MinK-related peptides (MiRPs) are single-span membrane proteins that assemble with specific voltage-gated K+ (Kv) channel alpha-subunits to establish gating kinetics, unitary conductance, expression level, and pharmacology of the mixed complex. MiRP3 (encoded by the KCNE4 gene) has been shown to alter the behavior of some Kv alpha-subunits in vitro but its natural partners and physiologic functions are unknown. Seeking in vivo partners for MiRP3, immunohistochemistry was used to localize its expression to a unique subcellular site, the apical membrane of renal intercalated cells, where one potassium channel type has been recorded, the calcium- and voltage-gated channel BK. Overlapping staining of these two proteins was found in rabbit intercalated cells, and MiRP3 and BK subunits expressed in tissue culture cells were found to form detergent-stable complexes. Electrophysiologic and biochemical evaluation showed MiRP3 to act on BK to reduce current density in two fashions: shifting the current-voltage relationship to more depolarized voltages in a calcium-dependent fashion ( approximately 10 mV at normal intracellular calcium levels) and accelerating degradation of MiRP3-BK complexes. The findings suggest a role for MiRP3 modulation of BK-dependent urinary potassium excretion.

Background

The 2P domain potassium (K2P) channels are a recently discovered ion channel superfamily. Structurally, K2P channels are distinguished by the presence of two pore forming loops within one channel subunit. Functionally, they are characterized by their ability to pass potassium across the physiologic voltage range. Thus, K2P channels are also called open rectifier, background, or leak potassium channels. Patch clamp studies of renal tubules have described several open rectifier potassium channels that have as yet eluded molecular identification. We sought to determine the segment-specific expression of transcripts for the 14 known K2P channel genes in human nephron to identify potential correlates of native leak channels.

Methods

Human kidney samples were obtained from surgical cases and specific nephron segments were dissected. RNA was extracted and used as template for the generation of cDNA libraries. Real-time polymerase chain reaction (PCR) (TaqMan) was used to analyze gene expression.

Results

We found significant (P < 0.05) expression of K2P10 in glomerulus, K2P5 in proximal tubule and K2P1 in cortical thick ascending limb of Henle's loop (cTAL) and in distal nephron segments. In addition, we repeatedly detected message for several other K2P channels with less abundance, including K2P3 and K2P6 in glomerulus, K2P10 in proximal tubule, K2P5 in thick ascending limb of Henle's loop, and K2P3, K2P5, and K2P13 in distal nephron segments.

Conclusion

K2P channels are expressed in specific segments of human kidney. These results provide a step toward assigning K2P channels to previously described native renal leaks.

MiRP3, the single-span membrane protein encoded by KCNE4, is localized by immunofluorescence microscopy to the transverse tubules of murine cardiac myocytes. MiRP3 is found to co-localize with Kv4.2 subunits that contribute to cardiac transient outward potassium currents (I(to)). Whole-cell, voltage-clamp recordings of human MiRP3 and Kv4.2 expressed in a clonal cell line (tsA201) reveal MiRP3 to modulate Kv4.2 current activation, inactivation and recovery from inactivation. MiRP3 shifts the half-maximal voltage for activation (V(1/2)) approximately 20 mV and slows time to peak approximately 100%. In addition, MiRP3 slows inactivation approximately 100%, speeds recovery from inactivation approximately 30%, and enhances restored currents so they 'overshoot' baseline levels. The cytoplasmic accessory subunit KChIP2 also assembles with Kv4.2 in tsA201 cells to increase peak current, shift V(1/2) approximately 5 mV, slow time to peak approximately 10%, slow inactivation approximately 100%, and speed recovery from inactivation approximately 250% without overshoot. Simultaneous expression of all three subunits yields a biophysical profile unlike either accessory subunit alone, abolishes MiRP3-induced overshoot, and allows biochemical isolation of the ternary complex. Thus, regional heterogeneity in cardiac expression of MiRP3, Kv4.2 and KChIP2 in health and disease may establish the local attributes and magnitude of cardiac I(to).

Myocardial repolarization capacity varies with sex, age, and pathology; the molecular basis for this variation is incompletely understood. Here, we show that the transcript for KCNE4, a voltage-gated potassium (Kv) channel β subunit associated with human atrial fibrillation, was 8-fold more highly expressed in the male left ventricle compared with females in young adult C57BL/6 mice (P < 0.05). Similarly, Kv current density was 25% greater in ventricular myocytes from young adult males (P < 0.05). Germ-line Kcne4 deletion eliminated the sex-specific Kv current disparity by diminishing ventricular fast transient outward current (Ito,f) and slowly activating K(+) current (IK,slow1). Kcne4 deletion also reduced Kv currents in male mouse atrial myocytes, by >45% (P < 0.001). As we previously found for Kv4.2 (which generates mouse Ito,f), heterologously expressed KCNE4 functionally regulated Kv1.5 (the Kv α subunit that generates IKslow1 in mice). Of note, in postmenopausal female mice, ventricular repolarization was impaired by Kcne4 deletion, and ventricular Kcne4 expression increased to match that of males. Moreover, castration diminished male ventricular Kcne4 expression 2.8-fold, whereas 5α-dihydrotestosterone (DHT) implants in castrated mice increased Kcne4 expression >3-fold (P = 0.01) to match noncastrated levels. KCNE4 is thereby shown to be a DHT-regulated determinant of cardiac excitability and a molecular substrate for sex- and age-dependent cardiac arrhythmogenesis.

Introduction

Information on the economic burden of focal segmental glomerulosclerosis (FSGS) is sparse. This study characterized health care resource utilization (HCRU) and costs in patients with FSGS, and evaluated the impact of nephrotic range proteinuria on these outcomes.

Methods

This retrospective, observational cohort study used administrative claims data from the Optum Clinformatics Data Mart Database from October 2015 to December 2019. Patients with FSGS (n = 844; first claim = index event) between April 2016 and December 2018 were matched on index date, age, sex, and race to non-FSGS controls (n = 1688). FSGS nephrotic range (urine protein/creatinine ratio >3000 mg/g or albumin/creatinine ratio >2000 mg/g) and non-nephrotic subpopulations were identified. Baseline comorbidities, 12-month post-index all-cause HCRU and costs (per patient per year [PPPY]), and immunosuppressant prescriptions were compared between matched cohorts and between FSGS subpopulations.

Results

Comorbidity burden was higher in FSGS. Of 308 patients with available urine protein/creatinine ratio/albumin/creatinine ratio results, 36.4% were in nephrotic range. All-cause HCRU was higher in FSGS across resource categories (all P < 0.0001); 50.6% of FSGS and 23.3% of controls were prescribed glucocorticoids (P < 0.0001). Mean total medical costs were higher in FSGS ($59,753 vs. $8431 PPPY; P < 0.0001), driven by outpatient costs. Nephrotic range proteinuria was associated with higher all-cause inpatient, outpatient, and prescription costs versus nonnephrotic patients (all P < 0.0001), resulting in higher total costs ($70,481 vs. $36,099 PPPY; P < 0.0001).

Conclusions

FSGS is associated with significant clinical and economic burdens; the presence of nephrotic range proteinuria increased the economic burden. New treatment modalities are needed to reduce proteinuria, help improve patient outcomes, and reduce HCRU and associated costs.