice. Mice lacking CAII had equivalent cortical interstitial osmolarity to wild-type mice: however, they had reduced medullary interstitial osmolarity. We propose therefore that reduced water flux through aquaporin-1 in the TDL in the absence of CAII prevents the generation of a maximally concentrated medullary interstitium. This, in turn, limits urinary concentration in CAII deficient mice.

Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and plays an important role in the maintenance of body water homeostasis. However, whether and how glucocorticoid regulates AQP2 remains unclear. In this study, we examined the direct effect of dexamethasone on AQP2 protein expression and activity. Dexamethasone increased AQP2 protein abundance in rat inner medullary collecting duct suspensions. This was confirmed in HEK293 cells transfected with AQP2 cDNA. Cell surface protein biotinylation showed an increase of dexamethasone-induced cell membrane AQP2 expression and this effect was blocked by glucocorticoid receptor antagonist RU486. Functionally, dexamethasone treatment of oocytes injected with an AQP2 cRNA increased water transport activity as judged by cell rupture time in a hypo-osmotic solution (66 ± 13s in dexamethasone vs 101 ± 11s in control, n=15). We further found that dexamethasone treatment reduced AQP2 degradation, which could result in an increase of AQP2 protein. Interestingly, dexamethasone promoted cell membrane AQP2 moving to less buoyant lipid raft submicrodomains. Taken together, our data demonstrate that dexamethasone promotes AQP2 protein expression and increases water permeability mainly via inhibition of AQP2 protein degradation. The increase in AQP2 activity promotes water reabsorption, which may contribute to glucocorticoid-induced water retention and hypertension.

Uncontrolled diabetes mellitus results in osmotic diuresis. Diabetic patients have lowered nitric oxide (NO) which may exacerbate polyuria. We examined how lack of NO affects the transporters involved in urine concentration in diabetic animals. Diabetes was induced in rats by streptozotocin. Control and diabetic rats were given L-NAME for 3 weeks. Urine osmolality, urine output, and expression of urea and water transporters and the Na-K-2Cl cotransporter were examined. Predictably, diabetic rats presented with polyuria (increased urine volume and decreased urine osmolality). Although metabolic parameters of control rats were unaffected by L-NAME, treated diabetic rats produced 30% less urine and osmolality was restored. UT-A1 and UT-A3 were significantly increased in diabetic-rat inner medulla. While L-NAME treatment alone did not alter UT-A1 or UT-A3 abundance, absence of NO prevented the upregulation of both transporters in diabetic rats. Similarly, AQP2 and NKCC2 abundance was increased in diabetic animals however, expression of these transporters were unchanged by L-NAME treatment of diabetes. Increased expression of the concentrating transporters observed in diabetic rats provides a compensatory mechanism to decrease solute loss despite persistent glycosuria. Our studies found that although diabetic-induced glycosylation remained increased, total protein expression was decreased to control levels in diabetic rats treated with L-NAME. While the role of NO in urine concentration remains unclear, lowered NO associated with diabetes may be deleterious to the transporters’ response to the subsequent osmotic diuresis.