Search

Final urine volume and concentration are defined by water reabsorption through the water channel proteins aquaporin (AQP)-2, -3 and -4 in the collecting duct. However, the transcriptional regulation of these AQPs is not well understood. The Hippo/Yes-associated protein 1 (YAP) pathway plays an important role in organ size control and tissue homeostasis. When the Hippo pathway including the Mst1/Mst2 kinases is inhibited, YAP is activated and functions as a transcription co-activator. Our previous work revealed a pathological role of tubular YAP activation in chronic kidney disease, but the physiological role of YAP in the kidney remains to be established. Here, we found that tubule-specific Yap knockout mice showed increased urine output and decreased urinary osmolality. Decreases in Aqp2, -3 and -4 mRNA and protein abundance in the kidney were evident in Yap knockout mice. Analysis of Mst1/Mst2 double knockout and Mst1/Mst2/Yap triple knockout mice showed that expression of Aqp2 and Aqp4 but not Aqp3 was dependent on YAP. Furthermore, YAP was recruited to the promoters of the Aqp2 and Aqp4 genes and stimulated their transcription. Interestingly, YAP was found to interact with transcription factors GATA2, GATA3 and NFATc1. These three factors promoted Aqp2 transcription in a YAP dependent manner in collecting duct cells. These three factors also promoted Aqp4 transcription whereas only GATA2 and GATA3 enhanced Aqp3 transcription. Thus, our results suggest that YAP promotes Aqp2 and Aqp4 transcription, interacts with GATA2, GATA3 and NFATc1 to control Aqp2 expression, while Aqp-2, -3 and -4 exploit overlapping mechanisms for their baseline transcriptional regulation., (Copyright © 2022 International Society of Nephrology. All rights reserved.)

Hypertonicity in the renal medulla stimulates local cyclooxygenase 2 expression, leading to abundant PGE2 production. Here we found that mRNA expression by the PGE2-activated G-protein-coupled receptors, EP3 and EP4 in the renal medulla was decreased by furosemide treatment, a procedure that reduces medullary hypertonicity. When HepG2 cells were cultured in hypertonic conditions by addition of salt or sorbitol, EP3 expression was induced. A specific EP3 agonist inhibited cAMP production, indicating receptor functionality, and this led to a substantial increase in cell survival in hypertonic media. Survival was independent of the SLC5A3 inositol transporter and aldose reductase expression, suggesting that EP3 promoted cell survival under hypertonic conditions independent of cellular organic osmolyte accumulation. Reduced cAMP production did not contribute to increased survival. EP4 expression was stimulated by hypertonicity in MDCK and HepG2 cells, which was associated with increased cAMP production in response to an EP4 agonist. Our study shows that local hypertonicity promotes PGE2 signaling in the renal medulla by stimulating cognate receptor and cyclooxygenase 2 expression that likely regulates local hemodynamics and tubular transport.Kidney International (2009) 75, 278–284. doi:10.1038/ki.2008.498 [ABSTRACT FROM AUTHOR]

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, −20, and −80°C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at −20°C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at −80°C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at −20 or −80°C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at −80°C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.Kidney International (2006) 69, 1471–1476. doi:10.1038/sj.ki.5000273; published online 22 February 2006 [ABSTRACT FROM AUTHOR]

Vasopressin-independent renal urinary concentration: Increased rBSC1 and enhanced countercurrent multiplication. Background. A close association between the expression of the sodium transporter, rat bumetanide sensitive cotransporter (rBSC), in thick ascending limb of Henle and urinary concentration has been reported. However, direct evidence for this association and the mechanism of rBSC1 expression are still to be elucidated. Methods. Brattleboro (BB) rats weighing approximately 200 g were dehydrated by water restriction for 4 hours, which induced around a 5% body weight reduction. Although plasma arginine vasopressin (AVP) was undetectable even after the water restriction, BB rats concentrated urine from 182 ± 23 (mean ± SD) at baseline to 404 ± 65 mOsm/kg · H2 O. Results. Urinary volume was reduced from 5.8 ± 1.8 to 1.4 ± 0.6 mL/h. This treatment significantly increased sodium and urea accumulation in the renal medulla and reduced urinary sodium excretion. rBSC1 signals for both mRNA and protein were increased in dehydrated rats, although aquaporin type 2 (AQP2) expression was not enhanced in dehydrated BB rats. Subcutaneous infusion of desmopressin acetate (DDAVP) intensified rBSC1 signals of BB rats more than those in dehydrated condition. Conclusion. Dehydration increased rBSC1 expression and enhanced countercurrent multiplication even in AVP deficiency. These results supply strong evidence for the association between rBSC1 expression and urinary concentration, and indicate the presence of an AVP-independent mechanism for urine concentration. [ABSTRACT FROM AUTHOR]

To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of β1- and β2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of β3-adrenergic receptor (β3-AR) in mouse kidney. The β3-AR is expressed in most of the nephron segments that also express the type 2 vasopressin receptor (AVPR2), including the thick ascending limb and the cortical and outer medullary collecting duct. Ex vivo experiments in mouse kidney tubules showed that β3-AR stimulation with the selective agonist BRL37344 increased intracellular cAMP levels and promoted 2 key processes in the urine concentrating mechanism. These are accumulation of the water channel aquaporin 2 at the apical plasma membrane in the collecting duct and activation of the Na-K-2Cl symporter in the thick ascending limb. Both effects were prevented by the β3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of β3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of β3-AR significantly reduced urine excretion of water and the same electrolytes. Moreover, BRL37344 promoted a potent antidiuretic effect in AVPR2-null mice. Thus, our findings are of potential physiologic importance as they uncover the antidiuretic effect of β3-AR stimulation in the kidney. Hence, β3-AR agonism might be useful to bypass AVPR2-inactivating mutations., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)