Abstract
Mechanisms underlying the frequent association between salt-sensitive hypertension and type 2 diabetes remain obscure. We previously found that protein kinase C (PKC) activation phosphorylates Kelch-like 3 (KLHL3), an E3 ubiquitin ligase component, at serine 433. We investigated whether impaired KLHL3 activity results in increased renal salt reabsorption via NaCl cotransporter (NCC). We used the db/db diabetes mouse model to explore KLHL3's role in renal salt handling in type 2 diabetes and evaluated mechanisms of KLHL3 dysregulation in cultured cells. We observed PKC activity in the db/db mouse kidney and phosphorylation of serine 433 in KLHL3 (KLHL3S433-P). This modification prevents binding of with-no-lysine (WNK) kinases; however, total KLHL3 levels were decreased, indicating severely impaired KLHL3 activity. This resulted in WNK accumulation, activating NCC in distal convoluted tubules. Ipragliflozin, a sodium glucose cotransporter 2 (SGLT2) inhibitor, lowered PKC activity in distal convoluted tubule cells and reduced KLHL3S433-P and NCC levels, whereas the thiazolidinedione pioglitazone did not, although the two agents similarly reduced in blood glucose levels. We found that, in human embryonic kidney cells expressing KLHL3 and distal convoluted tubule cells, cellular glucose accumulation increased KLHL3S433-P levels through PKC. Finally, the effect of PKC inhibition in the kidney of db/db mice confirmed PKC's causal role in KLHL3S433-P and NCC induction. Dysregulation of KLHL3 is involved in the pathophysiology of type 2 diabetes. These data offer a rationale for use of thiazide in individuals with diabetes and provide insights into the mechanism for cardiorenal protective effects of SGLT2 inhibitors.
Highlights
Mechanisms underlying the frequent association between salt-sensitive hypertension and type 2 diabetes remain obscure
Ipragliflozin, a sodium glucose cotransporter 2 (SGLT2) inhibitor, lowered protein kinase C (PKC) activity in distal convoluted tubule cells and reduced KLHL3S433-P and NaCl cotransporter (NCC) levels, whereas the thiazolidinedione pioglitazone did not, the two agents reduced in blood glucose levels
In human embryonic kidney cells expressing Kelch-like 3 (KLHL3) and distal convoluted tubule cells, cellular glucose accumulation increased KLHL3S433-P levels through PKC
Summary
We used the db/db diabetes mouse model to explore KLHL39s role in renal salt handling in type 2 diabetes and evaluated mechanisms of KLHL3 dysregulation in cultured cells. For the Western blotting analysis of antiKLHL3S433-P, we used mouse monoclonal anti-KLHL3S433-P created and characterized in the laboratory as described previously (in the work by Ishizawa et al.[22]). Anti-KLHL3 antibody (Sigma-Aldrich) was characterized as previously described,[31] and it was further validated in this study. The antibody was produced using amino acids 2–51 of human KLHL3 as an immunogen, and the sequence identity in this region between KLHL2 and KLHL3 is ,50% (Supplemental Figure 2A). This antibody recognized a strong signal in the cortex but not in the medulla (where KLHL2 is predominantly present),[39] confirming that the antibody does not recognize KLHL2 (Supplemental Figure 2B). Other antibodies included anti-Flag (Sigma-Aldrich), anti-tubulin (SigmaAldrich), anti-WNK4,22 anti-WNK1 (Cell Signaling Technology), anti-phospho-SPAK/OSR1 (Millipore),[40] anti-SPAK (Abcam), anti-NCC,[31] anti-NCC phosphorylated at Thr[53] (NCCT53-P),[41] anti-phospho-PKC (bearing phosphorylation at Ser[660] in PKCbII; the antibody detects phosphorylated PKCa, -bI, -d, -«, -h, and -u; Cell Signaling Technology; we previously confirmed that PKC activation sharply increases the signal detected by this antibody),[22] anti-phospho-PKCa/b (bearing phosphorylation at Thr638/641), anti-total PKC, anti-phospho-Akt, and anti-Akt (Cell Signaling Technology)
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