Emerging Targets of Diuretic Therapy

Abstract

Diuretics are commonly prescribed for treatment in patients with hypertension, edema, or heart failure. Studies on hypertensive and salt-losing disorders and on urea transporters have contributed to better understanding of mechanisms of renal salt and water reabsorption and their regulation. Proteins involved in the regulatory pathways are emerging targets for diuretic and aquaretic therapy. Integrative high-throughput screening, protein structure analysis, and chemical modification have identified promising agents for preclinical testing in animals. These include WNK-SPAK inhibitors, ClC-K channel antagonists, ROMK channel antagonists, and pendrin and urea transporter inhibitors. We discuss the potential advantages and side effects of these potential diuretics.

Figure 1. The mechanisms of salt reabsorption in the distal nephron and the related new classes of diuretics

The WNK-SPAK/OSR1-N(K)CC pathway regulates the activity of NCC in the distal convoluted tubule (DCT) and NKCC2 in the loop of Henle through a kinase-dependent phosphorylation cascade. SPAK dominates NCC phosphorylation in DCT while both OSR1 and SPAK activate NKCC2. WNK1 was reported to enhance ENaC in the connecting tubule and cortical collecting duct through SKG1-mediated Nedd4-2 phosphorylation and inactivation, which inhibits the ubiquitination and endocytosis of ENaC. This stimulatory effect of WNK1 on SGK1 is kinase activity independent and may need to cooperate with 3-Phosphoinositide-dependent protein kinase (PDK). The ClC-Kb chloride channel is expressed in the basolateral membrane of TAL, DCT, and non-principal cells of connecting tubule and cortical collecting duct and functionally couples with Na⁺ transporters in the apical membrane. The potassium Kir4.1/5.1 channel is also localized in the basolateral membrane of the distal nephron and is essential to keep the normal resting membrane potential (MP) and Na⁺, K⁺ ATPase activity. ROMK channel provides K⁺ efflux and recycling to maintain NKCC2 activity in TAL and K⁺ secretion in the connecting tubule (CNT) and cortical collecting duct (CCD). The Cl^--HCO3^- exchanger pendrin modulates NaCl reabsorption via ENaC in principal cells and NDCBE in type B intercalated cells. Potentially novel diuretics are listed in the right lower corner, and the molecular targets of these agents are marked in the figure (+P: phosphorylation, ↑: stimulation, ┬: inhibition).

Figure 2. The activation cascade of the WNK-SPAK/OSR1-N(K)CC pathway and the related novel diuretics

(A) Domain structures of WNKs, SPAK/OSR1, and NKCC1/NKCC2/NCC are shown. Autophosphorylation of WNK kinase (S382 and S335 in WNK1 and WNK4 respectively) is required for WNK activation and subsequent phosphorylation of SPAK and OSR1 (T233 and T185 in the activation loop and S373 and S325 in the S-motif of SPAK and OSR1 respectively). This process requires the interaction between RFxV motifs of WNKs and the CCT domain of SPAK/OSR1. The activated SPAK/OSR1 binds to the N-terminal RFxV/I motifs on their substrates via the CCT domain and phosphorylates a cluster of conserved threonine and serine residues. WNK inhibitors prevent the autophosphorylation of WNKs. WNK-SPAK disrupters interfere with the interaction between WNK and SPAK/OSR1. SPAK inhibitors inhibit SPAK kinase activity and N(K)CC phosphorylation and activation. These novel diuretic agents are highlighted in blue font. The red arrow denotes kinase-dependent phosphorylation. Black arrow represents protein-protein interactions. The blue line indicates pharmacological inhibition.

Figure 3. Regulation of the WNK-SPAK/OSR1-NCC pathway in the distal convoluted tubule

Autophosphorylation on the T-loop serine (S382 in WNK1, S335 in WNK4) activates WNK kinases. This process is inhibited by the Cl^- ion binding to the Cl^- sensing pocket. The plasma K⁺ level affects membrane potential (MP) and Cl^- efflux via the ClC-Kb channel. Hypokalemia (↓[K]o) releases the Cl^--sensing inhibition on WNK by hyperpolarizing MP, increasing Cl^- efflux and reducing intracellular Cl^- level ([Cl^-]_i). Hyperkalemia (↑[K]o) is supposed to do the opposite or inactivates NCC through an unknown SPAK/OSR1-independent protein phosphatase (PP) pathway (blue dashed arrow). Kir4.1/5.1 functions to maintain normal MP of distal convoluted tubule (DCT), together with the Na⁺, K⁺ ATPase and ClC-Kb channel. Inhibition of Kir4.1 results in a depolarized MP and increased intracellular Cl^- concentration. Activated WNKs switch on SPAK/OSR1-NCC signaling through a phosphorylation cascade. Other kinases may phosphorylate NCC since Spak and Osr1 double knockout mice still preserved some phosphorylated NCC. KS-WNK1 may exert competitive inhibition on WNK1 through the interaction with WNK1 downstream substrates. Red lines and blue lines denote stimulatory and inhibitory regulations on NCC, respectively. +P: phosphorylation; -P: dephosphorylation.

Figure 4. New allosteric WNK and SPAK/OSR1 inhibitors

(A) The Cl^- binding pocket of WNK1 (PDB: 3FPQ) is surrounded by the glycine-rich loop (yellow), αC-helix (cyan), and activation loop (red). The Cl^- (orange dot) forms hydrophobic interactions with Leu369 and Leu371 (green sticks) in the DLG motif (green) and with Phe283 and Leu299 in β3 & β4 helix, respectively. The catalytic lysine (Lys233) and T-loop serine (Ser382) are shown in blue and red sticks. (B) The crystal structure of WNK1 complexed with WNK463 (PDB: 5DRB). WNK463 (magenta) contacts the hinge region (orange loop) of the ATP-binding site and fits perfectly into the narrow tunnel of the catalytic site and interferes with ATP binding of WNKs. (C) ATP non-competitive WNK473 (magenta) binds only to the back pocket (surrounded by DLG sequence, activation loop, and αC-helix) of WNKs but not the ATP-binding site (PDB: 5TF9). (D) The OSR1 CCT domain (PDB: 2V3S) shows the primary pocket (blue arrow) for RFQV peptide docking and secondary pocket where novel SPAK/OSR1 inhibitors bind (red arrow). We drew these figures using Pymol and the open PDB files.

Figure 5. The tissue expression pattern of the novel diuretic target genes

Heat map represents the abundance (red: presence, white: absence) of 11 potential targets of novel diuretics or aquretics (columns) across 22 human tissues (rows) based on public RNA-seq data (the Genotype-Tissue Expression (GTEx) Project, etc.). Color intensity represents fractional density expression of each mRNA across all tissues.

Figure 6. The mechanisms of NaCl and urea accumulation in renal medulla and the related new classes of aquaretics

The urinary NaCl concentration (black arrow) increases along the thin descending limb. When urine enters the hairpin loop and thin ascending limb, the highly-concentrated urinary NaCl enters the inner medulla via ClC-Ka on the apical and basolateral membranes. NaCl further accumulates in outer medulla via the active reabsorption by NKCC2 in thick ascending limb. The urinary urea (red arrow) is concentrated along the nephron. The concentrated urea in inner medullary collecting duct is reabsorbed via UT-A1 and UT-A3 under the stimulation of antidiuretic hormone. Urea leaving the inner medulla via ascending vasa recta (AVR) can be brought back to inner medulla via UT-B on descending vasa recta (DVR) or UT-A2 on thin descending limb. This phenomenon is called urea recycling. UT-A2 may reabsorb urinary urea into adjacent AVR (dashed arrow) and then reenter into DVR or general circulation, although the physiological function of UT-A2 is still unclear. Vascular bundle (dashed, pink cylinder) where close proximity of the DVR and AVR in the center and the AVR and thin descending limb in the periphery prevents removal of medullary urea into the circulation; see text for more details. The dashed line marked the separations of renal cortex, outer medulla, and inner medulla. Each colored line represents the localization of proteins that are involved in NaCl or urea accumulation. For the sake of simplicity, only a long loop of Henle is shown. The classes of potential novel diuretics are highlighted in blue font.