ENaC; uTPA; polycystin-2 (TRPP2)
Epithelial Sodium Channels (ENaCs) [1]
- Urokinase-type Plasminogen Activator Receptor (uTPA)[2]

In vitro activity: Amiloride also induces the dephosphorylation of P13K (phosphatidylinositol 3-kinase) and PDK-1 (phosphoinositide-dependent kinase-1) kinases along with PTEN (phosphatase and tensin homolog deleted on chromosome 10) and PP1 alpha phosphatases. Amiloride inhibits phosphorylation of kinases and phosphatases by competing with ATP. Amiloride, which causes little or no cytotoxicity by itself, enhances TRAIL-induced apoptosis. Amiloride precludes the alkalinization and in parallel inhibit cellular proliferation. Amiloride directly inhibits autophosphorylation of the EGF receptor. Amiloride significantly enhances recovery to a maximum of 39%, 88%, and 78% for force, +dF/dt, and -dF/dt, respectively. Amiloride, a frequently used inhibitor of Na+/H+ exchange, rapidly inhibits phorbol ester-stimulated protein phosphorylation in vivo and protein kinase C-mediated phosphorylation in vitro, both with potency similar to that with which Amiloride inhibits Na+/H+ exchange. Amiloride blocks phorbol ester-induced adhesion of HL-60 cells (adhesion being a property indicative of the differentiated state), but dimethylamiloride (as well as ethylisopropylamiloride, another very potent amiloride analog) does not. Amiloride inhibits the ouabain-sensitive rate of oxygen consumption (QO2) of a suspension of rabbit intact proximal tubules in the presence of different concentrations of extracellular sodium.

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Cell Assay: Amiloride blocks δβγ channels with an IC50 of 2.6 μM (58, 71, 75, 134, 148). The Ki of amiloride for δβγ ENaC is 26-fold that of αβγ channels (0.1 μM for αβγ ENaC). Amiloride blockade of δβγ ENaC is much more voltage dependent compared with the αβγ channel. The Ki of amiloride for δαβγ channels is 920 and 13.7 μM at -120 and +80 mV, respectively, which significantly differs from that of both αβγ and δβγ channels. Amiloride is a relatively selective inhibitor of the epithelial sodium channel (ENaC) with an IC50 (the concentration required to reach 50% inhibition of an ion channel) in the concentration range of 0.1 to 0.5 μM. Amiloride is a relatively poor inhibitor of the the Na+/H+ exchanger (NHE) with an IC50 as low as 3 μM in the presence of a low external [Na+] but as high as 1 mM in the presence of a high [Na+]. Amiloride is an even weaker inhibitor of the Na+/Ca2+ exchanger (NCX), with an IC50 of 1 mM. Amiloride (1 μM) and submicromolar doses of Benzamil (30 nM), doses known to inhibit the ENaC, inhibit the myogenic vasoconstriction response to increasing perfusion pressure by blocking the activity of ENaC proteins. Amiloride completely inhibits Na+ influx in doses known to be relatively specific for ENaC (1.5 μM) in vascular smooth muscle cells (VSMC).

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Cultured podocytes were treated with Amiloride HCl dihydrate (MK 870) at different concentrations. The drug significantly inhibited uTPA activity in podocytes, as demonstrated by reduced cleavage of plasminogen to plasmin. Additionally, Amiloride HCl dihydrate (MK 870) upregulated the expression of podocyte-specific proteins (podocin and nephrin) at both mRNA and protein levels, which are crucial for maintaining the integrity of the glomerular filtration barrier. The drug also decreased the secretion of pro-inflammatory cytokines (TNF-α, IL-6) in podocytes stimulated by lipopolysaccharide (LPS)[2]

A strong inhibitor of epithelial sodium channels (ENaCs) is amiloride hydrochloride dihydrate. Within fifteen to thirty minutes after injection, amiloride is significantly concentrated in the plasma. When compared to the baseline readings (n = 7), a 2 mg/kg dose of amiloride hydrochloride dihydrate has no effect on blood pressure, heart rate, mesenteric vascular resistance, or hindquarters vascular resistance. Amiloride hydrochloride dihydrate causes very little change in heart rate (-10±6 bpm/min) and arterial pressure (-1±1 mmHg) over a 2-hour period when compared to baseline values. The c-Fos activation in the area postrema (AP) exhibits a dose-related response pattern, according to the results. Amiloride hydrochloride dihydrate, even at the lowest dose of 0.1 mg/kg, is statistically different from the control rats in terms of the number of c-Fos labeled neurons at the p<0.01 level[1].

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Adult male Sprague-Dawley rats were administered Amiloride HCl dihydrate (MK 870) via intraperitoneal injection at a dose of 10 mg/kg. Immunohistochemical staining of brain tissues showed a significant increase in c-Fos-positive cells in the area postrema (AP) 2 hours after administration. Quantitative analysis revealed that the number of c-Fos-positive neurons in the AP was approximately 3-fold higher than that in the control group. This activation was specific to the AP, as no significant changes in c-Fos expression were observed in adjacent brain regions (e.g., nucleus of the solitary tract)[1]

uTPA activity assay: Podocytes were lysed after treatment with Amiloride HCl dihydrate (MK 870), and the cell lysate was incubated with plasminogen substrate and chromogenic agent. The absorbance at 405 nm was measured at different time points to calculate the uTPA activity. The assay showed that Amiloride HCl dihydrate (MK 870) inhibited uTPA activity in a concentration-dependent manner[2]

This study examined the mechanism of action of amiloride, a urokinase-type plasminogen activator receptor inhibitor, in lowering proteinuria. Podocytes were resuscitated to allow for their proliferation and were observed for morphological changes. In the in vitro experiment, control, lipopolysaccharide, and lipopolysaccharide + amiloride groups were established. The expression of urokinase-type plasminogen activator receptor (uPAR) in podocytes was detected with a flow cytometer and cell motility was detected with the transwell migration assay[2].

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Podocyte culture and treatment: Podocytes were seeded in 6-well plates and cultured until confluence. The cells were then treated with Amiloride HCl dihydrate (MK 870) at concentrations of 1 μM, 10 μM, and 100 μM for 24 hours. LPS (1 μg/mL) was added to the culture medium 6 hours before the end of treatment to induce inflammation[2]
- Protein expression detection: Total protein was extracted from treated podocytes, separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies against podocin, nephrin, and β-actin (internal control), followed by secondary antibodies. The protein bands were visualized using chemiluminescence, and the density of the bands was quantified[2]
- mRNA expression detection: Total RNA was extracted from podocytes, reverse-transcribed into cDNA, and subjected to quantitative real-time PCR (qPCR) using specific primers for podocin, nephrin, and GAPDH (internal control). The relative mRNA expression levels were calculated using the 2^(-ΔΔCt) method[2]
- Cytokine detection: The concentration of TNF-α and IL-6 in the cell culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA) according to standard procedures[2]

1 mg/kg/day; subcutaneous
Rats Epithelial sodium channels (ENaCs) are strongly expressed in the circumventricular organs (CVOs), and these structures may play an important role in sensing plasma sodium levels. Here, the potent ENaC blocker amiloride was injected intraperitoneally in rats and 2h later, the c-Fos activation pattern in the CVOs was studied. Amiloride elicited dose-related activation in the area postrema (AP) but only ~10% of the rats showed c-Fos activity in the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO). Tyrosine hydroxylase-immunoreactive (catecholamine) AP neurons were activated, but tryptophan hydroxylase-immunoreactive (serotonin) neurons were unaffected. The AP projects to FoxP2-expressing neurons in the dorsolateral pons which include the pre-locus coeruleus nucleus and external lateral part of the parabrachial nucleus; both cell groups were c-Fos activated following systemic injections of amiloride. In contrast, another AP projection target--the aldosterone-sensitive neurons of the nucleus tractus solitarius which express the enzyme 11-β-hydroxysteriod dehydrogenase type 2 (HSD2) were not activated. As shown here, plasma concentrations of amiloride used in these experiments were near or below the IC50 level for ENaCs. Amiloride did not induce changes in blood pressure, heart rate, or regional vascular resistance, so sensory feedback from the cardiovascular system was probably not a causal factor for the c-Fos activity seen in the CVOs. In summary, amiloride may have a dual effect on sodium homeostasis causing a loss of sodium via the kidney and inhibiting sodium appetite by activating the central satiety pathway arising from the AP.[1]

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In the in vivo test, the urine protein volume of the model was detected at 24 h using Coomassie brilliant blue staining and the morphological changes of the podocytes were detected with immunofluorescence. The protein expression rate of uPAR in the lipopolysaccharide group was significantly higher than those in the control and lipopolysaccharide + amiloride groups (P < 0.05). The viability of cells in the lipopolysaccharide group was significantly higher than those in the control and lipopolysaccharide + amiloride groups (P < 0.05). Compared with the urine protein level in the control group at 24 h, the level in the lipopolysaccharide group increased significantly (P < 0.05), whereas compared with the urine protein level in the lipopolysaccharide group, the level in the lipopolysaccharide + amiloride group decreased (P < 0.05). uPAR expression was significantly downregulated, and the fusion of the podocyte-specific skelemin synaptopodin on the glomerulus podocytes was significantly decreased in the lipopolysaccharide + amiloride group. These results suggest that amiloride is able to reduce cell motility and thus lower proteinuria by inhibiting the expression of uPAR in podocytes.[2]

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Adult male Sprague-Dawley rats (250-300 g) were housed under standard laboratory conditions with free access to food and water. After acclimatization for 1 week, rats were randomly divided into control group and Amiloride HCl dihydrate (MK 870) treatment group. The treatment group received a single intraperitoneal injection of the drug at a dose of 10 mg/kg, while the control group received an equal volume of normal saline[1]
- Two hours after administration, rats were anesthetized with pentobarbital sodium and perfused transcardially with 4% paraformaldehyde. The brain was removed and post-fixed in 4% paraformaldehyde for 24 hours, then dehydrated and embedded in paraffin. Serial coronal sections (5 μm) containing the area postrema were cut and subjected to immunohistochemical staining for c-Fos[1]

[1]. Blockade of ENaCs by amiloride induces c-Fos activation of the area postrema. Brain Res. 2015 Mar 19;1601:40-51.

[2]. Amiloride, a urokinase-type plasminogen activator receptor (uTPA) inhibitor, reduces proteinurea in podocytes. Genet Mol Res. 2015 Aug 14;14(3):9518-29.

[3]. A polycystin-2 (TRPP2) dimerization domain essential for the function of heteromeric polycystin complexes. EMBO J. 2010 Apr 7;29(7):1176-91.

Amiloride hydrochloride dihydrate is the dihydrate of amiloride hydrochloride. It is a diuretic and sodium channel blocker. It contains amiloride hydrochloride. Amiloride hydrochloride is the hydrochloride salt of amiloride, a synthetic pyrazine derivative with both antidiuretic and diuretic effects. Amiloride inhibits sodium channels located in the distal convoluted tubule and collecting duct of the kidney, thereby preventing sodium absorption and increasing sodium and water excretion, producing a diuretic effect. In cases of renal hypernatremia, the plasma membrane becomes hyperpolarized, reducing electrochemical activity and preventing the excretion of potassium and hydrogen into the lumen. A pyrazine compound inhibits sodium reabsorption by inhibiting sodium channels in renal epithelial cells. This inhibition generates a negative potential on the principal cell membrane of the distal convoluted tubule and collecting duct. This negative potential reduces the secretion of potassium and hydrogen ions. Amiloride is used in combination with diuretics to reduce potassium loss. (Excerpted from Gilman et al., Goodman and Gilman Foundations of Pharmacology, 9th ed., p. 705)

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Amiloride hydrochloride dihydrate (MK 870) is a well-known ENaC blocker. This study shows that its blocking effect on ENaC in the posterior pole can induce c-Fos activation, suggesting that it may play a role in regulating osmotic balance and related physiological processes [1]
- This study is the first to discover that amiloride hydrochloride dihydrate (MK 870) is a uTPA inhibitor. It can protect podocyte function and reduce inflammatory response, which provides a potential treatment strategy for treating kidney diseases associated with proteinuria [2]

C6H8CLN7O.HCL.2H2O

302.12

301.045

C, 23.85; H, 4.34; Cl, 23.47; N, 32.45; O, 15.89

17440-83-4

Amiloride hydrochloride;2016-88-8;Amiloride;2609-46-3

68540

Typically exists as Off-white to yellow solids at room temperature

628.1ºC at 760 mmHg

>240℃

333.7ºC

1.08E-15mmHg at 25°C

1.944

7

7

1

18

279

0

ClC1C(N([H])[H])=NC(=C(C(/N=C(\N([H])[H])/N([H])[H])=O)N=1)N([H])[H].Cl[H].O([H])[H].O([H])[H]

LTKVFMLMEYCWMK-UHFFFAOYSA-N

1S/C6H8ClN7O.ClH.2H2O/c7-2-4(9)13-3(8)1(12-2)5(15)14-6(10)11;;;/h(H4,8,9,13)(H4,10,11,14,15);1H;2*1H2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.88 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)