Introduction

Erectile dysfunction (ED) is the impotence to get and keep an erection firm enough for sex. It has a high incidence worldwide, with over 50% of men aged 40–70 years diagnosed with ED particularly1,2. ED is an early warning signal of cardiovascular disease3, which seriously affects patients’ quality of life and causes infertility. At present, oral phosphodiesterase type 5 inhibitors (PDE5I) have become the first choice for treating ED, but some patients, especially those with diabetes mellitus erectile dysfunction (DMED), do not respond well to PDE5 inhibitors4,5,6,7. Studies published in JAMA showed that using PDE5 inhibitors increases the risk of melanoma8,9. Therefore, elucidating the exact etiology of ED and identifying modifiable risk factors are critical steps toward developing targeted therapeutic strategies for patients refractory to existing treatments.

Uric acid (UA) is the end-product of purine catabolism mediated by Xanthine Oxidoreductase (XOR) in humans. When UA crystal deposits to a certain extent, it will result in the known pathologic process of gout. Overproduction or excretion failure of UA causes hyperuricemia (HUA), where serum uric UA measures up to 420 μmol/L. Hyperuricemia is another crucial metabolic disorder, besides diabetes, hypertension, and hyperlipidemia, closely associated with the mobility and mortality of patients with cardiovascular diseases. Recent retrospective studies have shown that the incidence of ED increased in gouty patients10. Another study showed that HUA is a risk factor for ED in the elderly, accompanied by diabetes. However, it is unclear whether ED is directly induced by hyperuricemia.

We know that cavernous corpus smooth muscle cells (CCSMC) are fundamental units for penis corpus cavernosal relaxation and contraction. ED occurs when CCSMC contracts continuously or fails to relax normally1. Decreased nitric oxide (NO) levels11,12 and activated MLCK-MLC2 pathway13 are vital for smooth muscle relaxation and contraction dysfunction. Moreover, many studies found that excess UA crystals in the blood vessels of gout patients often reduce eNOS activation in endothelial cells and the proliferation of vascular smooth muscle cells to promote vascular diseases like atherosclerosis14,15,16.

Here, we determined that hyperuricemia caused erectile dysfunction in the early stage without NO change, and UA interacted with MLCK, leading to increased MLCK levels and activity accompanied by contraction of CCSMC in vivo and in vitro. Besides, UA-lowering drugs and MLCK inhibitors prevented hyperuricemia-induced ED. This study reveals the key molecules and mechanisms of hyperuricemia-induced ED, which provides evidence for hyperuricemia or gout patients to control UA levels and prevent ED.

Results

Hyperuricemia increases the incidence of ED by three folds in young patients

To investigate the potential association between hyperuricemia and erectile dysfunction, we first collected clinical retrospective data on patients (20–49 years old) at the physical examination center of SYSU third hospital (Study I), which included patients with both non-ED (Control) and ED diagnosed by the IIEF5 questionnaire (Fig. S1A). Considering selection bias and uncertain ED diagnostic criteria, we retrospectively collected clinical data on non-ED (Control) or ED patients diagnosed with the chief complaint and divided ED patients into two subgroups that were ED with hyperuricemia (HUAED) or not (ED w/o HUA) at andrology department of SYSU first hospital (Study II) (Fig. S1B). We found that serum uric acid (UA) levels increased significantly among young ED populations in Study I (455.8 μM) and Study II (416.1 μM) (Fig. 1A, B and Table S1–S2). IIEF5 scores decreased further in HUAED patients (Fig. 1C and Table S3). Notably, an increase of 100 μM in serum UA level was associated with an over 2.5-fold increase in the risk of ED (Odds Ratios (OR) = 2.522 and 2.951) (Fig. 1D). Besides, serum UA level was uncorrelated with common risk factors for ED (Table S4), which revealed that hyperuricemia was a dominantly independent risk factor for ED.

Fig. 1: Serum UA level was correlated with erectile dysfunction.
figure 1

A, B Dot plot of UA levels from ED (n = 33) and control group (n = 43) in study I, and from ED (n = 94) and control group (n = 67) in study II. C Dot plot of IIEF5 scores from ED patients without hyperuricemia (ED w/o HUA, n = 53) and with hyperuricemia (HUAED, n = 41) in study II. The two-tailed unpaired t-test was used in (A–C). (D) Table showing the odd ratio (OR), 95%CI, and P values of serum UA to ED in studies I and II analyzed by the Logistic regression. E Dot plot of IIEF5 scores in non-hyperuricemia (Control, n = 21), mild (n = 20), moderate (n = 23), and severe (n = 20) hyperuricemia group in Study III analyzed by one-way ANOVA and Dunnett-t-test. F Table showing IIEF5 scores and incidence of ED (ED%) analyzed by Chi-square test. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01. Bars correspond to Mean ± SD.

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Furthermore, male patients (20–49 years old) were recruited from the metabolism department of Shanghai 10th Hospital (Study III) and divided into non (Control), mild, moderate, and severe hyperuricemia diagnosed by serum UA level (Fig. S1C). Then, erectile function was evaluated cross-sectionally through an online IIEF5 questionnaire. We did not observe differences in ED common risk factors among the four groups (Table S5). However, IIEF5 scores reduced gradually as the growing severity of HUA (Fig. 1E), and the incidence of ED increased significantly by approximately three folds (Fig. 1F). These results suggest that hyperuricemia might directly induce ED in young patients.

High uric acid impairs the erectile function of rats directly at an early stage

Next, we used the Urate oxidase (Uox) gene knockout rats as a spontaneous hyperuricemia animal model (Fig. 2A). Heterozygous rats’ sequencing showed overlapping peaks. Homozygous rats had no CTTCG bases around 210 bp without overlapping peaks in sequencing compared to wild-type (WT) rats (Fig. S2). Serum UA level increased significantly by about 3.5 times and maintained at a high level in homozygous rats compared to WT rats (Fig. 2B). Next, we marked homozygous rats as Uox-KO rats in context. Then, we assessed intracavernous pressure (ICP) to imply penis erectile function at 16, 20, 30, and 43 weeks (Fig. 2A). We found that ICP diminished and exasperated worse in Uox-KO rats from 20 weeks (Fig. 2C–F). As well as the ratios of maximal ICP to mean arterial pressure (Max ICP/MAP) and area under the curve (AUC) of ICP were significantly lower progressively in Uox-KO rats from 20 weeks (Fig. 2G–H). Previous reports showed that obesity, androgen deficiency, hypertension, hyperlipidemia, diabetes, etc., were known ED risk factors17. There was no difference in the above risk factors in Uox-KO rats from 16 to 30 weeks (Fig. S2). These results confirmed that high UA independently induced erectile dysfunction in the early stage.

Fig. 2: Erection was impaired in Uox-KO rats from 20 weeks.
figure 2

A Schematic diagram of hyperuricemia model construction using Uox-KO SD rats and the timepoint of the erection function test. Wk weeks. B The line chart of serum UA levels of Uox-KO and WT rats at 16, 20, 30, and 43 weeks was analyzed by the two-tailed unpaired t-test at each timepoint. n = 5 rats per group. Mean ± SD. C–F Representative images of intracavernous pressure (ICP) at 16, 20, 30, and 43 weeks Uox-KO and WT rats. G, H Dot plots of Max ICP/MAP and AUC at 16, 20, 30, and 43 weeks Uox-KO (n = 5) and WT (n = 6) rats were analyzed by the two-tailed unpaired t-test at each time point. Bars correspond to Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Panel a images created in BioRender; Wencan, C. (2025), https://BioRender.com/6xs09se.

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High uric acid-induced CCSMC contraction leads to erectile dysfunction

In Study II, color Doppler duplex ultrasonography (CDDU) showed that flow-mediated vasodilation values (FMD) were unchanged in HUAED patients. Still, the peak systolic velocity (PSV) of the left and right penile cavernosal arteries (PCA) was reduced (Table S3). Furthermore, in penile cavernosal arteries of Uox-KO rats, the lumen diameter narrowed significantly, but wall thickness did not alter during the initial 20 weeks (Fig. 3A–D). Besides, no apparent structural damage during the early 20 weeks, such as muscle fiber loss, although Masson staining revealed structural damage at 43 weeks, where muscle fiber lost (Fig. S3). These results provided clues for further study of the high UA-induced contraction.

Fig. 3: HUA enhanced corpus cavernosum smooth muscle contractility.
figure 3

A Representative Masson staining images of the cavernous artery. Scale bars, 100 μm. B–D The histogram of vessel wall thickness (mm), lumen diameter (mm), and the ratio of wall thickness to lumen diameter (%). n = 3 corpus cavernosum sections from rats per group. E Schematic diagram of cell contraction assay proceeding from cell mixture to generate the polymerization gel, stressing, treatment, and release. F Cell contraction assay showing CCSMC contraction induced by high uric acid (450 μM) or control (extra 0 μM uric acid) with or without SNP (0.1 mM). The digital gel size change (contraction index) images were taken at 0 and 3 days. G The histogram of the gel area ratio of day 3 to day 0. H Representative image of CCSMC stained by Phalloidin-FITC (Green). Scale bars: 25 μm. I The histogram of relative cell surface area (%). n = 3 independent biological replicates in F-I. Bars correspond to Mean ± SD. *P < 0.05, ***P < 0.001 analyzed by the two-tailed unpaired t-test.

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The corpus cavernosum comprises cavernous endothelial cells, fibroblasts, and corpus cavernosum smooth muscle cells (CCSMC), which account for 38.5-52.0% of cavernosal cells and function as the effector of relaxation and contraction18. In vitro, we first cultured and identified the primary CCSMC from rats (Fig. S4). Then, the cell contraction assay was constructed (Fig. 3E) where sodium nitroprusside (SNP), as a nitric oxide donor, could not wholly reverse the contraction enhanced by HUA (Fig. 3F, G) in CCSMC-mixed gel. Moreover, CCSMC emerged with contractionary morphology stained by phalloidin-FITC in the HUA condition (Fig. 3H-I). All these above suggest that HUA-induced ED resulted from increased CCMSC contraction.

MLCK increased by intracellular UA contributes to CCSMC contraction

Cellular transport proteins are essential for the absorption and excretion of UA, including urate anion transporter 1 (URAT1) and glucose transporter 9 (GLUT9). Variations of URAT1 and GLUT9 are currently considered the primary genetic risk factors of hyperuricemia19. URAT1 and GLUT9 were expressed in rats’ corpus cavernosum, and the expression of URAT1 was upregulated in vivo and in vitro (Fig. S5A-D). Verinurad (RDEA3170, 3170) is the most promising drug for inhibiting URAT1, and it is currently in Phase II clinical trials to treat gout and asymptomatic hyperuricemia20. We found that 3170 suppressed intracellular UA levels (Fig. S5E, F), intimating that it might be intracellular UA that enhanced the contraction of CCSMC.

Erectile dysfunction occurs when the CCSMC enhanced contraction or weakened relaxation becomes disabled. MLC2 phosphorylation by MLCK and MLC2 dephosphorylation by myosin light chain Phosphatase (MLCP) are critical and recognized mechanisms of smooth muscle relaxation and contraction13,21,22. This process is mediated by nitric oxide (NO), which involves endothelial NO synthase (eNOS), neuronal NO synthase (nNOS)14,15,16, and Xanthine Oxidoreductase (XOR) activity23,24,25 (Fig. 4A). Firstly, we found that serum nitrite/nitrate levels did not alter yet in HUAED patients compared with ED w/o HUA patients in Study II (Table S3), as well as the serum and intracavernous nitrite/nitrate levels in Uox-KO rats did not differ from that in WT rats before 20 weeks (Fig. 4B, C). Moreover, we found that p-eNOS/eNOS expression was reduced. While XOR and p-nNOS/nNOS were reinforced in the corpus cavernosum of 20-week-old HUA rats (Fig. 4D), explaining why plasma nitrite/nitrate levels are unchanged at this time-point.

Fig. 4: MLCK mediated the contraction induced by HUA in CCSMC.
figure 4

A Schematic diagram of CCSMC contraction and relaxation involving MLCK/MLCP-MLC2 pathway, intracellular calcium, and nitrite/nitrate levels regulated by nNOS/eNOS/XOR. B Statistical graphs of serum and intracavernous nitrite/nitrate levels of WT and Uox-KO rats. n = 5 per group. C The dotplot of serum and intracavernous nitrite/nitrate levels of WT (n = 6) and Uox-KO (n = 5) rats. D Representative WB images of p-eNOS, eNOS, p-nNOS, nNOS, and XOR protein levels. E p-MYPT1 and MYPT1. F Representative WB images of p-MLC2, MLC2, MLCK protein levels from corpus cavernosum tissues. n = 6 rats per group. Protein lysates from two rats were pooled randomly to generate each biological replicate (n = 3 pooled samples per group). G, H Representative IF images of p-MLC2 (Red), and MLCK (Green) levels in cavernous tissues. Cell nuclei were stained with DAPI (Blue). Scale bars, 100 μm. n = 3 corpus cavernosum sections from rats per group. I, J Representative WB images and the histogram of MLCK, p-MLC2, and MLC2 protein levels in CCSMC induced by HUA (450 μM) with 0, 0.5, or 5 μM ML-7 for 24 h. n = 3 independent biological replicates. Bars correspond to Mean ± SD. *P < 0.05 was analyzed by the two-tailed unpaired t-test.

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Besides, myosin phosphatase target subunit 1 (MYPT1), a subunit of MLCP, in turn inhibits MLCP activity26. We found that MLCK expression and phosphorylated MLC2 increased, while MYPT1 expression and phosphorylated MYPT1 were constant (Fig.4E, F) in the corpus cavernosum of Uox-KO rats from 20 weeks detected by Western blot and immunofluorescence (Fig. 4G, H). We carried out an experiment to address the correlation between elevated uric acid concentration and alterations in key indicators. We established a concentration gradient of uric acid levels in the cell model by adding 0, 100, 200, 300, 400, and 500 μM of uric acid to 10% FBS DMEM to culture primary CCSMC from rats for 48 h (Fig. S6A). We found that MLCK protein levels and MLC2 phosphorylation in CCSMC increased via uric acid concentration dependent. Furthermore, it has been reported that serum MLCK increased in type 2 diabetes patients44, and we found serum MLCK levels increased significantly among HUA patients and HUAED patients, as well as in Uox-KO rats (Fig. S6B, C). ML-7 is a specific cell membrane-permeable MLCK inhibitor that interacts directly with the ATP binding site of the MLCK active center27. In vitro, MLCK expression and phosphorylated MLC2 down-regulated significantly when we utilized ML-7 in CCSMC (Fig. 4I, J). Meanwhile, HUA-induced CCSMC contraction was reversed by ML-7 in CCSMC (Fig. S6D–G).

These results suggested that the increase of contraction of CCSMC caused by HUA is achieved by increasing MLCK instead of NO or MYPT1.

UA interacting with MLCK diminishes its degradation

Previous reports defined that MLCK activity depended on intracellular Ca2+ concentration, changing protein structure to expose the ATP binding site28. However, we found it hard to trigger more calcium influx by HUA in CCSMC (Fig. S7A), which suggests that MLCK may be allosterically regulated by UA. Besides, MLCK mRNA levels did not fluctuate significantly when exposed to HUA (Fig. S7B). Whether it is as an allosteric regulator of MLCK or a small molecule affecting MLCK levels, UA is very likely to interact with MLCK. Cellular Thermal Shift Assay (CTSA) was constructed (Fig. 5A), one typical assay to verify the direct binding of two molecules. It showed that HUA increased the MLCK melting point (~52 °C) compared to the control group ( ~ 49 °C) by about 3 °C (Fig. 5B, C), indicating that UA interacts with MLCK.

Fig. 5: UA interacting with MLCK diminished its degradation.
figure 5

A Schematic diagram of cellular thermal shift assay (CTSA) in CCSMC pre-treated by HUA, then equiparting and heating with 3 °C gradient temperature from 37 to 64 °C. The soluble MLCK levels in the supernatant were detected by WB. B Representative WB images of remaining MLCK levels after heating in CCSMC supernatant detected by CTSA. C Protein quantification of the melting point where 50% MLCK remained from (B). D, E Uric acid structure chart and the schematic diagram of the amino acid from MLCK for UA-interacting. F Representative WB images showing remaining MLCK after heating in the supernatant of ARPE19 cells transfected with plasmid overexpressing WT MLCK, N803A/K799A mutated MLCK (MLCK-WT, N803A, and K799A) for 48 h. Heating with 3 °C gradient temperature from 37 to 58 °C among the range in (B). G Protein quantification of the melting point where 50% MLCK remained in (F). H Representative WB images showing remaining MLCK after heating in the supernatant of CCSMC transfected with plasmid overexpressing control, WT MLCK, N803A/K799A mutated MLCK (Vector, MLCK-WT, N803A, K799A) for 48 h under HUA condition or not. Heating with 3°C gradient temperature from 40 to 55 °C among the range in (B). I Protein quantification of the melting point where 50% MLCK remained in (H). J Representative WB images of MLCK protein levels collected at 0, 4, 8, or 12 h after CHX or CHX + MG132 treatment in CCMC under HUA condition or not. K The line chart of the relative MLCK protein levels from (J). (L) Table showing the P values of (J) in different groups and hours analyzed by the two-tailed unpaired t-test. CHX = 50 μM. MG132 = 50 μM. n = 3 independent biological replicates. HUA means 10% DMEM with an extra 450 μM uric acid for 48 h after 4 h starvation.

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In silico (Fig. 5D, E), we found that lysine (K799) and leucine (L797) residues of smooth muscle MLCK interacted with the 2th carbonyl group at 2 sites of UA; as well as asparagine (N803) and serine (S767) residues bound to carbonyl group at 6 sites in UA. Thus, we selectively focused on K799 and N803 from each carbonyl group to construct mutant MLCK plasmids (MLCK-K799A and MLCK-N803A). Then, we highly efficiently transfected CCSMC with WT and mutant plasmids (Fig. S7C-D). We found that the N803A and K799A single-site mutation did not alter the melting curves of MLCK protein without high uric acid treatment (Fig. 5F, G). Furtherly, after high uric acid treatment for 48 h, the melting curve of MLCK-N803A was different from MLCK-WT and MLCK-K799 (Fig. 5H, I). As a result, the N803 mutation (N803A), but not the K799 mutation (K799A), curbed MLCK interacting with UA and abated the melting point validly (Fig. 5F–I). Furthermore, Surface Plasmon Resonance (SPR) was used to measure the equilibrium dissociation constant (KD) to show the affinity between UA and MLCK WT protein (KD = 257 μM) and N803A mutational protein (KD = 312 μM) (Fig. S7E). These data showed that N803 of MLCK might be the critical binding site for UA.

NEDD4L is the E3 ligase responsible for MLCK ubiquitination and degradation

The previous report determined that MLCK degradation depends on ubiquitin-proteasome systems29. Consistently, we found that MG132 alleviated MLCK degradation significantly, and high UA could further weaken its degradation (Fig. 5J–L). Furthermore, it showed that high UA blocked ubiquitin interacting with MLCK (Fig. 6A) detected by co-immunoprecipitation (co-IP). As UA could interact with MLCK at the N803 site, MLCK WT and N803A plasmid were used to assess the role of N803 in regulating MLCK levels. As shown (Fig. 6B, C), high UA increased MLCK levels when CCSMCs were transfected with MLCK-WT plasmid, while failing to prevent MLCK degradation in the MLCK-N803A group, which could not provide an interacting site for UA. Meanwhile, HUA-induced contractionary morphology extended validly when CCSMCs were transfected with MLCK-N803A, which were stained by phalloidin-FITC in the HUA condition (Fig. S7F, G).

Fig. 6: NEDD4L is the E3 ligase responsible for MLCK ubiquitination and degradation.
figure 6

A MLCK ubiquitination in CCSMC regulated by HUA using Co-IP under MG132 treatment for 12 h. B Representative WB images of MLCK protein levels in CCSMC transfected with MLCK-WT or N803A plasmid for 48 h under HUA conditions or not. C The dot plot of MLCK protein levels in CCSMC analyzed by the two-tailed unpaired t-test from (M) Bars corresponds to Mean ± SD. *P < 0.05, **P < 0.01. n = 3 independent biological replicates per group. D MLCK ubiquitination in ARPE19 transfected with MLCK-WT or N803A plasmid for 48 h under HUA condition or not using Co-IP under MG132 treatment for 12 h. E Network view for retrieved E3 ligases for the query protein: MYLK (species: H.sapiens) using an in-silico ubiquitination prediction tool, UbiBrowser. F Representative WB images of NEDD4L protein levels from CCSMC treated with HUA or not. n = 3 independent biological replicates per group. G Representative WB images of NEDD4L and MLCK protein levels in CCSMC treated with HUA (or control) transfected with NEDD4L plasmid (or negative control plasmid) for 48 h. H The interaction between endogenous MLCK and NEDD4L in CCSMC for 48 h under MG132 treatment for 12 h by Co-IP. I The interaction between MLCK, NEDD4L and ubiquitin in ARPE19 with Flag-NEDD4L and either wild-type (WT) MLCK or the N803A mutant for 48 h under MG132 treatment for 12 h byCo-IP. HUA means 10% DMEM with an extra 450 μM uric acid for 48 h after 4 h starvation. MG132, 50 μM.

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In addition, we cultured ARPE19, one retinal cell line with MLCK in lower levels (proteomicsdb.org), to be transfected with MLCK WT and N803A plasmids. We found no difference in ubiquitylation levels between MLCK-WT and N803A transfecting (lanes 1 and 2 in Fig. 6D) detected by co-IP. High UA inhibited MLCK ubiquitylation levels when overexpressing MLCK-WT while it failed to stop MLCK ubiquitylation because UA lost the interacting site from MLCK N803A (lanes 3 and 4 in Fig. 6D). All these data determined that directly targeting MLCK by UA inhibited its ubiquitylation and subsequently increased its amount and activity.

We identified NEDD4L as a potential E3 ligase interacting with MLCK using an in-newsilico ubiquitination prediction tool, UbiBrowser (Fig. 6E). NEDD4L, a HECT-domain E3 ligase, has recently emerged as a key regulator of vascular dysfunction30,31. In hyperuricemic CCSMCs, we observed a significant reduction in NEDD4L protein levels (Fig. 6F). Strikingly, overexpression of NEDD4L in hyperuricemic CCSMCs restored MLCK protein levels (Fig. 6G), suggesting that NEDD4L negatively regulates MLCK stability under hyperuricemic conditions.

Then, co-IP assays in CCSMCs confirmed a direct physical interaction between endogenous MLCK and NEDD4L (Fig. 6H). To further dissect the role of the N803 residue, we transfected low-MLCK-expressing ARPE19 cells with Flag-NEDD4L and either MLCK WT or the N803A mutant. Co-IP revealed that the N803A mutation abolished the MLCK-NEDD4L interaction (Fig. 6I). Importantly, ubiquitination assays showed that the N803A mutation significantly reduced polyubiquitination levels of MLCK (Fig. 6I), indicating that the N803 residue is critical for NEDD4L-mediated ubiquitination.

These findings strongly support NEDD4L as the E3 ligase responsible for MLCK ubiquitination and degradation in hyperuricemia. The N803 residue appears to serve as a key structural determinant for NEDD4L binding and subsequent ubiquitination.

FBST, BZMR, 3170, and ML-7 improves HUA-induced erectile function

The above results indicate that high UA causes erectile dysfunction and the underlying mechanism. Therefore, we attempted to use UA-lowering therapy and key molecular inhibitors to clarify the improvement effect of these drugs on erectile dysfunction.

We intragastrically administrated Uox-KO rats with Febuxostat (FBST) and Benzbromarone (BZMR) in 0.5% CMC-Na and intracavernously injected Uox-KO rats with 3170 and ML-7 in 0.1% DMSO from 16 weeks to 20 weeks (Fig. 7A). As shown, the erectile function was improved distinctly by these interventions (Fig. 7B–D). Serum UA levels decreased after FBST, BZMR, and 3170 treatments, while ML-7 did not lower serum UA levels (Fig. 7E). In addition, MLCK expression and activity decreased uniformly in these intervention groups detected by Western blot (Fig. 7F–I) and immunofluorescence (Fig. 7J). Meanwhile, we initially and briefly monitored the safety of these interventions, including body weight, blood routine, and liver and kidney function (Table S6). Furthermore, we found that the decreased MLCK ubiquitylation in the corpus cavernosum of HUA rats up-regulated by FBST for UA lowering therapy detected by Co-IP (Fig. S7H).

Fig. 7: FBST, BZMR, 3170, and ML-7 improved erectile dysfunction.
figure 7

A Schematic diagram of rats administering febuxostat (n = 5), benzbromarone (n = 5), URAT1 inhibitor 3170 (n = 5), and MLCK inhibitor ML-7 (n = 5) compare to Uox-KO (n = 6) and WT rats (n = 6) from 16 to 20 weeks. Blood was collected at 16 and 20 weeks. Erection function tested at 20 weeks. ML-7, 10 μM. 3170, 1 μM. B Representative images of ICP from rats after the intervention. C, D Dot plots of Max ICP/MAP and AUC in different groups. A two-tailed unpaired t-test was used. *P < 0.05, **P < 0.01, administration vs. Uox-KO + CON; ###P < 0.001, Uox-KO + CON vs.WT + CON. E The statistical graph of serum UA levels at baseline of 16 weeks (pale blue) and after treatment of 20 weeks (light pink). The oblique lines match the same mouse at baseline and after treatment. *P < 0.05, **P < 0.01 analyzed by the two-tailed paired t-test. F–I Representative WB images of MLCK and p-MLC2 protein levels in penis tissues of Uox-KO + CON and Uox-KO+administrations. Protein lysates from two rats were pooled to generate each biological replicate (n = 3 pooled samples per group). J Representative IF images of MLCK (Green) and p-MLC2 (Red) protein levels in penis tissues of Uox-KO + CON and Uox-KO+administrations. Cell nuclei were stained with DAPI (Blue). Scale bars, 100 μm.

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These results suggest that these interventions are potential new strategies for treating erectile dysfunction induced by high UA.

Discussion

Erectile dysfunction is associated with metabolic diseases, including diabetes mellitus, atherosclerosis, hypertension, dyslipidemia, and obesity32,33. A recent retrospective study showed an increased incidence of ED in gout patients compared with controls10, but it failed to link hyperuricemia to ED directly because these gout patients were older and accompanied with other metabolic disorders, like diabetes. Herein, younger and non-diabetic participants were enrolled to demonstrate that hyperuricemia was an independent risk factor for erectile dysfunction (Fig. 1). Our clinical data highlight that hyperuricemia correlated with ED directly because the risk factors mentioned above were excluded strictly. Most importantly, we used a stable spontaneous hyperuricemia animal model to determine that erection function was damaged significantly by elevated UA at the early stage (Fig. 2), when there was no abnormal glucose metabolism, lipid metabolism, body weight, and hormones (Fig. S2). Besides, considering sample selection bias, not only andrology outpatients but also participants from Physical Examination Center were enrolled in this study. Due to the lack of a gold diagnosis standard, we adopted both the IIEF5 questionnaire and chief complaint as diagnostic criteria for ED.

Thus, we speculate that UA-lowering therapy can improve hyperuricemia-induced erectile dysfunction. Allopurinol is the first choice for UA-lowering therapy, but patients with a positive HLA-B*5801 gene will develop lethal exfoliative dermatitis after taking the drug. The positive rate of the HLA-B*5801 gene in the Asian population is as high as 7.4%34, so we preferentially utilized other first-line UA-lowering drugs FBST and BZMR. We found that FBST and BZMR improved erectile dysfunction in hyperuricemia rats (Fig. 7). These data indicate that early UA-lowering therapy may effectively ameliorate erectile function in hyperuricemia patients.

During the initial 20 weeks, only changes in lumen diameter were observed, with no thickening of the wall (Fig. 3A–D), and no apparent structural damage, such as muscle fiber loss (Fig. S3). Masson staining revealed structural damage at 43 weeks, with loss of muscle fiber (Fig. S7). Early intervention with uric acid before 20 weeks notably improved erectile function in rats (Fig. 7C, D). Whether intervention starting at 43 weeks can ameliorate structural damage and improve ED needs to be demonstrated in the future through additional groups and experiments. Previous studies recommended that hyperglycemia should be controlled before structural changes occur in the ED and diabetes duration since diagnosis increased an increasingly greater risk of ED (RR = 1.7, 95% CI 1.1–2.7, for men diagnosed >20 years previously, trend test P < 0.0001)35,36,37. Therefore, we hypothesize that late-stage treatments may not be as effective as early uric acid intervention once structural damage occurs, even if uric acid levels are controlled.

Cavernous smooth muscle cells (CCSMC) are fundamental units for corpus cavernosal relaxation and contraction, and ED occurs when CCSMC contract continuously or fails to relax normally1. We have shown that penile cavernosal arteries were significantly narrowed down in young patients and 20-week-old HUA rats. Furthermore, high UA treatment triggered CCSMC contraction in vitro (Fig. 3). Additional thoracic aorta rings experiments from rats were conducted and indicated that vascular rings from hyperuricemic rats exhibit a significantly greater contraction amplitude and heightened sensitivity to Phenylephrine (Fig. S3), providing complementary evidence of systemic vascular hyperreactivity. Therefore, we confirmed that the erectile dysfunction induced by HUA at the early stage resulted from HUA enhancing the contractility of CCSMC.

Genome-wide association studies have demonstrated that gene mutations such as URAT1 and GLUT9 can affect the risk of hyperuricemia19. New studies have shown that vascular smooth muscle cells express URAT1 and GLUT938, and URAT1 is responsible for transporting UA into cells, but the role of GLUT9 is uncertain. Umbilical vein endothelial cells do not express URAT1 but express GLUT939, which plays a role in the absorption of UA. However, whether CCSMC expresses URAT1 and GLUT9 is unclear. Our study found that URAT1 and GLUT9 are expressed in the corpus cavernosum and increased URAT1 uptake of more UA into CCSMC cells in vitro and in vivo (Fig. S5). Previous studies showed that UA crystals inhibited URAT1 expression through upregulating alpha kinase 140 and prevented URAT1 lysosome degradation induced by Numb41. These explained why URAT1 protein levels increased in hyperuricemia rats.

Previous studies have suggested that nitric oxide deficiency is the early stage of erection dysfunction, produced from endothelial cells mediated by p-eNOS/eNOS and cavernous nerves aroused by p-nNOS/nNOS14,15,16. However, we noticed no significant fluctuation in NO levels when the erection was impaired in younger ED patients (Table. S3) and at 20 weeks in Uox-KO rats (Fig. 4B, C). It is easy to overlook that xanthine oxidoreductase (XOR) promoted NO synthesis23,24,25, besides functioning as one crucial enzyme for UA production. We found that XOR and p-nNOS/nNOS were reinforced, although p-eNOS/eNOS was reduced in the corpus cavernosum (Fig. 4D). These data explained that hyperuricemia induced erectile dysfunction in another way at an early stage.

MLC phosphorylation mediated by MLCK or MYPT1 is one of the most critical steps in smooth muscle contraction42,43. It has been reported that the expression of MLCK increased in the aortic endothelial cells of STZ-induced diabetic rats44. We detected that UA activated MLCK, not MYPT1, to phosphorylate MLC during CCSMC contraction. However, how UA affects MLCK has not been reported. Regulation of enzymatic activity is a rapid regulation of the rate of enzymatic reactions, including allosteric regulation and chemical modification regulation of enzymes. After excluding the possibility that calcium influx (Fig. S7A) triggered the contraction of CCSMCs, we used CTSA, molecular docking, and SPR to clarify that UA can bind to the asparagine at the 803 site of MLCK (Fig. 5 and Fig. S7E), mediating the contraction of CCSMCs. During SPR, we calculated that KD values were 257 μM and 317 μM, exceeding the highest added UA concentration of 100 μM when UA bound to MLCK WT protein and N803A protein, respectively. Because UA is a weak diprotic organic acid with limited solubility in water, we dissolved UA powders into a sodium hydroxide solution, referred to the instructions from Sigma (#U0881). However, considering the pH and solvent interference, more precise and accurate KD values need to be optimized further for the proposal for SPR.

Herein, we used MLCK inhibitor ML-7 in vivo, and we found that ML-7 improved erectile dysfunction in hyperuricemia (Fig. 6). Multiple studies have shown that elevated MLCK can mediate the dysregulation of tight junctions and barrier loss in colonic mucosal epithelial cells, resulting in colitis45. Moreover, vascular lumen stenosis43, increased blood pressure46, gastrointestinal motility disorders47, lung injury, and asthma48 are all associated with increased MLCK. Therefore, therapies targeting MLCK have broad application prospects. We also initially and briefly monitored the drugs and inhibitors’ safety (Table S6), including body weight, blood routine, liver, and kidney function tests, which did not show pathological changes. Besides, it has been reported that serum MLCK increased in type 2 diabetes patients49, and we found serum MLCK levels increased significantly among HUA patients and HUAED patients, as well as in Uox-KO rats (Fig. S6C, D), which suggested that serum MLCK levels might be a potential biomarker for diagnosing hyperuricemia-induced erectile dysfunction. In our hyperuricemic rat model, ML-7 did not significantly alter mean arterial pressure (MAP) (Fig. S8). For ED patients with concurrent hyperuricemia and hypertension—a subgroup often refractory to PDE5 inhibitors—ML-7’s ability to ameliorate HUA-driven cavernosal hypercontractility without exacerbating hypotension may offer a clinically advantageous niche. While our data suggest minimal systemic effects, we acknowledge that the localized administration route (intracavernosal injection) used here may limit extrapolation to systemic delivery. These results offer some preclinical evidence for hyperuricemia in preventing ED, but it deserves more data to prove.

In summary, our studies uncovered hyperuricemia-induced erectile dysfunction directly. Mechanistically, UA interacts with asparagine residues at the MLCK 803 site, where intracellular UA inhibits MLCK ubiquitylation degradation and promotes its activity to induce the contraction in CCSMC. This study provides evidence for hyperuricemia or gout patients to control UA levels and prevent ED, in addition to inhibition of MLCK and URAT1.

Methods

Study design

The overall objective of this study was to investigate the causative role of high uric acid in erectile dysfunction in patients and rat models by targeting MLCK, as well as to determine whether UA-lowering therapy of MLCK inhibitor could improve erectile function in rats. To address this, clinical information and serum of ED patients and healthy participants were collected from three different centers; High uric acid rat model (Uox-KO) was established alongside with wild-type rats as a control group; Pharmacologic inhibition of MLCK with ML-7 was also conducted in these rat model; Primary CCSMC was isolated and cultured for in vitro mechanistic study. Each rat group consisted of a minimum of five rats, and cell experiments were repeated three times. Sample sizes were determined based on our previous experiences and informed by similar non-randomized experiments, because the experimental groups were typically separated by genotypes. However, when a single genotype was divided into multiple groups, then randomization was used for allocation. Typically, investigators during data collection and analysis were not blinded to group allocation. In rat-based experiments, genotyping was identified before group allocation to ensure sufficient group size, resulting in investigators being unblinded. For cell-based experiments, the same investigators performed group allocation, data collection, and analysis thus, it is impractical to make blinding. Both sampling and experimental replicates were at least three times biological and specified in each figure legend. There were no exclusions to report.

Participations

We collected information and serum from three centers: (1) Study I: Males aged 24–49 years old who underwent physical examination were included, completed the IIEF-5 questionnaire after their routine physical examinations under the guidance from physicians, and were grouped by their IIEF-5 scores. (2) Study II: Participants aged 24–49 years from the Andrology Department were included and grouped by their presence or absence of ED chief complaints (3) Study III: Male participants from the Metabolism Department aged 24–49 years were included and grouped by their serum UA levels. Patients with cardiovascular disease, diabetes, hypertension, obesity, hormone imbalance, nerve injury, and penile sclerosis were excluded. All participants signed informed consent, and the experimental ethics committee approved the study protocol of Zhongshan School of Medicine, Sun Yat-sen University (ZSSOM-Medical Ethics-2020-037). All ethical regulations relevant to human research participants were followed.

IIEF5 questionnaire

Rosen designed IIEF5 to quickly assess the presence and severity of erectile dysfunction50. It is often used for screening and diagnosis in the ED. The maximum score is 25. A score ≥ 22 is considered normal, 17–21 is mild, 8–16 is moderate, and ≤ 7 implies a severe situation. The questionnaire was conducted one-on-one among participants by a certain researcher for minimizing potential confounders.

Animals

Urate oxidase (Uox) knockout rats were used as hyperuricemia models gifted from Prof. Haibing Chen. CRISPR/Cas9 technique was used to knock out 5 bp of CTTCG in the Uox sequence of Wistar rats51. All experiments have been reviewed and approved by the Ethics Committee of Laboratory Animals of Sun Yat-sen University (SYSU-IACUC-2020-B0041). All animals were raised in an SPF environment and used in accordance with the guidelines of Sun Yat-sen University. We have complied with all relevant ethical regulations for animal use.

Erection function evaluation

For hypothesis-testing studies, function evaluation is the primary outcome measure. Rats were anesthetized by intraperitoneal injection of 30 mg/kg of pentobarbital sodium. The BL-420A/F biometric signal acquisition and analysis system (Techman Software) recorded the carotid pressure to represent the intracavernous pressure (ICP) and mean arterial pressure (MAP) and released a bipolar electrode to stimulate the erection (constant voltage 5 V, frequency 20 Hz, stimulation wave width 0.2 ms) for 60 s each time.

Arterial tension measurement

After rats were euthanatized by intraperitoneal injection of 100 mg/kg of pentobarbital sodium, the thoracic aorta was excised and immediately placed in a physiological saline solution (PSS) equilibrated with 95% O2 and 5% CO2. The aorta was then sectioned into cylindrical rings (2–3 mm in width) after connective tissue and adventitia were carefully removed. The aortic rings were subsequently mounted in a 4-chamber myograph system (DMT 620 M, Aarhus, Denmark) for isometric tension recording. Each ring was suspended between two stainless-steel hooks within an organ bath containing PSS, maintaining a temperature of 37 °C. The chambers were continuously aerated with a gas mixture of 95% O2 and 5% CO2 to mimic physiological conditions. After a balance of 15 min, optimal resting tension (5mN) was applied gradually for over 60 min, with buffer replacement every 15 min. Vessels were depolarized and activated by 100 mM KCl in PSS. Subsequently, the rings were stimulated to induce the contraction using phenylephrine (PE, 10−9 to 10−4 M). After achieving a stable contraction for an additional 30 min, concentration-response curves for acetylcholine (ACh, 10−6 to 10−5 M) were obtained. The tension (mN) is continuously recorded and analyzed using the data acquisition and analysis software (LabChart, ADInstruments).

Drugs and inhibitors

According to the Reagan-Shaw dose conversion formula [human equivalent dose (mg/kg) = rat dose (mg/kg) × (6/37)]52, the dose of benzbromarone and febuxostat was 1 mg/mL intragastric administration, with the control group was given a 0.5% CMC-Na solution. According to the manufacturer instructions, 10 μM ML-7 (#S8388, Selleck) and 50 μM 3170 (#S8736, Selleck) in 100 μL 0.1% DMSO solution, respectively, were injected into the cavernous tissue with an equal volume of 0.1% DMSO as control. Uox-KO rats of the same age were put together in one huge cage gently and then allocated to control and treatment groups randomly. All the rats were administrated once every two days.

Glucose/insulin tolerance test (GTT/ITT)

GTT: The rats were weighed and starved 16 h in advance. The end of the tail was cut off, and the fasting blood glucose was measured after wiping the first drop of blood with a cotton ball. Then the 20% glucose (#G7021, Sigma) working solution was intraperitoneally injected at the ratio of 2 g/kg body weight. Then, blood glucose levels were monitored after 15 min, 30 min, 45 min, 60 min, 90 min, and 120 min, respectively. ITT: The rats were weighed and starved 4 h in advance. The tail end of the rat was cut off, and the first drop of blood was wiped with a cotton ball before the fasting blood glucose was detected. The insulin (Novo Nordisk, 100 U/mL) working solution should be prepared for immediate use and kept away from light. The rats were injected intraperitoneally with insulin at the ratio of 0.75U/kg body weight. Blood glucose levels were monitored at 15 min, 30 min, 45 min, 60 min, 90 min and 120 min, respectively.

Cell culture

Primary cavernous smooth muscle cells were extracted and cultured from 7-week-old wild-type male Sprague-Dawley rats according to the previous description53,54. In detail, the entire penis was dissected under aseptic conditions. After several washing times with PBS, the mucosa tissue, urethra tissue, and glans head were removed in a medium dish. After rinsing with PBS several times, the corpus cavernosum tissue was placed in a new sterile container and cut into 1–2 mm pieces. The bottom of the flask was moistened in advance with FBS, and the tissue pieces were attached to the bottom with tweezers apart by 0.5 cm to 1 cm. About 2 mL DMEM was added to the culture bottle, and then the bottle was placed with the bottom side facing up in the incubator. After 2–4 h, the bottle was turned back to face up. After 3–5 days, cells appeared around the tissue blocks, which could be overgrown in about 15 days. During this period, DMEM was added when the culture medium turned yellow. After overgrowth, cells were digested with 0.25% Trypsin-EDTA (Gibco) and cultured in DMEM with 10% FBS and 1% penicillin-streptomycin solution. Besides, a human retinal pigment epithelial cell line (ARPE19, #CRL-2302, ATCC) was used as the low-MLCK-expressing cell model. Cells were digested with 0.25% Trypsin-EDTA (Gibco) and cultured in DMEM with 10% FBS and 1% penicillin-streptomycin solution.

Plasmids and recombined proteins

The overexpression plasmid pCDNA3.1-MYLK was constructed by cloning a 2994 bp fragment from MYLK cDNA (XM_017006469.2 → XP_016861958.1, myosin light chain kinase, smooth muscle isoform X7, 992aa) into the EcoRI and XhoI sites of control plasmid pcDNA3.1. Plasmids were constructed by Hanyi Biotechnology Co., LTD (#B4547-2, MLCK-WT; #B4624-2, MLCK-N803A; # K37188, MLCK-K799). The recombined WT MLCK protein was purchased from Abcam (#ab55674), and N803A MLCK protein was constructed by Genscript Biotechnology Co., LTD (#SC1810). The pcNDA3.1-Flag-NEDD4L plasmid was kindly provided by JY Zhang and K Li’s laboratory.

Transfection

According to the manufacturer’s instructions, the transfection of plasmids was performed at about 70% confluency of cells using Advanced DNA/RNA Transfection Reagent (#AD6000150, Zeta Life). In detail, directly mix at a ratio of 1 μg DNA with 1 μL transfection reagent or 1 μL RNA (20 μM) with 1 μL transfection reagent. Homogenize the mixture through 15 cycles of vigorous pipetting, followed by incubation at room temperature for 15 min to allow complex formation. Add the resulting complexes dropwise to the cell culture medium and distribute them by gentle swirling. After 24 h of standard incubation, aspirate the medium and replace it with fresh, pre-warmed culture medium.

Histological and immunofluorescence (IF) analyses

After being harvested, each rat corpus cavernosum tissue was equally divided into three aliquots. The middle ones were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 3–4 μm sections with sequence number. Sections of the same number were selected for histological staining. Vessel wall thickness and luminal diameter by Masson’s trichrome staining were quantified using ImageJ with a calibrated scale bar. Measurements were averaged across 3 non-consecutive sections per animal to ensure representativeness. Besides, after dewaxing, hydration, and quenching, sections were incubated with goat serum at room temperature for 1 h. Then, the penis tissue slices and cell slides were incubated with primary antibody at 4 °C for 16 h and then with secondary antibody at 37 °C for 2 h, protected from light. Finally, slices were incubated with 0.5ug/mL DAPI at room temperature for 10 min. Olympus BX63 microscope was used to observe and photograph. The antibodies and reagents used are shown in Table S7-S8.

Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was prepared using the RNA Purification Kit (#B0004D, EZBioscience). Quantification and integrity analysis of total RNA were performed by running 2 μL of each sample on a Nanodrop ultraviolet spectrophotometer. The cDNA was prepared by reverse transcription using the Evo M-MLV RT Premix (#AG11706, Accurate Biology).RT-qPCR was performed using the NovoStart® Universal Fast SYBR qPCR SuperMix (#E401, Novoprotein Scientific Inc.) and Bio-Rad CFX 96 fluorescent quantitative PCR instrument. Primers are described in Table S9.

Western blot (WB)

Before performing WB, an equal weight of tissues was lysed by the cell lysates with protease and phosphatase inhibitors on the ice. Then, we homogenized, sonicated, and centrifuged at 12,000 r/min for 30 min at 4°. The supernatant was extracted for protein quantification and WB. Due to limited protein yield from corpus cavernosum tissues, samples from two rats were pooled at equal concentrations for immunoblotting. This approach is consistent with prior studies using low-abundance tissues55. Equal proteins were separated by SDS–PAGE and transferred to PVDF membranes. Membranes were incubated overnight at 4 °C with diluted primary antibodies for 16 h and incubated at 4 °C with diluted second antibodies for 4 h. The Western blot was finally exposed using ECL substrate (#310212, Zeta Life) and imaged with a Bio-Rad imaging system. The antibodies used are listed in Table S7.

Immunoprecipitation

The cells were lysed with 500 μL of weak RIPA lysate overnight on a 4 °C shaker. Then, the lysate was centrifuged at 12,000 rpm at 4 °C for 20 min, and the supernatant was taken for protein quantification. After taking 500 μg protein into the new centrifuge tube, add the corresponding IP antibody at 1:100 and incubate on a 4 °C shaker overnight. Protein A/G PLUS Agarose was washed with weak RIPA lysate three times and added to the antibody-incubated samples overnight, and incubated again. On the second day, the mixture was centrifuged at 2000 RPM at 4 °C for 5 min, and the supernatant was removed. After washing with 300 μL weak RIPA lysate 3 times, the residue was lysed by SDS and detected by WB. The antibodies and reagents used are shown in Table S7-S8.

Nitrite/nitrate levels assay

The nitric oxide (NO) end products nitrite/nitrate in the serum of participants/rats and corpus cavernosum of rats were determined by the nitrite/nitrate levels assay kit (#S0021, Beyotime). Firstly, centrifuge human or rat fresh blood at 1500–2000 × g for 10 min and collect the serum. Collect the rat corpus cavernosum tissue supernatant after homogenizing the tissue in ice-cold PBS and centrifuging at 12,000 × g for 15 min at 4 °C. Then, prepare the standard curve (0–100 μM). Add 50 μL of standards or samples, 50 μL Griess Reagent I, and 50 μL Griess Reagent II immediately to each well. Mix gently and incubate away from light at RT for 15 min. Finally, read absorbance at 540 nm and calculate nitrite/nitrate concentrations from the standard curve.

MLCK levels detection

The MLCK levels in the serum of participants and rats were determined using the human and rat MLCK ELISA kits (#HM11395 and #RA20766, Bioswamp). Firstly, collect the serum of the participants and rats. Then, add 50 μL of standards (in duplicate) or 40 μL of serum and 10 μL of biotinylated antibody, and 50 μL of HRP-conjugate to each well (except the blank) step by step. Incubate for 30 min at 37 °C and wash 5 times with 1× wash buffer (300 μL/well) between each step. Add 50 μL Chromogen A/B, incubate for 10 min in the dark, and stop with 50 μL stop solution. Finally, read OD at 450 nm within 15 min (blank well as zero) and calculate the MLCK levels according to the standard curve.

Cell contraction assay

According to previous studies and kit instructions (#CBA-201, Cell Biolabs)56,57. In detail, primary CCSMCs at the same cell passage number, less than four, were collected to make cell suspension at the concentration of 2–5 × 106 cells/mL, and the suspension was mixed with collagen gel in a ratio of 1:4. Next, 0.5 mL of cell collagen mixture was placed in each well of the 24-well plate and incubated at 37 °C for 1 h. After collagen polycondensation, place 1 mL of medium in each well and gently separate the collagen block from the edge of the dish with the sterile blunt tablet. The cells were cultured continuously in the dish with DMEM (10%FBS) with or without high uric acid (450 μm) for 48 h and photographed at different times.

Molecular docking

LigandScout software simulated the binding site between uric acid (PubChem CID: 1175) and MLCK protein (Uniport: Q15746). By optimizing the position and conformation, the biangular surface of the rotatable bond, the side chain of the amino acid residues, and the skeleton of the target protein58,59,60, the conformation with the lowest energy was found when MLCK bound to uric acid in its active region.

Cellular thermal shift assay (CTSA)

The method works by heating cells to different temperatures to denature and precipitate proteins. After repeated freezing and thawing, the cells were lysed. After the cell lysates were centrifuged to precipitate, the soluble proteins in the supernatant were quantified by Western Blot or mass spectrometry. According to the corresponding relationship between protein content and temperature, the temperature corresponding to 50% protein content can be obtained, that is, the melting point. CTSA is a biophysical detection method based on the principle of ligand-induced thermal stability of target protein, which means that the melting point of the protein will change with the interaction of ligand, i.e., the direct binding of the small molecule to protein will affect the melting point of the protein61,62,63,64.

Surface plasmon resonance (SPR)

Following the manufacturer’s instructions, Biacore T100 and CM7 sensor chips were used. Data were analyzed by Biacore T100 Evaluation software (GE Healthcare). All assays were performed at 25 °C. In brief, the CM7 sensor chip was activated, and MLCK WT or N803A protein was immobilized on the chip’s surface. Next, the chip was blocked and equilibrated with PBS. Next, the uric acid stock solution (0.1 mM) was diluted in PBS to a series of concentrations (50, 25, 12.5, and 6.25 μM). Then, the uric acid solution flowed at 30 μl min–1 for 150 s each run. Finally, the Steady model calculated the dissociation constants (KD).

Live-cell imaging

1×105 cells/Well were inoculated on 24-well plates. After synchronous treatment the next day, an automatic high-throughput live cell imaging analysis system (BioTek-Lionheart FX) was used to take pictures every 30 min for 48 h to observe the cell contraction.

Statistics and reproducibility

SPSS24 software and GraphPad Prism 8 were used for statistical analysis and graphing. Continuous quantitative data conforming to normal distribution were represented by mean ± standard deviation (Mean ± SD). The two-tailed unpaired and paired t-test were used to compare the differences between the two groups under appropriate circumstances. One-way ANOVA and Dunnett’s multiple comparisons test were used for the different comparisons of multiple groups of continuous variables. The Chi-square test was applied to compare event rates. Risk factors for the disease were investigated using Logistic regression analysis to calculate OR and 95% CI. P-value < 0.05 was considered statistically significant. Sample sizes (n), defined as biologically independent units (e.g., individual participants or animals) are stated for each experiment in figure legends. Biological replicates (n) represent independently processed biological samples under identical conditions. All experiments were independently repeated at least three times with consistent results and are stated in figure legends.