The C-terminal tail of polycystin-1 suppresses cystic disease in a mitochondrial enzyme-dependent fashion.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent potentially lethal monogenic disorder. Mutations in the PKD1 gene, which encodes polycystin-1 (PC1), account for approximately 78% of cases. PC1 is a large 462-kDa protein that undergoes cleavage in its N and C-terminal domains. C-terminal cleavage produces fragments that translocate to mitochondria. We show that transgenic expression of a protein corresponding to the final 200 amino acid (aa) residues of PC1 in two Pkd1-KO orthologous murine models of ADPKD suppresses cystic phenotype and preserves renal function. This suppression depends upon an interaction between the C-terminal tail of PC1 and the mitochondrial enzyme Nicotinamide Nucleotide Transhydrogenase (NNT). This interaction modulates tubular/cyst cell proliferation, the metabolic profile, mitochondrial function, and the redox state. Together, these results suggest that a short fragment of PC1 is sufficient to suppress cystic phenotype and open the door to the exploration of gene therapy strategies for ADPKD.

ß3 adrenergic receptor as potential therapeutic target in ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) disrupts renal parenchyma through progressive expansion of fluid-filled cysts. The only approved pharmacotherapy for ADKPD involves the blockade of the vasopressin type 2 receptor (V2R). V2R is a GPCR expressed by a subset of renal tubular cells and whose activation stimulates cyclic AMP (cAMP) accumulation, which is a major driver of cyst growth. The ß3-adrenergic receptor (ß3-AR) is a GPCR expressed in most segments of the murine nephron, where it modulates cAMP production. Since sympathetic nerve activity, which leads to activation of the ß3-AR, is elevated in patients affected by ADPKD, we hypothesize that ß3-AR might constitute a novel therapeutic target. We find that administration of the selective ß3-AR antagonist SR59230A to an ADPKD mouse model (Pkd1fl/fl ;Pax8rtTA ;TetO-Cre) decreases cAMP levels, producing a significant reduction in kidney/body weight ratio and a partial improvement in kidney function. Furthermore, cystic mice show significantly higher ß3-AR levels than healthy controls, suggesting a correlation between receptor expression and disease development. Finally, ß3-AR is expressed in human renal tissue and localizes to cyst-lining epithelial cells in patients. Thus, ß3-AR is a potentially interesting target for the development of new treatments for ADPKD.

Everything You Always Wanted to Know about ß3-AR * (* But Were Afraid to Ask).

The beta-3 adrenergic receptor (ß3-AR) is by far the least studied isotype of the beta-adrenergic sub-family. Despite its study being long hampered by the lack of suitable animal and cellular models and inter-species differences, a substantial body of literature on the subject has built up in the last three decades and the physiology of ß3-AR is unraveling quickly. As will become evident in this work, ß3-AR is emerging as an appealing target for novel pharmacological approaches in several clinical areas involving metabolic, cardiovascular, urinary, and ocular disease. In this review, we will discuss the most recent advances regarding ß3-AR signaling and function and summarize how these findings translate, or may do so, into current clinical practice highlighting ß3-AR's great potential as a novel therapeutic target in a wide range of human conditions.

Human ß3-Adrenoreceptor is Resistant to Agonist-Induced Desensitization in Renal Epithelial Cells.

BACKGROUND/AIMS: We recently showed that the ß3-adrenoreceptor (ß3AR) is expressed in mouse kidney collecting ducts (CD) cells along with the type-2 vasopressin receptor (AVPR2). Interestingly, a single injection of a ß3AR selective agonist promotes a potent antidiuretic effect in mice. Before considering the feasibility of chronic ß3AR agonism to induce antidiuresis in vivo, we aimed to evaluate in vitro the signaling and desensitization profiles of human ß3AR. METHODS: Human ß3AR desensitization was compared with that of human AVPR2 in cultured renal cells. Video imaging and FRET experiments were performed to dissect ß3AR signaling under acute and chronic stimulation. Plasma membrane localization of ß3AR, AVPR2 and AQP2 after agonist stimulation was studied by confocal microscopy. Receptors degradation was evaluated by Western blotting. RESULTS: In renal cells acute stimulation with the selective ß3AR agonist mirabegron, induced a dose-dependent increase in cAMP. Interestingly, chronic exposure to mirabegron promoted a significant increase of intracellular cAMP up to 12 hours. In addition, a slow and slight agonist-induced internalization and a delayed downregulation of ß3AR was observed under chronic stimulation. Furthermore, chronic exposure to mirabegron promoted apical expression of AQP2 also up to 12 hours. Conversely, long-term stimulation of AVPR2 with dDAVP showed short-lasting receptor signaling, rapid internalization and downregulation and apical AQP2 expression for no longer than 3 h. CONCLUSIONS: Overall, we conclude that ß3AR is less prone than AVPR2 to agonist-induced desensitization in renal collecting duct epithelial cells, showing sustained cAMP production, preserved membrane localization and delayed degradation after 12 hours agonist exposure. These results may be important for the potential use of chronic pharmacological stimulation of ß3AR to promote antidiuresis overcoming in vivo renal concentrating defects caused by inactivating mutations of the AVPR2.

The expression of Lamin A mutant R321X leads to endoplasmic reticulum stress with aberrant Ca2+ handling.

Mutations in the Lamin A/C gene (LMNA), which encodes A-type nuclear Lamins, represent the most frequent genetic cause of dilated cardiomyopathy (DCM). This study is focused on a LMNA nonsense mutation (R321X) identified in several members of an Italian family that produces a truncated protein isoform, which co-segregates with a severe form of cardiomyopathy with poor prognosis. However, no molecular mechanisms other than nonsense mediated decay of the messenger and possible haploinsufficiency were proposed to explain DCM. Aim of this study was to gain more insights into the disease-causing mechanisms induced by the expression of R321X at cellular level. We detected the expression of R321X by Western blotting from whole lysate of a mutation carrier heart biopsy. When expressed in HEK293 cells, GFP- (or mCherry)-tagged R321X mislocalized in the endoplasmic reticulum (ER) inducing the PERK-CHOP axis of the ER stress response. Of note, confocal microscopy showed phosphorylation of PERK in sections of the mutation carrier heart biopsy. ER mislocalization of mCherry-R321X also induced impaired ER Ca2+ handling, reduced capacitative Ca2+ entry at the plasma membrane and abnormal nuclear Ca2+ dynamics. In addition, expression of R321X by itself increased the apoptosis rate. In conclusion, R321X is the first LMNA mutant identified to date, which mislocalizes into the ER affecting cellular homeostasis mechanisms not strictly related to nuclear functions.

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.

ß3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function.

To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of ß1- and ß2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of ß3-adrenergic receptor (ß3-AR) in mouse kidney. The ß3-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 ß3-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 ß3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of ß3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of ß3-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 ß3-AR stimulation in the kidney. Hence, ß3-AR agonism might be useful to bypass AVPR2-inactivating mutations.