Familial Hyperkalemic Hypertension

Abstract

The rare disease Familial Hyperkalemic Hypertension (FHHt) is caused by mutations in the genes encoding Cullin 3 (CUL3), Kelch-Like 3 (KLHL3), and two members of the With-No-Lysine [K] (WNK) kinase family, WNK1 and WNK4. In the kidney, these mutations ultimately cause hyperactivation of NCC along the renal distal convoluted tubule. Hypertension results from increased NaCl retention, and hyperkalemia by impaired K ⁺ secretion by downstream nephron segments. CUL3 and KLHL3 are now known to form a ubiquitin ligase complex that promotes proteasomal degradation of WNK kinases, which activate downstream kinases that phosphorylate and thus activate NCC. For CUL3, potent effects on the vasculature that contribute to the more severe hypertensive phenotype have also been identified. Here we outline the in vitro and in vivo studies that led to the discovery of the molecular pathways regulating NCC and vascular tone, and how FHHt-causing mutations disrupt these pathways. Potential mechanisms for variability in disease severity related to differential effects of each mutation on the kidney and vasculature are described, and other possible effects of the mutant proteins beyond the kidney and vasculature are explored. © 2024 American Physiological Society. Compr Physiol 14:5839-5874, 2024.

Figure 1.. Distal convoluted tubule (DCT) is a part of the distal nephron.

Na⁺ and K⁺ filtered from the glomeruli are primarily reabsorbed along the proximal tubule and thick ascending limb of the Henle’s loop. However, the final concentration of urinary Na⁺ and K⁺ is determined by the distal nephron, including the distal convoluted tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD).

Figure 2.. The mechanism of Familial Hyperkalemic Hypertension (FHHt).

The effect of FHHt-causing mutations on the kidney (Left) and blood vessels (Right). (Left) Along the distal convoluted tubule, the Na⁺-Cl^– cotransporter (NCC) is phosphorylated and activated primarily by Ste20-related Proline Alanine Kinase (SPAK) but also to a minor degree by Oxidative Stress Response Kinase 1 (OSR1). SPAK and OSR1 are both in turn activated by WNK kinases. CUL3 and KLHL3 are components of a ubiquitin ligase complex that mediates WNK kinase degradation (not shown, see Figure 6). In FHHt, WNK kinases accumulate since the CUL3-KLHL3-WNK complex cannot form, ultimately resulting in NCC hyperactivation. This leads to direct effects on Na⁺ retention and indirect effects on plasma [K⁺] and [HCO₃⁻]. These effects on the kidney are observed with FHHt-causing mutations in the CUL3, KLHL3, WNK1, and WNK4 genes. (Right) In vascular smooth muscle cells, mutations in CUL3 cause increased abundances of RAS homolog family, member A (RhoA) leading to activation of Rho-associated protein kinase (ROCK) which then phosphorylates myosin phosphatase targeting subunit 1 (MYPT1) promoting vasoconstriction. Mutant CUL3 also impairs vasodilation through effects on nitric oxide generation (not shown, see Figure 7).

Figure 3.. FHHt-causing mutations in WNK1, WNK4, KLHL3, and CUL3.

WNK1 mutations are large deletions in the first intron that do not alter the coding sequence but lead to ectopic WNK1 expression. WNK4 mutations are missense mutations found within two discrete regions. The first region is located within an acidic motif adjacent to the first coiled coil (CC) domain (e.g. WNK4 Q565) and the second is adjacent to the second coiled coil domain. Kelch-like 3 (KLHL3) mutations are clustered in short segments within the Kelch propeller (e.g. KLHL3 S433) and the BTB-BACK domains, which are implicated in substrate and CUL3 binding, respectively. The stars indicate the locations where mutations have been reported. Most FHHt-causing Cullin 3 (CUL3) mutations are located in sites associated with splicing of exon 9, including intron 8 (Branch point and Splice acceptor), exon 9 (ES enhancer), and intron 9 (Splice donor) leading to deletion of exon 9 (CUL3-Δ9). This causes the deletion of the 57 amino acid 4HB domain adjacent to the α/β1 domain, which interacts with the CUL3 regulator CSN5 (also called JAB1). A single patient has been identified with a mutation in exon 10 that leads to deletion of four amino acids (CUL3-Δ474–477) in the α/β1 domain. Both CUL3-Δ9 and CUL3-Δ474–477 display reduced interaction with CSN5. The stars indicate the locations where mutations have been reported.

Figure 4.. Mechanism of hyperkalemia in FHHt.

In connecting segment and cortical collecting duct (CNT/CCD) of the kidney, Epithelial Na⁺ Channel (ENaC)-mediated Na⁺ reabsorption generates the electrical driving force for K⁺ secretion through the Renal Outer Medullary K⁺ channel (ROMK) into the tubule lumen (dashed circle emphasizes this coupling). NCC activity along the distal convoluted tubule (DCT) plays a critical role in the physiological maintenance of plasma [K⁺] since it meters delivery of Na⁺ to the CNT/CCD, with NCC activated when plasma [K⁺] decreases (see Figure 5). When NCC is hyperactivated in FHHt, Na⁺ delivery to ENaC is reduced, lowering the drive for ROMK-mediated K⁺ secretion, leading to hyperkalemia. FHHt-causing mutations prevent NCC from being inactivated, as would occur physiologically to correct the hyperkalemia (see Figure 5). NCC hyperactivation in FHHT may also cause hypertrophy of the DCT, and atrophy of the CNT/CCD (not shown), further reducing the capacity for K⁺ secretion.

Figure 5.. NCC regulatory pathway in response to physiological reductions in plasma [K⁺]. NCC activation by WNK-SPAK pathway.

WNK kinases are inhibited by direct binding of chloride (Cl^–). Intracellular [Cl^–] is determined by coupling of basolateral K⁺ and Cl^– efflux via Kir4.1/5.1 and CLC-KB respectively. As plasma [K⁺] decreases, increased K⁺ and Cl^– efflux lowers intracellular [Cl^–], leading to the activation of WNK-SPAK-NCC pathway. When serum [K⁺] increases, the WNK-SPAK pathway is switched off by increased intracellular [Cl^–] and the action of phosphatases (not shown but see (185) for a detailed review). As shown in Figure 4, higher NCC activity reduces Na⁺ delivery to downstream K⁺ secreting segments of the tubule and thus lowers K⁺ secretion to normalize plasma [K⁺]. FHHt-causing mutations override this leading to persistent hyperkalemia.

Figure 6.. WNK4 degradation by the CUL3-KLHL3 ubiquitin ligase complex.

The scaffold Cullin 3 (CUL3) assembles with a BTB substrate-binding adaptor (e.g. KLHL3), a RING ubiquitin ligase, and E2 to form a CRL3, facilitating substrate ubiquitination and subsequently proteasomal degradation. WNK4 is a direct target for ubiquitination by the CUL3–KLHL3 CRL3 complex. Angiotensin II (Ang II) signaling through protein kinase C and hypokalemia promote KLHL3 phosphorylation at S433, preventing WNK4 degradation and increasing NCC phosphorylation. This promotes Na⁺ retention and lowers K⁺ secretion (see Figure 4) to maintain plasma volume or plasma [K⁺].

Figure 7.. Effects of WNKs and CUL3 on vascular tone.

(Left) In the vascular smooth muscle cells, the WNK-SPAK pathway may activate vascular NKCC1 promoting vasoconstriction. WNK1 and WNK3 accumulation in the vasculature may therefore contribute to hypertension through this mechanism. (Right) In the vascular endothelium, Phosphodiesterase 5 (PDE5), a substrate of the CUL3-RhoBTB1 ubiquitin ligase complex, hydrolyzes cGMP to inactive 5′-GMP, counteracting NO-mediated vasodilatory effects. In FHHt caused by mutant CUL3, both enhanced vasoconstriction via increased activation of the RhoA-ROCK-MYPT1 cascade and impaired vasodilation due to increased PDE5 may contribute to the hypertensive phenotype.

Figure 8.. Molecular effects of WNK1, WNK4, KLHL3, and CUL3 mutations.

Intron 1 deletion in WNK1 results in an increased expression of a long ubiquitously expressed and catalytically active isoform (L-WNK1) along the distal convoluted tubule and connecting segment. WNK4 mutations at acidic motif impair WNK4 and CUL3-KLHL3 interactions, inhibiting WNK4 ubiquitination by the KLHL3-CUL3 complex. KLHL3 mutations in the Kelch propeller (e.g. KLHL3 S433) or BTB-BACK domains impair KLHL3 binding to WNK kinases or CUL3, respectively. This prevents WNK kinase ubiquitination, leading to WNK accumulation. In vitro, CUL3-Δ9 and CUL3^Δ474−477 display multiple defects (1) loss of cycling of CRL3 assembly and disassembly due to decreased interaction with CSN5 (JAB1), (2) enhanced binding to adaptors, (3) autoubiquitination and degradation, (4) impaired dimerization with WT CUL3, and (5) inappropriate adaptor degradation (demonstrated for KLHL3). Defects (1) to (4) have been reported for both CUL3-Δ9 and CUL3^Δ474−477, while mechanism (5) has only been reported for CUL3-Δ9.

Figure 9.. CUL3 neddylation and deneddylation.

Stability and activity of the CUL3 Ring Ligase complex (CRL3) depends upon covalent addition (neddylation) and removal (deneddylation) of the ubiquitin-like protein NEDD8 (N8). NEDD8 is conjugated to CUL3 in an ATP-dependent manner to activate the CRL3. Deneddylation is mediated by the COP9 signalosome (CSN) and allows CRL3 disassembly.