Activation of the Calcium-Sensing Receptor Corrects the Impaired Mitochondrial Energy Status Observed in Renal Polycystin-1 Knockdown Cells Modeling Autosomal Dominant Polycystic Kidney Disease

Abstract

Autosomal Dominant Polycistic kidney Disease (ADPKD) is a renal channelopathy due to loss-of-function mutations in the PKD1 or PKD2 genes, encoding polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 is a large protein found predominantly on the plasma membrane where interacts with different proteins, including PC2. PC2 is a smaller integral membrane protein also expressed in intracellular organelles, acting as a non-selective cation channel permeable to calcium. Both PC1 and PC2 are also localized to the primary cilium of renal epithelial cells serving as mechanosensor that controls calcium influx through the plasma membrane and regulates intracellular calcium release from the endoplasmic reticulum. The mechanisms by which PC1/2 dysfunction leads to ADPKD needs still to be clarified. We have recently reported that selective Calcium-Sensing Receptor (CaSR) activation in human conditionally immortalized Proximal Tubular Epithelial cells deficient for PC1 (ciPTEC-PC1KD), deriving from urine sediments reduces intracellular cAMP and mTOR activity, and increases intracellular calcium reversing the principal ADPKD dysregulations. Reduced cellular free calcium found in ADPKD can, on the other hand, affect mitochondrial function and ATP production and, interestingly, a relationship between mitochondria and renal polycystic diseases have been suggested. By using ciPTEC-PC1KD as experimental tool modeling of ADPKD, we show here that, compared with wild type cells, ciPTEC-PC1KD have significantly lower mitochondrial calcium levels associated with a severe deficit in mitochondrial ATP production, secondary to a multilevel impairment of oxidative phosphorylation. Notably, selective CaSR activation with the calcimimetic NPS-R568 increases mitochondrial calcium content close to the levels found in resting wild type cells, and fully recovers the cell energy deficit associated to the PC1 channel disruption. Treatment of ciPTEC-PC1KD with 2-APB, an IP3R inhibitor, prevented the rescue of bioenergetics deficit induced by CaSR activation supporting a critical role of IP3Rs in driving ER-to-mitochondria Ca2+ shuttle. Together these data indicate that, besides reversing the principal dysregulations considered the most proximal events in ADPKD pathogenesis, selective CaSR activation in PKD1 deficient cells restores altered mitochondrial function that, in ADPKD, is known to facilitate cyst formation. These findings identify CaSR as a potential therapeutic target.

The Impact of Acetyl-CoA and Aspartate Shortages on the N-Acetylaspartate Level in Different Models of Cholinergic Neurons

“…Moreover, we investigated the impact of 2-APB (an antagonist of the IP3 receptor, intracellular Ca 2+ release from ER to the mitochondria) on acetyl-CoA and NAA levels ( Figure 4 A and Figure 6 E). One of the commonly observed side effects of 2-APB is the significant depletion of ATP level, also noted in our study ( Figure 6 C) [ 15 ]. Our further studies revealed that such a shortage is associated with a drop in acetyl-CoA availability ( Figure 6 E).…”

“…To establish if acetyl-CoA shortages have the same impact on both NAA and acetylcholine productions, SN56 cells were exposed to mecamylamine (Mec, an antagonist of the nicotinic acetylcholine receptor), nifedipine (NF, an antagonist of the L-type voltage calcium channel) or 2-aminoethoxydiphenyl borate (2-APB, an antagonist of the IP3 receptor). Mec was used to reduce acetylcholine release [ 14 ], nifedipine to reduce free radical production [ 12 ] and 2-APB to evoke ATP shortages [ 15 ].…”

“…The 2-APB antagonist has been reported as a factor triggering the reduction of ATP levels, although the exact mechanism of the 2-APB influence has not been established yet [ 15 ]. We noted that 2-APB treatment can disturb acetyl-CoA production by the inhibition of pyruvate dehydrogenase complex activity ( Figure 6 E and Figure 7 A).…”

“…The acute approach showed energy disorders evoked by Zn 2+ uptake taking place under depolarizing conditions [ 12 ]. To distinguish different Zn 2+ -related biochemical changes, we confirmed them using different inhibitors: mecamylamine to suppress cholinergic neurotransmission [ 14 ], nifedipine to control calcium influx and free radical production [ 12 ] as well as 2-aminoethoxydiphenyl borate to affect ATP level [ 15 ]. The chronic Zn 2+ treatment showed the long-term impact on the NAT8L activity [ 4 ].…”

Vasopressin–aquaporin-2 pathway: recent advances in understanding water balance disorders

“…The disruption in calcium and cAMP signaling cascades activates pathways causing cell proliferation, increased fluid secretion, and interstitial inflammation 116, 117 . Our recent in vitro study demonstrated that in human conditionally immortalized proximal tubular epithelial cells silenced for PKD1 (ciPTEC-PC1KD) or generated from a patient with ADPKD1 (ciPTEC-PC1Pt), selective activation of the calcium-sensing receptor increases cytosolic calcium, reduces intracellular cAMP and mTOR activity 118 , and rescues defective ATP mitochondrial production 119 , reversing the principal ADPKD dysregulations.…”