Prednisone-induced sustained remission in a patient with familial fibronectin glomerulopathy (GFND).

Glomerulopathy with Fibronectin Deposits (GFND) is a rare, autosomal dominant disease characterized by proteinuria, hematuria and progressive renal failure associated with glomerular deposition of fibronectin, frequently resulting in end-stage renal disease (ESRD). There is no established treatment for this condition beyond conservative measures such as blood pressure control and the use of angiotensin-converting enzyme (ACE) inhibitors. We present a case of GFND associated with progressive chronic kidney disease (CKD) and nephrotic range proteinuria showing a sustained response to prednisone treatment. A 27-year-old G2P2 Caucasian female presented with 3 g/day of proteinuria, serum creatinine (Cr) 0.7 mg/dL, inactive urinary sediment and normotension without medication. She was part of a large family with glomerular disease, including three members who died of cerebral hemorrhage or stroke in their thirties. The patient's kidney biopsy showed mesangial deposition of fibronectin consistent with GFND. No interstitial fibrosis was seen. Genotyping revealed the Y973C FN1 gene mutation. Despite maximal tolerable ACE inhibition, proteinuria increased to 4-6 g/g Cr and serum Cr increased to 1.0 mg/dL. She was treated with prednisone 60 mg (~ 1 mg/Kg) daily for 2 mos and then tapered by ~ 0.2 mg/Kg every month for 6 mos of total therapy. Proteinuria decreased to ~ 1 g/g Cr for > 5 years and serum Cr stabilized in the 1.2 mg/dL range with treatment. No significant side effects were encountered. In conclusion, this protocol should be considered in GFND patients with nephrotic range proteinuria despite maximal angiotensin system inhibition who have relatively preserved renal function.

Potassium conservation is impaired in mice with reduced renal expression of Kir4.1.

To better understand the role of the inward-rectifying K channel Kir4.1 (KCNJ10) in the distal nephron, we initially studied a global Kir4.1 knockout mouse (gKO), which demonstrated the hypokalemia and hypomagnesemia seen in SeSAME/EAST syndrome and was associated with reduced Na/Cl cotransporter (NCC) expression. Lethality by ~3 wk, however, limits the usefulness of this model, so we developed a kidney-specific Kir4.1 "knockdown" mouse (ksKD) using a cadherin 16 promoter and Cre-loxP methodology. These mice appeared normal and survived to adulthood. Kir4.1 protein expression was decreased ~50% vs. wild-type (WT) mice by immunoblotting, and immunofluorescence showed moderately reduced Kir4.1 staining in distal convoluted tubule that was minimal or absent in connecting tubule and cortical collecting duct. Under control conditions, the ksKD mice showed metabolic alkalosis and relative hypercalcemia but were normokalemic and mildly hypermagnesemic despite decreased NCC expression. In addition, the mice had a severe urinary concentrating defect associated with hypernatremia, enlarged kidneys with tubulocystic dilations, and reduced aquaporin-3 expression. On a K/Mg-free diet for 1 wk, however, ksKD mice showed marked hypokalemia (serum K: 1.5 ± 0.1 vs. 3.0 ± 0.1 mEq/l for WT), which was associated with renal K wasting (transtubular K gradient: 11.4 ± 0.8 vs. 1.6 ± 0.4 in WT). Phosphorylated-NCC expression increased in WT but not ksKD mice on the K/Mg-free diet, suggesting that loss of NCC adaptation underlies the hypokalemia. In conclusion, even modest reduction in Kir4.1 expression results in impaired K conservation, which appears to be mediated by reduced expression of activated NCC.

Mutations in cytoplasmic loops of the KCNQ1 channel and the risk of life-threatening events: implications for mutation-specific response to ß-blocker therapy in type 1 long-QT syndrome.

BACKGROUND: ß-Adrenergic stimulation is the main trigger for cardiac events in type 1 long-QT syndrome (LQT1). We evaluated a possible association between ion channel response to ß-adrenergic stimulation and clinical response to ß-blocker therapy according to mutation location. METHODS AND RESULTS: The study sample comprised 860 patients with genetically confirmed mutations in the KCNQ1 channel. Patients were categorized into carriers of missense mutations located in the cytoplasmic loops (C loops), membrane-spanning domain, C/N terminus, and nonmissense mutations. There were 27 aborted cardiac arrest and 78 sudden cardiac death events from birth through 40 years of age. After multivariable adjustment for clinical factors, the presence of C-loop mutations was associated with the highest risk for aborted cardiac arrest or sudden cardiac death (hazard ratio versus nonmissense mutations=2.75; 95% confidence interval, 1.29-5.86; P=0.009). ß-Blocker therapy was associated with a significantly greater reduction in the risk of aborted cardiac arrest or sudden cardiac death among patients with C-loop mutations than among all other patients (hazard ratio=0.12; 95% confidence interval, 0.02-0.73; P=0.02; and hazard ratio=0.82; 95% confidence interval, 0.31-2.13; P=0.68, respectively; P for interaction=0.04). Cellular expression studies showed that membrane spanning and C-loop mutations produced a similar decrease in current, but only C-loop mutations showed a pronounced reduction in channel activation in response to ß-adrenergic stimulation. CONCLUSIONS: Patients with C-loop missense mutations in the KCNQ1 channel exhibit a high risk for life-threatening events and derive a pronounced benefit from treatment with ß-blockers. Reduced channel activation after sympathetic activation can explain the increased clinical risk and response to therapy in patients with C-loop mutations.

Ion channel mechanisms related to sudden cardiac death in phenotype-negative long-QT syndrome genotype-phenotype correlations of the KCNQ1(S349W) mutation.

UNLABELLED: BACKGROUND: Data regarding possible ion channel mechanisms that predispose to ventricular tachyarrhythmias in patients with phenotype-negative long-QT syndrome (LQTS) are limited. METHODS AND RESULTS: We carried out cellular expression studies for the S349W mutation in the KCNQ1 channel, which was identified in 15 patients from the International LQTS Registry who experienced a high rate of cardiac events despite lack of significant QTc prolongation. The clinical outcome of S349W mutation carriers was compared with that of QTc-matched carriers of haploinsufficient missense (n = 30) and nonsense (n = 45) KCNQ1 mutations. The channels containing the mutant S349W subunit showed a mild reduction in current (<50%), in the haploinsuficient range, with an increase in maximal conductance compared with wild-type channels. In contrast, expression of the S349W mutant subunit produced a pronounced effect on both the voltage dependence of activation and the time constant of activation, while haploinsuficient channels showed no effect on either parameter. The cumulative probability of cardiac events from birth through age 20 years was significantly higher among S349W mutation carriers (58%) as compared with carriers of QTc-matched haploinsufficent missense (21%, P = 0.004) and nonsense (25%, P = 0.01) mutations. CONCLUSIONS: The S349W mutation in the KCNQ1 potassium channel exerts a relatively mild effect on the ion channel current, whereas an increase in conductance compensates for impaired voltage activation of the channel. The changes observed in voltage activation of the channel may underlie the mechanisms predisposing to arrhythmic risk among LQTS patients with a normal-range QTc.

Molecular basis of decreased Kir4.1 function in SeSAME/EAST syndrome.

SeSAME/EAST syndrome is a channelopathy consisting of a hypokalemic, hypomagnesemic, metabolic alkalosis associated with seizures, sensorineural deafness, ataxia, and developmental abnormalities. This disease links to autosomal recessive mutations in KCNJ10, which encodes the Kir4.1 potassium channel, but the functional consequences of these mutations are not well understood. In Xenopus oocytes, all of the disease-associated mutant channels (R65P, R65P/R199X, G77R, C140R, T164I, and A167V/R297C) had decreased K(+) current (0 to 23% of wild-type levels). Immunofluorescence demonstrated decreased surface expression of G77R, C140R, and A167V expressed in HEK293 cells. When we coexpressed mutant and wild-type subunits to mimic the heterozygous state, R199X, C140R, and G77R currents decreased to 55, 40, and 20% of wild-type levels, respectively, suggesting that carriers of these mutations may present with an abnormal phenotype. Because Kir4.1 subunits can form heteromeric channels with Kir5.1, we coexpressed the aforementioned mutants with Kir5.1 and found that currents were reduced at least as much as observed when we expressed mutants alone. Reduction of pH(i) from approximately 7.4 to 6.8 significantly decreased currents of all mutants except R199X but did not affect wild-type channels. In conclusion, perturbed pH gating may underlie the loss of channel function for the disease-associated mutant Kir4.1 channels and may have important physiologic consequences.

A novel mitochondrial K(ATP) channel assay.

RATIONALE: The mitochondrial ATP sensitive potassium channel (mK(ATP)) is implicated in cardioprotection by ischemic preconditioning (IPC), but the molecular identity of the channel remains controversial. The validity of current methods to assay mK(ATP) activity is disputed. OBJECTIVE: We sought to develop novel methods to assay mK(ATP) activity and its regulation. METHODS AND RESULTS: Using a thallium (Tl(+))-sensitive fluorophore, we developed a novel Tl(+) flux based assay for mK(ATP) activity, and used this assay probe several aspects of mK(ATP) function. The following key observations were made. (1) Time-dependent run down of mK(ATP) activity was reversed by phosphatidylinositol-4,5-bisphosphate (PIP(2)). (2) Dose responses of mK(ATP) to nucleotides revealed a UDP EC(50) of approximately 20 micromol/L and an ATP IC(50) of approximately 5 micromol/L. (3) The antidepressant fluoxetine (Prozac) inhibited mK(ATP) (IC(50)=2.4 micromol/L). Fluoxetine also blocked cardioprotection triggered by IPC, but did not block protection triggered by a mK(ATP)-independent stimulus. The related antidepressant zimelidine was without effect on either mK(ATP) or IPC. CONCLUSIONS: The Tl(+) flux mK(ATP) assay was validated by correlation with a classical mK(ATP) channel osmotic swelling assay (R(2)=0.855). The pharmacological profile of mK(ATP) (response to ATP, UDP, PIP(2), and fluoxetine) is consistent with that of an inward rectifying K(+) channel (K(IR)) and is somewhat closer to that of the K(IR)6.2 than the K(IR)6.1 isoform. The effect of fluoxetine on mK(ATP)-dependent cardioprotection has implications for the growing use of antidepressants in patients who may benefit from preconditioning.

Quantification of K+ secretion through apical low-conductance K channels in the CCD.

Outward and inward currents through single small-conductance K(+) (SK) channels were measured in cell-attached patches of the apical membrane of principal cells of the rat cortical collecting duct (CCD). Currents showed mild inward rectification with high [K(+)] in the pipette (K(p)(+)), which decreased as K(p)(+) was lowered. Inward conductances had a hyperbolic dependence on K(p)(+) with half-maximal conductance at approximately 20 mM. Outward conductances, measured near the reversal potential, also increased with K(p)(+) from 15 pS (K(p)(+) = 0) to 50 pS (K(p)(+) = 134 mM). SK channel density was measured as the number of conducting channels per patch in cell-attached patches. As reported previously, channel density increased when animals were on a high-K diet for 7 days. Addition of 8-cpt-cAMP to the bath at least 5 min before making a seal increased SK channel density to an even greater extent, although this increase was not additive with the effect of a high-K diet. In contrast, increases in Na channel activity, assessed as the whole cell amiloride-sensitive current, due to K loading and 8-cpt-cAMP treatment were additive. Single-channel conductances and channel densities were used as inputs to a simple mathematical model of the CCD to predict rates of transepithelial Na(+) and K(+) transport as a function of apical Na(+) permeability and K(+) conductance, basolateral pump rates and K(+) conductance, and the paracellular conductance. With measured values for these parameters, the model predicted transport rates that were in good agreement with values measured in isolated, perfused tubules. The number and properties of SK channels account for K(+) transport by the CCD under all physiological conditions tested.

Basolateral K+ conductance in principal cells of rat CCD.

Whole cell K+ current was measured by forming seals on the luminal membrane of principal cells in split-open rat cortical collecting ducts. The mean inward, Ba2+-sensitive conductance, with 40 mM extracellular K+, was 76 +/- 12 and 141 +/- 22 nS/cell for animals on control and high-K+ diets, respectively. The apical contribution to this was estimated to be 3 and 16 nS/cell on control and high-K+ diets, respectively. To isolate the basolateral component of whole cell current, we blocked ROMK channels with either tertiapin-Q or intracellular acidification to pH 6.6. The current was weakly inward rectifying when bath K+ was > or =40 mM but became more strongly rectified when bath K+ was lowered into the physiological range. Including 1 mM spermine in the pipette moderately increased rectification, but most of the outward current remained. The K+ current did not require intracellular Ca2+ and was not inhibited by 3 mM ATP in the pipette. The negative log of the acidic dissociation constant (pKa) was approximately 6.5. Block by extracellular Ba2+ was voltage dependent with apparent Ki at -40 and -80 mV of approximately 160 and approximately 80 microM, respectively. The conductance was TEA insensitive. Substitution of Rb+ or NH4+ for K+ led to permeability ratios of 0.65 +/- 0.07 and 0.15 +/- 0.02 and inward conductance ratios of 0.17 +/- 0.03 and 0.57 +/- 0.09, respectively. Analysis of Ba2+-induced noise, with 40 mM extracellular K+, yielded single-channel currents of 0.39 +/- 0.04 and -0.28 +/- 0.04 pA at voltages of 0 and -40 mV, respectively, and a single-channel conductance of 17 +/- 1 pS.

Carboxy-terminal determinants of conductance in inward-rectifier K channels.

Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at V(m) = -100 mV from 34 to 21 pS. The slope conductance measured between -60 and -140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba(2+), suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl(+):K(+), accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K(+) transport.