Part2: 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

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4. Discussion

Fluid transport across the tubular epithelium is one of the key events directly correlated to the majority of the distinct renal functions[49]. Among the different solutes handled in the kidney, Nat is especially important. Due to its abundance in the extracellular fluid, it is imperative to understand the mechanisms by which Nat is reabsorbed or excreted in urine because alterations in the interstitial and intravascular Na+ concentrations lead to changes in volume and might cause disturbances in blood pressure[50]. The regulation of Nat levels in body liquid compartments are principally driven by primary active transporters with the classical Nat + Kt-ATPase [51] being involved in mass transport, while the"second" Nat pump or Nat-ATPase is involved in the fine-tuned regulation of Nat reabsorption [52,53]There are many reports in the literature concerning the regulation of Nat + K+-ATPase by hormones and autacoids[50,54] as well as the role of regulatory phosphorylation [50,55].In contrast, fewer groups quote the regulation ofNa+-ATPase as there is still some controversy concerning its identity, even though reports were showing a furosemide-sensitive Nat-ATPase activity from mammals[41,56,57] to protozoa [58,59] as well as the cloning of the pump from different organisms [60-63]. From the results presented here, our group showed that not only are hormones and autacoids able to trigger cell signaling pathways that modulate the Nat-ATPase, but the effect of ClP exogenously added to the BLM fractions points to either a possible interaction of the bioactive lipid with the hydrophobic domains of regulators, or the pump itself; or, there must be a kind of C1P receptor in the BLM, not identified yet [20,22,23]. Figure 6 shows a proposed scheme to summarize these possible ClP ways of action. The ability of ceramides to trigger kinases and elicit a different physiological response was reported in liver cells [24]. The authors not only showed the ability of ceramide-dependent modulation of the Nat/K+-ATPase and kinase regulation, but they also reported biphasic responses considering time-dependent stimulation of those cell lines, which illustrates that there must be complex crosstalk between ceramide-dependent signaling and other signaling systems present in the cell membranes. membranes (Figure 2). Moreover, the results presented here in combination with previous results from our group clearly show that Cer [33] and ClP modulated the BLM-associated PKA and PKC, which appeared to mediate the effects elicited by ceramides on Nat-ATPase When the BLM fractions were treated with 100 nM C1P, inhibition of the pump could not be attributed to PKA (Figure 4)since the pre-incubation with the PKAi did not affect the C1P inhibitory action. On the other hand, the significative inhibition of the Nat-ATPase activity upon ClP treatment is fully prevented by calphostin C(Figure 3). Previous reports have shown that PKA stimulates the renal Nat-ATPase activity in a pathway that links Gs-coupled receptors with the pump activity [41]. The signaling systems aborded here in our manuscript—PKA and PKC—have been well studied, so there is plenty of information in the literature showing the concentrations for positive controls as used here (cAMP and phorbol myristate acid PMA, respectively). As our focus was not a dose-response study either with classic activators or inhibitors for the different signaling systems, we only used the concentrations of activators or inhibitors already effectively used in other studies from our or other groups.

Figure6. Proposed mechanism for the ClP action on the Na+-ATPase activity from BLM. (1)C1P(red lipid) inhibits PKC through a yet unknown G-protein coupled receptor, resulting in the inhibition of the Na+-ATPase(blue protein). The same route could activate BLM PKA(red arrow)with no effect on the pump. (2)CIP could directly bind to effectors present in the BLM, such as the PKC, leading to the inhibitory action on the Na+-ATPase activity described. Our results showed that although PKA is present in the BLM and ready to modulate the pump by cAMP(green circles and green arrow), its activation by CIP did not affect the pump (red dashed arrow). (3)CIP directly binds to the Nat-ATPase or is enriched in the lipidic membrane microenvironment surrounding the Nat-ATPase, thus modulating it. From the results presented here, we were not able to detect any modulatory action of ClP on the Nat/K+-ATPase(purple protein) present in the BLM fractions.

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The detection of a hierarchy in the phosphorylation of the Nat-ATPase was suggested when we stimulated the BLM with Cer [33] and we observed that the blockage of the BLM-PKCZ, blunted the inhibitory action of Cer on the pump and allowed cAMP-activated PKA to stimulate it. Despite an increasing number of reports showing different roles for Cer activated kinases [44,64-66], this is the first report of the physiological action of C1P on BLM-resident kinases, which certainly plays a role in the regulation of Nat handling in the kidney. We cannot definitively affirm that the Nat-ATPase is directly phosphorylated by the studied kinases, as there are no available experimental tools for this, mainly because the complete structure of the second sodium pump has not been identified yet. However, it is well known that the Nat-ATPase is a P-type ATPase, and this fact allows us to postulate that it can be directly modulated by phosphorylation in putative regulatory sites [46,55]We cannot rule out that the activation of the respective kinases would lead to the phosphorylation of another protein in this regulatory network, which would be responsible for ATPase inhibition.

Among the different bioactive sphingolipids, C1P is less documented in the different physiological systems. On the other hand, sphingosine-1-phosphate, which may be the most important bioactive lipid, is very well studied in kidney physiology and pathophysiology [2,6,7,67]. As S1P and ClP members are a type of family of signaling molecules with common precursors and sharing mutual interconversion steps, it is possible to imagine that ClP would also be present along the entire nephron. The work by Sugiura et al.,2002[11], a hallmark in the field, showed that CerK is highly expressed in kidneys. Despite robust data in the literature showing the role of ClP in podocytes and glomerular disease[68,69], there has not been a work individually showing CerK expression and ClP action in any of the nephron segments. Otherwise, ClP is likely to be present in all the renal cells either by the presence of sphingomyelin, which would allow the salvage route for ClP synthesis, or by the fact that the ClP can be provided to the renal tissue by the bloodstream. These are very plausible ways to think that this signaling lipid would be playing different roles in the other nephron segments, although we did not explore it here.

It was substantially demonstrated that Cer levels were significantly elevated during renal injury due to an already reported increase in Ca2t-stimulated sphingomyelinase activity after cellular Ca2t homeostasis is disrupted [2,4,24,70]. This observation allows us to postulate that the kinase-mediated modulation of the BLM Nat-ATPase activity by Cer and C1P could be especially relevant in the injured renal tissue, which could open a new role for ceramides in the establishment and progression of nephropathies. In the presence of C1P, the BLMkinases studied were differentially modulated (Figure 5). While ClPinhibited the PKC, leading to significant inhibition of the Nat-ATPase activity, C1P-dependent activation of PKA did not affect the Nat-ATPase activity. This allows us to postulate that there is a hierarchy involving different kinases, which results in a complex switch in/off to the Nat-ATPase. This switch may involve different isoforms of PKC and PKA, or, there must be crosstalk with the potential conversion of C1P to Cer and other bioactive lipids triggering other signaling pathways harbored in the BLM, which has already been described in liver cells [24].

The balance between Cer and ClP could be a new promising target for the development of new classes of drugs with more efficient action to try to stop the progress of renal failure, or at least, raise the life quality for renal patients.

5. Conclusions

From the results presented here, a signaling cascade starting with Cer production and further phosphorylation to ClP is an efficient pathway for inhibiting Nat-ATPase in renal proximal tubules cells through BLM-associated PKA and PKC as effectors[17]. These observations reveal the importance of protein kinases in the fine control of Nat fluxes in a nephron segment where more than two-thirds of the glomerular ultrafiltrate is reabsorbed, the proximal tubule. To the best of our knowledge, the present work shows for the first time that ClP can modulate the BLM-Nat-ATPase from kidney proximal tubule cells by triggering membrane-associated kinases (PKA and PKC). Thus, the above results ad evidence to the view that Cer and ClP participate in the regulatory network of bioactive sphingolipids and glycerolipids resident in this nephron segment, which should be true for the overall nephron. This hypothesis can be supported by the view that Cer is a lipid virtually present in all the nephron segments as well as that the Cer availability would allow for the formation of C1P.