Diabetic nephropathy (DN) is a prevalent complication of diabetes and a significant contributor to end-stage renal failure.1 The incidence of DN ranges from 30% to 40% and has been progressively increasing in recent years.2 3 Currently, renin-angiotensin system inhibitors are commonly employed for treatment. However, there is an occurrence of aldosterone escape during the course of treatment, which hinders complete inhibition of the renin-angiotensin system.4 Antidiabetic drugs such as glucagon-like peptide-1 receptor agonists are also frequently used but exhibit certain side effects, with gastrointestinal reactions being the most prevalent.5 Hence, there exists an urgent necessity to identify a more efficacious treatment approach. Proteinuria serves as an early clinical manifestation of DN primarily caused by podocyte apoptosis.6 7 Consequently, an escalating number of studies are concentrating on ameliorating kidney damage associated with DN through inhibition of podocyte apoptosis.

Low-intensity pulsed ultrasound (LIPUS) is a form of physical therapy primarily recognized for its non-thermal effects, which are advantageous for tissue healing. Extensive research has demonstrated its efficacy in promoting bone healing,8 treating soft tissue injuries,9 and restoring ovarian function in cases of failure.10 11 The mechanism by which LIPUS alleviates premature ovarian failure is through the inhibition of apoptosis in ovarian granulosa cells. Furthermore, LIPUS has been found to inhibit apoptosis in human osteoarthritis cells,12 reduce apoptosis in periodontal ligament cells,13 and decrease apoptosis in renal tubular cells during acute kidney injury.14 Therefore, it is evident that LIPUS possesses the capability to effectively inhibit apoptosis. Importantly, podocyte apoptosis plays a significant role in the progression of DN, and inhibiting podocyte apoptosis can alleviate DN.15 16 Hence, we believe that LIPUS may offer a novel treatment option for DN and play a positive role in its management.

The process of apoptosis is primarily associated with the calcium ion (Ca2+). Research has demonstrated that ultrasound can activate calcium mechanosensitive channels, and Ca2+ exhibits a high responsiveness to pulse length (PL).17–20 PL refers to the duration of a pulse signal, which is measured in milliseconds (ms) as the time difference between its initiation and termination. In Liao et al’s study,20 significant intracellular Ca2+ changes were observed in Piezo 1 within the range of 10 ms to 100 ms, with peak values reached at a PL of 20 ms. However, exceeding a PL of 20 ms may elevate the risk of cell detachment and membrane perforation. Our previous studies have indicated that LIPUS with a PL of 0.2 ms effectively inhibits apoptosis.10 These findings suggest that PL plays an essential role in LIPUS treatment since different PLs yield varying effects. Therefore, it is crucial to compare different PLs (0.2 ms, 10 ms, and 20 ms) to determine the optimal PL for maximizing the effectiveness of LIPUS treatment.

In conclusion, the innovation of this study lies in proposing a non-invasive LIPUS therapy for direct renal irradiation in DN. The objective is to validate the role of LIPUS in inhibiting podocyte apoptosis and improving DN, while also investigating the optimal PL to maximize the efficacy of LIPUS.

The objective of this study was to investigate the reparative effects of LIPUS with different PLs on renal structure and function in DN. The findings indicated that LIPUS effectively enhanced the structural and functional performance of injured kidneys by mitigating lipid disorders. Moreover, it was observed that the most pronounced reparative effect on DN occurred when the PL was set at 0.2 ms.

The main parameters of ultrasonic research include Isata, which is the parameter used for alleviating acute kidney injury at a power density of 100 mW/cm2,14 promoting skeletal muscle regeneration at a power density of 60 mW/cm2,23 and targeted gene transfer at a power density of 30 mW/cm2.24 In our previous studies, we have determined that the appropriate irradiation parameter for most cells is 30 mW/cm2.25 26 Another crucial parameter in ultrasound research is PL, which has been extensively studied.17 20 27 28 Abnormal elevation of intracellular Ca2+ can result in cell apoptosis.29 30 Moreover, LIPUS has been found to inhibit cell apoptosis,10 11 and there is evidence suggesting that PL plays a significant role in regulating Ca2+ sensitivity,17–20 making it an essential screening parameter for inhibiting cell apoptosis in podocytes.

The findings of this study demonstrate that LIPUS with three different PLs can effectively suppress podocyte apoptosis and reduce renal injury. Compared with the three PLs, the results of um-ALB, PCX, BUN and UA showed that the optimal effect was achieved when PLs were 0.2 ms. Pathological examination showed that LIPUS could effectively repair the renal structure, and 0.2 ms was optimal in three PLs. Based on the above results, the effect of 0.2 ms among the three PLs is preferred, so 0.2 ms is selected for further investigation. However, a study by Yoo et al17 indicated that the calcium response reached its peak at 500 ms. Additionally, research conducted by Liao et al20 suggested that the optimal PL is 20 ms. These inconsistent results may be attributed to the varying effects of different PLs on mechanically sensitive ion channels,19 as well as the use of different experimental models requiring distinct PLs. Therefore, future research should explore more precise parameters for different tissues and cells to achieve accurate treatment with LIPUS.

There is an elevated concentration of UA in DN rats. However, the findings of this study indicate a significant decrease in UA concentration (p<0.05, figure 1E). It is speculated that this may be attributed to functional injury of the renal tubule, leading to reduced reabsorption or increased secretion of UA.31 Notably, previous research has demonstrated that LIPUS can inhibit apoptosis of renal tubule epithelial cells14 and improve damage to renal tubules, which aligns with our results (figure 2C). Consequently, following LIPUS irradiation, the level of UA in DN rats was observed to increase when compared with the DN group.

The metabonomic results demonstrate that LIPUS primarily affects the metabolic pathways related to alpha-linolenic acid and linoleic acid metabolism, along with beta-oxidation of very long chain fatty acids during renal injury repair. These findings imply that the mechanism underlying LIPUS-mediated relief from DN involves the modulation of lipolysis. Prior research has shown a high susceptibility of podocytes to free fatty acids (FFA), leading to podocyte apoptosis.32 33 Our study reveals ultrasound irradiation’s ability to modulate lipolysis and diminish FFA production, thus mitigating podocyte apoptosis. This observation is further supported by WB results indicating reduced expression levels of crucial lipolysis enzymes HSL and ATGL after exposure to LIPUS irradiation (p<0.05, figure 3E).

Su et al observed an increase in HSL and ATGL levels following treatment, resulting in the alleviation of DN. However, our findings demonstrate a divergent outcome, potentially attributed to variations in the lipid metabolism capacities of the DN models employed. In their study, diminished lipolysis ability within the DN model led to excessive lipid deposition and exacerbated kidney damage. Conversely, our experiment revealed abnormally elevated lipolysis within the DN model, leading to excessive production of FFA, podocyte apoptosis, and aggravated kidney injury. Consequently, LIPUS modulates lipolysis and ameliorates DN by restoring lipid metabolic equilibrium and reducing renal damage.

In the in vitro experiment, it was observed that LIPUS decreased intracellular Ca2+ levels and inhibited podocyte apoptosis. This contradicts the findings of Yoo et al,17 which demonstrated an increase in intracellular Ca2+ levels following LIPUS exposure. The disparity may be attributed to compromised cell membranes in apoptotic cells, resulting in aberrant Ca2+ influx and apoptosis. Subsequent to LIPUS irradiation, the damaged cell membrane structure was restored, leading to normal Ca2+ flow and a reduction in cellular apoptosis rates.

The inhibitory effect of LIPUS with different PLs on podocyte apoptosis was found to be significant. The lowest rate of apoptosis (16.7±1.7%) was observed at a PL of 0.2 ms, compared with the STZ group (52.9±7.2%) (p<0.05, figure 4C). Furthermore, intracellular Ca2+ FI was significantly lower at a PL of 0.2 ms (3891.6±486.7) compared with the STZ group (7169±301.0) (p<0 .05, figure 5A). These findings suggest that the impact of different PLs on apoptosis is associated with Ca2+, which aligns with previous studies demonstrating that elevated levels of Ca2+ induce apoptosis.29 30 Studies have shown that Bcl-2 regulates calcium uptake to prevent excessive levels.34 WB results confirmed that LIPUS reduces Bax expression and increases Bcl-2 expression (p<0.05, figure 5C), leading to decreased Ca2+ concentration and inhibition of apoptosis.

Studies have shown that increasing Ca2+ concentration can promote lipolysis.35 In this study, there was an excessive increase in lipolysis in DN rats, which needed to be restored to normal levels. And excessive Ca2+ flow, will lead to podocyte apoptosis aggravate DN.36 37 However, LIPUS is closely related to changes in Ca2+ concentration, and different parameters have different effects.38 39 After LIPUS irradiation, the decreased Ca2+ concentration returned to normal levels, and the abnormally increased lipolysis also returned to normal levels. It is consistent with the study of Zhang35 et al. Therefore, we believe that the possible mechanism is that LIPUS improves DN by regulating excessive Ca2+ flow and ameliorating lipid metabolism disorder.

There are certain limitations associated with this study. First, although the expected energy reaching the kidney was estimated to be 4710 J, an accurate measurement of the actual energy reaching the kidney was not conducted, highlighting the need for a more efficient method of precise measurement. Second, this study did not include a follow-up observation of DN rats after treatment to evaluate the duration of LIPUS effects. Third, the relationship between the dose of LIPUS and the concentration of calcium and the degree of recovery of DN has not been deeply studied. Therefore, further investigation is warranted.

In conclusion, we propose a novel non-invasive LIPUS method for the treatment of DN. This study validates the efficacy of LIPUS in repairing renal structure and function, as well as inhibiting podocyte apoptosis. All three types of PLs demonstrate the ability to repair renal structure and function, with optimal effectiveness observed at 0.2 ms. The mechanism by which LIPUS alleviates DN involves ameliorating lipid metabolism disorder, suggesting its potential as an innovative tool for DN.