The Pharmacological Inhibition of CaMKII Regulates Sodium Chloride Cotransporter Activity in mDCT15 Cells

The thiazide-sensitive sodium chloride cotransporter (NCC) in the distal convoluted tubule is responsible for reabsorbing up to one-tenth of the total filtered load of sodium in the kidney. The actin cytoskeleton is thought to regulate various transport proteins in the kidney but the regulation of the NCC by the actin cytoskeleton is largely unknown. Here, we identify a direct interaction between the NCC and the cytoskeletal protein filamin A in mouse distal convoluted tubule (mDCT15) cells and in the native kidney. We show that the disruption of the actin cytoskeleton by two different mechanisms downregulates NCC activity. As filamin A is a substrate of the Ca2+/calmodulin-dependent protein kinase II (CaMKII), we investigate the physiological significance of CaMKII inhibition on NCC luminal membrane protein expression and NCC activity in mDCT15 cells. The pharmacological inhibition of CaMKII with the compound KN93 increases the active form of the NCC (phospho-NCC) at the luminal membrane and also increases NCC activity in mDCT15 cells. These data suggest that the interaction between the NCC and filamin A is dependent on CaMKII activity, which may serve as a feedback mechanism to maintain basal levels of NCC activity in the distal nephron.

The Hydrogen-Coupled Oligopeptide Membrane Cotransporter Pept2 is SUMOylated in Kidney Distal Convoluted Tubule Cells

Protein post-translational modification by the Small Ubiquitin-like MOdifier (SUMO) on lysine residues is a reversible process highly important for transcription and protein stability. In the kidney, SUMOylation appears to be important for the cellular response to aldosterone. Therefore, in this study, we generated a SUMOylation profile of the aldosterone-sensitive kidney distal convoluted tubule (DCT) as a basis for understanding SUMOylation events in this cell type. Using mass spectrometry-based proteomics, 1037 SUMO1 and 552 SUMO2 sites, corresponding to 546 SUMO1 and 356 SUMO2 proteins, were identified from a modified mouse kidney DCT cell line (mpkDCT). SUMOylation of the renal hydrogen-coupled oligopeptide and drug co-transporter (Pept2) at one site (K139) was found to be highly regulated by aldosterone. Using immunolabelling of mouse kidney sections Pept2 was localized to DCT cells in vivo. Aldosterone stimulation of mpkDCT cell lines expressing wild-type Pept2 or mutant K139R-Pept2, post-transcriptionally increased Pept2 expression up to four-fold. Aldosterone decreased wild-type Pept2 abundance in the apical membrane domain of mpkDCT cells, but this response was absent in K139R-Pept2 expressing cells. In summary, we have generated a SUMOylation landscape of the mouse DCT and determined that SUMOylation plays an important role in the physiological regulation of Pept2 trafficking by aldosterone.

Activation of the Thiazide-Sensitive Sodium-Chloride Cotransporter by Beta3-Adrenoreceptor in the Distal Convoluted Tubule

We previously showed that the beta-3 adrenergic receptor (BAR3) is expressed in most segments of the nephron where its agonism promotes a potent antidiuretic effect. We localized BAR3 in distal convoluted tubule (DCT) cells expressing the thiazide-sensitive sodium-chloride cotransporter (NCC). Aim of this study is to investigate the possible functional role of BAR3 on NCC modulation in DCT cells. Here, we found that, in mice, the knockout of BAR3 was paralleled by a significant attenuation of NCC phosphorylation, paralleled by reduced expression and activation of STE-20/SPS1-related proline-alanine-rich kinase (SPAK) and WNKs the main kinases involved in NCC activation. Conversely, in BAR1/2 knockout mice, we found reduced NCC abundance with no changes in the phosphorylation state of NCC. Moreover, selective BAR3 agonism promotes both SPAK and NCC activation in wild-type mouse kidney slices. In conclusion, our findings suggest a novel role for BAR3 in the regulation of NCC in DCT.

Deletion of renal Nedd4-2 abolishes the effect of high K+ intake (HK) on Kir4.1/Kir5.1 and NCC activity in the distal convoluted tubule.

High-dietary K+ (HK) intake inhibits the basolateral Kir4.1/Kir5.1 activity in the distal convoluted tubule (DCT) and HK-induced inhibition of Kir4.1/Kir5.1 is essential for HK-induced inhibition of Na-Cl cotransporter (NCC). We now examine whether Nedd4-2-deletion compromises the effect of HK on basolateral Kir4.1/Kir5.1 and NCC in the DCT. Single- channel-recording and whole-cell-recording showed that neither HK decreased nor low-dietary-K+ (LK) increased the basolateral Kir4.1/Kir5.1 activity of the DCT in kidney-tubule-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. In contrast, HK inhibited and LK increased Kir4.1/Kir5.1 activity in the control mice (Nedd4lflox/flox). Also, HK-intake decreased the negativity of K+-current (IK) reversal potential in the DCT (depolarization) only in the control mice but not in Ks-Nedd4-2 KO mice. Renal clearance experiments showed that HK-intake decreased while LK intake increased hydrochlorothiazide (HCTZ)-induced renal Na+ excretion only in the control mice but this effect was absent in Ks-Nedd4-2 KO mice. Western blot also demonstrated that HK-induced inhibition of phosphor-NCC (pNCC at Thr53) and total NCC (tNCC) was observed only in the control but not in Ks-Nedd4-2 KO mice. Furthermore, the expression of all three subunits of epithelia-Na+-channel (ENaC) in the Ks-Nedd4-2 KO mice on HK was higher than in the control mice. Thus, plasma K+ concentrations were similar between Nedd4lflox/flox and Ks-Nedd4-2 KO mice on HK for 7 days despite high NCC expression. We conclude that Nedd4-2 plays a role in regulating HK-induced inhibition of Kir4.1/Kir5.1 and NCC in the DCT.

Hypomagnesemia and Hypokalemia: The Possible Role of Vasopressinase in This Rare Presentation of Gestational Diabetes Insipidus

Background: Gestational diabetes insipidus (GDI) is a rare but recognized complication of pregnancy. We are reporting the case of a 33-year-old patient who presented in her 3rd trimester with GDI, severe asymptomatic hypokalemia & hypomagnesemia. This presentation of GDI has not been reported yet to our knowledge. Clinical Case: A 33-year-old G2P1 patient who presented to the hospital due to the discovery of new onset severe asymptomatic hypokalemia on routine labs. Further evaluation revealed K of 2.8 mEq/L, Mg of 1.4 mg/dL & Na of 142mEq/L. She endorsed polyuria & polydipsia and a 24-hour urine collection of 9.35L confirmed diabetes insipidus. Labs also revealed a serum osmolality of 286mOsm/kg & urine K of 15mEq/L. Her urine osmolality was inappropriately low at 138mOsm/kg. She received K & Mg supplementation. In pregnant patients suspected of having GDI who have normal serum Na, water restriction test can be done for further evaluation. It must performed with close monitoring because dehydration can lead to uteroplacental insufficiency. Given her history of previous fetal demise & report of headaches with attempts of water restriction, we opted not to do the test. She was given subcutaneous 1 mcg of desmopressin overnight with her thirst & polyuria improving only briefly. Due to her suboptimal response, we began her on 10 mcg of intranasal spray of desmopressin once in the evening. Her symptoms resolved on this regimen after a few days of observation. Her Mg & eventually K levels reached and remained at normal levels after repletion. The rest of her pregnancy went on without complications and she delivered a healthy male infant via scheduled cesarean section at 36 weeks gestation. She discontinued intranasal desmopressin after 2 weeks and has remained asymptomatic. Labs at follow up 4 weeks later remained normal. It is known that vasopressinase which is made by placental trophoblasts during pregnancy degrade endogenous ADH1. We are hypothesizing that since ADH has been shown to be effective in conserving magnesium in rats by stimulating its uptake in the distal convoluted tubule cells2, a similar mechanism could be present in humans. We believe that the loss of endogenous ADH, leads to the depletion of Mg and consequently hypokalemia in susceptible patients. Conclusion: To our knowledge this is the first case presenting the possible role of vasopressinase in the development of hypomagnesemia & hypokalemia in a patient with GDI. Reference: 1. Gordge MP, Williams DJ, Huggett NJ, Payne NN, and Neild GH. Loss of biological activity of arginine vasopressin during its degradation by vasopressinase from pregnancy serum. Clinical Endocrinology, vol. 42, no. 1, pp. 51- 58, 1995. 2. Dai LJ, Bapty B, Ritchie G, Quamme GA. Glucagon and arginine vasopressin stimulate Mg2+ uptake in mouse distal convoluted tubule cells. Am J Physiol. 1998 Feb;274(2):F328-35. doi: 10.1152/ajprenal.1998.274.2.F328. PMID: 9486227.

Roles of WNK4 and SPAK in K+-mediated dephosphorylation of the NaCl cotransporter

Although a great deal is known about mechanisms by which thiazide-sensitive NaCl cotransporter is phosphorylated and activated, much less is known about dephosphorylation. Here, we show that rapid dephosphorylation by high K+ depends on the Cl− sensitivity of with-no-lysine 4 and the rapid dephosphorylation of STE20/SPS1-related proline-alanine-rich protein kinase, primarily along the early distal convoluted tubule.