Ceramide-1-Phosphate as a Potential Regulator of the Second Sodium Pump from Kidney Proximal Tubules by Triggering Distinct Protein Kinase Pathways in a Hierarchic Way

Abstract

Kidney proximal tubules are a key segment in the reabsorption of solutes and water from the glomerular ultrafiltrate, an essential process for maintaining homeostasis in body fluid compartments. The abundant content of Na+ in the extracellular fluid determines its importance in the regulation of extracellular fluid volume, which is particularly important for different physiological processes including blood pressure control. Basolateral membranes of proximal tubule cells have the classic Na+ + K+-ATPase and the ouabain-insensitive, K+-insensitive, and furosemide-sensitive Na+-ATPase, which participate in the active Na+ reabsorption. Here, we show that nanomolar concentrations of ceramide-1 phosphate (C1P), a bioactive sphingolipid derived in biological membranes from different metabolic pathways, promotes a strong inhibitory effect on the Na+-ATPase activity (C1P50 ≈ 10 nM), leading to a 72% inhibition of the second sodium pump in the basolateral membranes. Ceramide-1-phosphate directly modulates protein kinase A and protein kinase C, which are known to be involved in the modulation of ion transporters including the renal Na+-ATPase. Conversely, we did not observe any effect on the Na+ + K+-ATPase even at a broad C1P concentration range. The significant effect of ceramide-1-phosphate revealed a new potent physiological and pathophysiological modulator for the Na+-ATPase, participating in the regulatory network involving glycero- and sphingolipids present in the basolateral membranes of kidney tubule cells.

Opposite Effects of Diacylglycerol and Phosphatidic Acid in the Modulation of the Plasma Membrane Ca2+-ATPase from Kidney Proximal Tubules: A Regulatory Role for Diacylglycerol Kinase in Calcium Homeostasis?

“…The results presented here support the hypothesis that DG modulates BLM PMCA activity via specific post-translational mechanisms, potentially through the activation of DG-sensitive kinases, such as PKC classic and novel isoforms, already shown in the BLM from pig proximal tubules [57], which are abundantly expressed in kidney cells and capable of phosphorylating PMCA [8,10,29,31,49,50]. On the other hand, the conversion of DG to phosphatidic acid (PA) by DG kinase (DGK) could alter the local lipid environment and indirectly affect PMCA conformation, anchoring, and thus, activity.…”

“…Among the different renal functions, regulating ion and water reabsorption is particularly important. These processes are closely linked to the polarization of tubular cells and the differential distribution of ion transporters in the apical and basolateral membranes [31,40]. Calcium homeostasis is critical for both the entire organism and kidney epithelial cells because this ion modulates diverse cellular events.…”

“…Intriguingly, this DGK appears functionally coupled to membrane-bound PLC, enabling rapid PA production following DG generation. This coupling might spatially constrain DG availability and control PKC activation, particularly in caveolin-rich lipid microdomains where PMCA is localized [23,30,31,61]. The downstream effects of DG and PA interconversion extend to the regulation of membrane calcium transporters.…”

Opposite Effects of Diacylglycerol and Phosphatidic Acid in the Modulation of the Plasma Membrane Ca2+-ATPase from Kidney Proximal Tubules: A Regulatory Role for Diacylglycerol Kinase in Calcium Homeostasis?

“…The results presented here support the hypothesis that DG modulates BLM PMCA activity via specific post-translational mechanisms, potentially through the activation of DG-sensitive kinases, such as PKC classic and novel isoforms, already shown in the BLM from pig proximal tubules [57], which are abundantly expressed in kidney cells and capable of phosphorylating PMCA [8,10,29,31,49,50]. On the other hand, the conversion of DG to phosphatidic acid (PA) by DG kinase (DGK) could alter the local lipid environment and indirectly affect PMCA conformation, anchoring, and thus, activity.…”

“…Among the different renal functions, regulating ion and water reabsorption is particularly important. These processes are closely linked to the polarization of tubular cells and the differential distribution of ion transporters in the apical and basolateral membranes [31,40]. Calcium homeostasis is critical for both the entire organism and kidney epithelial cells because this ion modulates diverse cellular events.…”

“…Intriguingly, this DGK appears functionally coupled to membrane-bound PLC, enabling rapid PA production following DG generation. This coupling might spatially constrain DG availability and control PKC activation, particularly in caveolin-rich lipid microdomains where PMCA is localized [23,30,31,61]. The downstream effects of DG and PA interconversion extend to the regulation of membrane calcium transporters.…”

Characterization of Ceramide Kinase from Basolateral Membranes of Kidney Proximal Tubules: Kinetics, Physicochemical Requirements, and Physiological Relevance

“…Having demonstrated the presence of CerK activity in the BLM-enriched fractions, we proceeded to investigate the biochemical requirements necessary for maximum activity of this enzyme, beginning, for the very first time, its characterization in a well-defined compartment of renal proximal tubule cells. Initially, a curve of increasing protein concentrations ranging from 0.05 to 0.3 mg × mL −1 was performed, in an assay carried out for 20 min at 37 °C and pH 7.2, the maximum reaction time, temperature, and pH used in the experiments to measure ATPase activity and protein kinase activation in our previous works [ 3 , 4 , 5 ]. Figure 3 shows that the phosphorylation of endogenous Cer, by BLM-associated CerK, remained linear compared to the increase in protein concentration, and a concentration of 0.2 mg × mL −1 of protein was chosen for the further BLM- associated CerK characterization assays, since this was the optimal concentration used in the description of the method used for lipid phosphorylation [ 40 ], in addition to this concentration had been the protein concentration used in previous determinations of ATPase activities.…”

“…SM degradation inhibits the Na + /glucose and Na + /Pi co-transporters present in the apical membrane of renal epithelial cells, regardless of changes in membrane fluidity [ 49 ]. Our group has previously investigated the involvement of Cer and its phosphorylated derivative, C1P, using the same biological model presented here [ 3 , 4 , 5 ].…”

Opposite Effects of Diacylglycerol and Phosphatidic Acid in the Modulation of the Plasma Membrane Ca2+-ATPase from Kidney Proximal Tubules: A Regulatory Role for Diacylglycerol Kinase in Calcium Homeostasis?

“…The results presented here support the hypothesis that DG modulates BLM PMCA activity via specific post-translational mechanisms, potentially through the activation of DG-sensitive kinases, such as PKC classic and novel isoforms, already shown in the BLM from pig proximal tubules [57], which are abundantly expressed in kidney cells and capable of phosphorylating PMCA [8,10,29,31,49,50]. On the other hand, the conversion of DG to phosphatidic acid (PA) by DG kinase (DGK) could alter the local lipid environment and indirectly affect PMCA conformation, anchoring, and thus, activity.…”

“…Among the different renal functions, regulating ion and water reabsorption is particularly important. These processes are closely linked to the polarization of tubular cells and the differential distribution of ion transporters in the apical and basolateral membranes [31,40]. Calcium homeostasis is critical for both the entire organism and kidney epithelial cells because this ion modulates diverse cellular events.…”

“…Intriguingly, this DGK appears functionally coupled to membrane-bound PLC, enabling rapid PA production following DG generation. This coupling might spatially constrain DG availability and control PKC activation, particularly in caveolin-rich lipid microdomains where PMCA is localized [23,30,31,61]. The downstream effects of DG and PA interconversion extend to the regulation of membrane calcium transporters.…”

Characterization of Ceramide Kinase from Basolateral Membranes of Kidney Proximal Tubules: Kinetics, Physicochemical Requirements, and Physiological Relevance

“…Having demonstrated the presence of CerK activity in the BLM-enriched fractions, we proceeded to investigate the biochemical requirements necessary for maximum activity of this enzyme, beginning, for the very first time, its characterization in a well-defined compartment of renal proximal tubule cells. Initially, a curve of increasing protein concentrations ranging from 0.05 to 0.3 mg × mL −1 was performed, in an assay carried out for 20 min at 37 °C and pH 7.2, the maximum reaction time, temperature, and pH used in the experiments to measure ATPase activity and protein kinase activation in our previous works [ 3 , 4 , 5 ]. Figure 3 shows that the phosphorylation of endogenous Cer, by BLM-associated CerK, remained linear compared to the increase in protein concentration, and a concentration of 0.2 mg × mL −1 of protein was chosen for the further BLM- associated CerK characterization assays, since this was the optimal concentration used in the description of the method used for lipid phosphorylation [ 40 ], in addition to this concentration had been the protein concentration used in previous determinations of ATPase activities.…”

“…SM degradation inhibits the Na + /glucose and Na + /Pi co-transporters present in the apical membrane of renal epithelial cells, regardless of changes in membrane fluidity [ 49 ]. Our group has previously investigated the involvement of Cer and its phosphorylated derivative, C1P, using the same biological model presented here [ 3 , 4 , 5 ].…”

Opposite Effects of Diacylglycerol and Phosphatidic Acid in the Modulation of the Plasma Membrane Ca2+-ATPase from Kidney Proximal Tubules: A Regulatory Role for Diacylglycerol Kinase in Calcium Homeostasis?

“…The results presented here support the hypothesis that DG modulates BLM PMCA activity via specific post-translational mechanisms, potentially through the activation of DG-sensitive kinases, such as PKC classic and novel isoforms, already shown in the BLM from pig proximal tubules [57], which are abundantly expressed in kidney cells and capable of phosphorylating PMCA [8,10,29,31,49,50]. On the other hand, the conversion of DG to phosphatidic acid (PA) by DG kinase (DGK) could alter the local lipid environment and indirectly affect PMCA conformation, anchoring, and thus, activity.…”

“…Among the different renal functions, regulating ion and water reabsorption is particularly important. These processes are closely linked to the polarization of tubular cells and the differential distribution of ion transporters in the apical and basolateral membranes [31,40]. Calcium homeostasis is critical for both the entire organism and kidney epithelial cells because this ion modulates diverse cellular events.…”

“…Intriguingly, this DGK appears functionally coupled to membrane-bound PLC, enabling rapid PA production following DG generation. This coupling might spatially constrain DG availability and control PKC activation, particularly in caveolin-rich lipid microdomains where PMCA is localized [23,30,31,61]. The downstream effects of DG and PA interconversion extend to the regulation of membrane calcium transporters.…”

Characterization of Ceramide Kinase from Basolateral Membranes of Kidney Proximal Tubules: Kinetics, Physicochemical Requirements, and Physiological Relevance

“…Having demonstrated the presence of CerK activity in the BLM-enriched fractions, we proceeded to investigate the biochemical requirements necessary for maximum activity of this enzyme, beginning, for the very first time, its characterization in a well-defined compartment of renal proximal tubule cells. Initially, a curve of increasing protein concentrations ranging from 0.05 to 0.3 mg × mL −1 was performed, in an assay carried out for 20 min at 37 °C and pH 7.2, the maximum reaction time, temperature, and pH used in the experiments to measure ATPase activity and protein kinase activation in our previous works [ 3 , 4 , 5 ]. Figure 3 shows that the phosphorylation of endogenous Cer, by BLM-associated CerK, remained linear compared to the increase in protein concentration, and a concentration of 0.2 mg × mL −1 of protein was chosen for the further BLM- associated CerK characterization assays, since this was the optimal concentration used in the description of the method used for lipid phosphorylation [ 40 ], in addition to this concentration had been the protein concentration used in previous determinations of ATPase activities.…”

“…SM degradation inhibits the Na + /glucose and Na + /Pi co-transporters present in the apical membrane of renal epithelial cells, regardless of changes in membrane fluidity [ 49 ]. Our group has previously investigated the involvement of Cer and its phosphorylated derivative, C1P, using the same biological model presented here [ 3 , 4 , 5 ].…”