Introduction

Hyperhomocysteinemia (HHcy) is defined as a homocysteine (Hcy) level above 10 μmol/l in human blood [1]. Hypertension associated with HHcy has a high incidence in China, accounting for approximately three-quarters of patients with hypertension [2]. A study of 19 centers in nine European countries demonstrated that high Hcy levels and hypertension are synergistic factors that increase the risk of vascular disease [3]. Several recent studies have also found that the risk of stroke and death significantly increase when hypertension co-occurs with HHcy [4, 5]. However, little is known about the pathogenic role of HHcy in the progression of glomerular damage associated with hypertension. In a previous clinical study, we confirmed that plasma Hcy and the C677T polymorphism in the gene encoding its key metabolic enzyme, MTHFR, are independent risk factors for early renal damage in the hypertensive Chinese Han population [6]. A case–control study also indicated that HHcy may increase the susceptibility of subjects with essential hypertension to microalbuminuria [7]. However, the mechanisms of HHcy-associated hypertensive renal damage have not been fully elucidated.

Hypertension is an independent risk factor for glomerular injury. It is unclear whether HHcy increases renal damage by exacerbating hypertension or by other mechanisms independent of hypertension. Numerous studies have shown that HHcy plays a pathological role by enhancing oxidative stress in local tissues or cells. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) was the first enzyme found to produce reactive oxygen species. Nox is a multicomponent enzyme that includes seven subtypes: NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, and DUOX2 [8, 9]. NOX2 and NOX4 are widely expressed in glomerular cells (mesangial cells, podocytes), renal tubular cells, and renal interstitial cells [10, 11] and act as aerobic receptors. Recent studies have demonstrated that Nox is involved in progressive glomerular injury associated with HHcy [12, 13] and hypertension target organ damage [14]. However, it is unknown whether increased NOX2 and NOX4 expression in HHcy-associated hypertension is involved in podocyte injury and the associated pathological changes. Nephrin, a transmembrane protein located in the slit diaphragm, is an important structural molecule and signaling molecule of glomerular podocytes. It plays an important role in determining the shape and viability of podocytes and maintaining the integrity of the filtration function of the glomerulus [15, 16]. To confirm the effects of Hcy on podocyte injury in hypertension, we determined whether an increased plasma Hcy level induces the expression of Nox subunits and decreases the expression of nephrin, thereby affecting the survival of podocytes.

Currently, the safest and most effective means of reducing Hcy is folic acid (FA) supplementation, which can reduce Hcy levels in blood but also antagonize the toxic effects of Hcy [17, 18]. The China Stroke Primary Prevention Trial concluded that FA supplementation in hypertensive patients can slow chronic renal impairment [2]. Moreover, FA therapy may significantly reduce the mortality risk associated with heavy proteinuria among adults with hypertension [19]. However, the mechanism by which FA improves renal function in hypertensive patients associated with HHcy is unknown, and there is no consensus on the efficacy of FA at different doses [20,21,22]. Therefore, using an animal model of hypertension associated with HHcy, the present study investigated whether Hcy can induce downregulation of nephrin expression by enhancing oxidative stress, resulting in podocyte apoptosis and glomerular injury. The effects of high-dose (HFA) and low-dose FA (LFA) were also evaluated.

Materials and methods

Animals

Male spontaneously hypertensive rats (SHRs) (250–270 g, 12 weeks) were provided by the Beijing Vital River Laboratory Animal Center (Beijing, China). Four rats were housed per cage and given free access to food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee and carried out in full accordance with the institutional animal care and use protocols of Laboratory Animals of Shandong University.

The SHRs were randomized into a control group, HHcy group, HHcy + LFA group and HHcy + HFA group, with eight animals per group. All animals were given intraperitoneal injection for 12 weeks and gavage at the same time during the last 8 weeks of the experiment, and the SHRs were maintained as follows: (1) the control group was given physiological saline (PS, 5 ml/kg, twice a day) intraperitoneal injection for 12 weeks and PS (0.5 ml/d) by gavage for 8 weeks; (2) the HHcy group was given 2% DL-Hcy (5 ml/kg, twice a day, H4628, Sigma-Aldrich, St. Louis, USA) intraperitoneal injection for 12 weeks and PS (0.5 ml/d) by gavage for 8 weeks; (3) the HHcy + LFA group was given 2% DL-Hcy (5 ml/kg, twice a day) intraperitoneal injection for 12 weeks and FA (0.4 mg/kg/d, F7876, Sigma) by gavage for 8 weeks; and (4) the HHcy + HFA group was given 2% DL-Hcy (5 ml/kg, twice a day) intraperitoneal injection for 12 weeks and FA (4 mg/kg/d) by gavage for 8 weeks. FA was freshly dissolved in 0.5 ml of PS immediately before the experiment. Blood pressure (BP) and plasma Hcy levels were measured at weeks 4, 8, and 12. At week 12, the urine and vena cava blood of rats were collected for urine albumin, urine creatinine, plasma creatinine, serum malondialdehyde (MDA), and superoxide dismutase (SOD) detection, and renal tissues were collected for histopathological examination, immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and TUNEL detection.

Measurement of BP

The systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the tail artery were monitored in the morning with a noninvasive BP measurement system in the conscious state. To ensure the precision of the measurement, the temperature was controlled at 37–39 °C and BP was measured after the rats were quiet and the pulse was stable. The BP of each rat was measured three times and averaged.

Plasma Hcy and creatinine analysis

At weeks 4 and 8 of the experiment, rat tail blood was collected after centrifugation for the detection of plasma Hcy. At week 12 of the experiment, rats were fasted overnight and anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Blood was collected from the vena cava. The plasma Hcy and creatinine concentrations were detected using a Cobas8000 automatic biochemistry analyzer (Roche, Switzerland).

Serum oxidative stress biomarker analysis

Serum MDA and SOD levels were determined using commercial kits (Jiancheng Institute of Biological Technology, Nanjing, Jiangsu, China) according to the manufacturer’s instructions.

Urinary albumin measurement and glomerular filtration rate (GFR)

To collect urine samples, rats were placed in metabolic cages to collect urine for 24 h and record urine volume. Urinary albumin and urine creatinine were examined by using a Cobas8000 automatic biochemistry analyzer (Roche, Switzerland). To eliminate the impact of urine volume, urinary albumin was expressed as the urinary albumin creatinine ratio (UACR). The GFR was calculated by the following equation: GFR = (urine creatinine/plasma creatinine) × urine volume/body weight [23].

qRT-PCR

Total RNA was extracted from rat kidney tissue with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed to cDNA with a Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN, USA). Primer sequences (BioSune Biotechnology, Shanghai, China) are listed in Table 1. The mRNA levels were analyzed with an ABI ViiA 7 Real-Time PCR system (Applied Biosystems, Foster City, CA) using SYBR Green Real-Time PCR reagent (Roche, Indianapolis, IN, USA). Gene expression levels were normalized to GAPDH expression levels. The relative quantity of mRNA expression was calculated according to the cycle threshold (\(2^{-\Delta\Delta {\mathrm{Ct}}}\)) method.

Table 1 Primer sequences for quantitative real-time polymerase chain reaction (qRT-PCR) analysis
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Western blot analysis

The kidney cortical tissues were cut into pieces, and total protein was extracted using RIPA protein extraction solution. Homogenates were incubated on ice for 30 min and centrifuged at 12,000 × g for 10 min at 4 °C. Protein concentrations were measured using an enhanced BCA protein assay kit (Beyotime, Shanghai, China). First, 1/4 of the volume of loading buffer was added to the protein, and the samples were denatured in boiling water for 5 min and aliquoted and stored at −80 °C to avoid repeated freezing and thawing. The proteins were separated by electrophoresis using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 80 V for 30 min and 120 V for 60 min and transferred onto a polyvinylidene difluoride membrane at 280 mA for 75 min. The membranes were blocked with 5% skim milk at the room temperature for 2 h. Membranes were incubated overnight at 4 °C with primary antibodies against NOX2 and NOX4. The membranes were washed three times with Tris-buffered saline Tween for 10 min and incubated for 1 h at the room temperature with appropriate HRP-conjugated reporter antibodies. After three 10-min washes, a ChemiDoc™ Touch Gel imaging system (Bio-Rad, Hercules, CA, USA) was used to visualize immunoreactivity with a chemiluminescent HRP substrate (Vazyme, Nanjing, Jiangsu, China). The band intensities were determined using Image Lab software and expressed relative to GAPDH. All antibodies were purchased from Abcam (Cambridge, MA, USA).

Histopathological examination

Pieces of renal tissue were fixed overnight in 10% formalin solution, dehydrated in ethanol, embedded in paraffin, and then sectioned. The sections were stained with periodic acid-Schiff (PAS) for histological and morphometric analysis. Each sample slice was observed under a microscope (Olympus, Tokyo, Japan) at a magnification of ×400.

Structural glomerular injury was analyzed by the glomerular sclerosis index (GSI). The severity of sclerosis for each glomerulus was graded from 0 to 4+ based on the degree of glomerulosclerosis and mesangial matrix expansion. No lesion was graded as 0, sclerosis values of <25%, 25–50%, >50–75%, and >75% of the glomerulus were graded as 1, 2, 3, and 4, respectively. The GSI was obtained by averaging the scores from counted glomeruli [24].

Immunohistochemical analysis

Paraffin-embedded kidney sections were deparaffinized, hydrated in ethyl alcohol, blocked with blocking solution`for 15 min at the room temperature, and then incubated overnight at 4 °C with rabbit anti-nephrin (1:100; Abcam, Cambridge, MA, USA). Sections were incubated with HRP polymer anti-rabbit IgG at room temperature for 30 min. The slides were then examined under a light microscope at ×400 magnification. Brown areas were considered positive.

Transmission electron microscopy

Kidney tissue was cut (1 mm3) in electron microscope fixative solution, dehydrated in graded acetone and ethanol series, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate. The ultrastructure of podocytes was then observed by transmission electron microscopy.

TUNEL assay

TUNEL assays for renal cell apoptosis were performed on 0.5-μm sections of paraffin-embedded tissue using the Roche TUNEL kit. After conventional dewaxing and sealing treatment, the sections were incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and digoxigenin-11-dUTP for 1 h, rinsed in PBS three times, and incubated with ovalbumin-labeled horseradish peroxidase for 30 min. The sections were stained with DAB, and the nuclei were counterstained with hematoxylin buffer. The nuclei were stained brown in TUNEL-positive cells. Thirty glomerular sections were observed in each kidney, and the number of positive cells was calculated and averaged.

Statistical analysis

Statistical analysis was conducted using one-way ANOVA followed by the LSD test. The results are expressed as the means ± SE. All statistical analyses were performed using SPSS software version 20. P < 0.05 was considered statistically significant.

Results

Blood pressure

The diagnostic criteria for hypertension in rats are SBP ≥ 150 mmHg and/or DBP ≥ 100 mmHg [25]. As shown in Fig. 1a, b, SBP and DBP were measured at week 4, week 8, and week 12. There was no significant difference in BP among the groups during the treatment period. These results suggest that Hcy and FA had no effect on the BP of SHRs.

Fig. 1
figure 1

The levels of systolic blood pressure (SBP), diastolic blood pressure (DBP), plasma homocysteine (Hcy), serum superoxide dismutase (SOD) and serum malondialdehyde (MDA) in control, hyperhomocysteinemia (HHcy), HHcy + low-dose folic acid (LFA) and HHcy +  high-dose folic acid (HFA) rats. a The levels of SBP at week 4, week 8, and week 12, b the levels of DBP at week 4, week 8, and week 12, c the levels of Hcy at week 4, week 8, and week 12, d serum SOD concentrations, and e serum MDA concentrations. *P < 0.05 vs control, #P < 0.05 vs HHcy (n = 8)

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Plasma Hcy content

SHRs developed HHcy after intraperitoneal injection of DL-Hcy for 4 weeks. These rats had much higher levels of plasma Hcy (25.60 ± 0.70, 26.60 ± 0.77, and 25.50 ± 0.81 μmol/L in the HHcy, HHcy + LFA and HHcy + HFA groups, respectively) than the levels in the control group (7.33 ± 0.74 μmol/l). The HHcy, HHcy + LFA, and HHcy + HFA groups were then given intraperitoneal injections of DL-Hcy for 8 weeks, and different doses of FA were given to the HHcy + LFA group and HHcy + HFA group. Both HFA and LFA significantly reduced the plasma Hcy level (at the 8th week, 26.54 ± 0.69, 13.74 ± 0.83, and 12.51 ± 0.84 μmol/l in the HHcy, HHcy + LFA and HHcy + HFA groups; at the 12th week, 27.75 ± 0.59, 12.90 ± 0.93, and 10.93 ± 0.83 μmol/l in the HHcy, HHcy + LFA, and HHcy + HFA groups, respectively). However, there was no significant difference in the plasma Hcy level between the HHcy + LFA and HHcy + HFA groups (Fig. 1c).

Serum SOD and MDA

Figure 1d, e shows the effects of HHcy and FA on the levels of serum SOD and MDA in all groups. A significant decrease in the serum SOD level was observed in HHcy rats (361.44 ± 11.74 U/ml) compared with control rats (432.01 ± 7.55 U/ml), and both LFA (462.6 ± 8.32 U/ml) and HFA (479.04 ± 7.29 U/ml) counteracted the HHcy-induced reduction in SOD levels. A significant increase was also observed in the serum MDA level in the HHcy group (9.16 ± 0.40 nmol/ml) compared with the control group (7.66 ± 0.29 nmol/ml), and both LFA (6.24 ± 0.36 nmol/ml) and HFA (6.72 ± 0.37 nmol/ml) counteracted the HHcy-induced increase in the MDA level. However, the changes observed in the HHcy + HFA group were insignificant compared with the HHcy + LFA group (P > 0.05).

UACR and GFR

To assess the effects of HHcy and the protective action of FA on the kidneys, the UACR and GFR levels were measured. As shown in Fig. 2a, the UACR was higher in the HHcy group (343.50 ± 6.68 mg/g) than in the control group (263.89 ± 7.37 mg/g). Supplementation with FA significantly reduced the UACR compared with the HHcy group. However, HFA (206.21 ± 3.00 mg/g) did not reduce the UACR to a greater degree than LFA (214.65 ± 7.53 mg/g). Figure 2b shows that GFR significantly decreased in the HHcy group (6.04 ± 0.36 ml/g. day) compared with the control group (8.15 ± 0.43 ml/g. day), and GFR increased in both the HHcy + HFA (8.06 ± 0.44 ml/g. day) and HHcy + LFA (7.64 ± 0.43 ml/g. day) groups, but there was no significant difference between the two groups. These results suggested that LFA treatment significantly reduced HHcy-induced increases in UACR and increased the HHcy-induced decrease in GFR, indicating a protective function against HHcy-induced kidney injury.

Fig. 2
figure 2

Changes in renal function and morphology among control, HHcy, HHcy + LFA, and HHcy + HFA rats. a The levels of urinary albumin creatinine ratio (UACR), b the levels of the glomerular filtration rate (GFR), c the glomerular sclerosis index, *P < 0.05 vs control, #P < 0.05 vs HHcy (n = 8), and d representative images of periodic acid-Schiff (PAS) staining showing glomerular injury (original magnification, ×400)

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Histopathological examination

Figure 2c shows that GSI significantly increased in the HHcy group (1.75 ± 0.10) compared with the control group (1.06 ± 0.07), and GSI decreased in both the HHcy + HFA (1.16 ± 0.06) and HHcy + LFA (1.20 ± 0.07) groups, but there was no significant difference between high and low doses of FA. Representative images of PAS staining showing glomerular injury are shown in Fig. 2d. The glomerular capillary loops were wrinkled, and the glomerular balloon spaces widened in the control group (hypertensive). Compared with the control group, the glomerular capillary loops in the HHcy group were significantly wrinkled and thickened, and the glomerular extracellular matrix was increased. Notably, the mesangial matrix increases in glomeruli in LFA and HFA rats could be alleviated compared with HHcy rats.

Expression levels of NOX2 and NOX4 in renal tissues

To examine the expression of NOX2 and NOX4 in the renal cortex, qRT-PCR and western blot analysis were performed. qRT-PCR indicated increased mRNA levels of NOX2 and NOX4 in the glomeruli of SHRs with HHcy compared with control SHRs. Both LFA and HFA inhibited the HHcy-induced expression of NOX2 and NOX4 in glomeruli (Fig. 3a, b). Western blot analysis demonstrated that NOX2 and NOX4 protein levels were significantly higher in the HHcy group than in the control group and that this effect was reversed by treatment with both HFA and LFA (Fig. 3c, d). Furthermore, NOX2 expression was significantly lower in the HHcy + HFA group than in the HHcy + LFA group, although there was no significant difference in Hcy between the two groups (Fig. 3a, c). Oxidative stress parameters in blood and tissues suggested that LFA can alleviate the HHcy-induced oxidative stress response and that FA may have an antioxidant effect independent of Hcy.

Fig. 3
figure 3

The mRNA and protein expression levels of NOX2 and NOX4 in renal tissues. a Relative mRNA levels of NOX2 by quantitative real-time polymerase chain reaction (qRT-PCR) analysis in renal tissues among control, HHcy, HHcy + LFA and HHcy + HFA rats. b Relative mRNA levels of NOX4 by qRT-PCR analysis. c Western blot analyses of NOX2 protein levels in renal tissues, including representative blot images and a densitometric summary of the blot analysis after normalization to GAPDH. d Western blot analyses of NOX4 protein levels in renal tissues, including representative blot images and a densitometric summary of the blot analysis after normalization to GAPDH. *P < 0.05 vs control, #P < 0.05 vs HHcy, †P < 0.05 vs HHcy + LFA (n = 8)

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Expression levels of nephrin in renal tissues

qRT-PCR demonstrated that nephrin mRNA levels were significantly downregulated in SHRs with HHcy compared with control rats and that this effect was reversed by treatment with FA (Fig. 4a). However, there was no significant difference in nephrin mRNA levels between the HHcy + LFA and HHcy + HFA groups. Immunohistochemistry showed that nephrin protein expression in the kidneys was significantly lower in the HHcy group than in the control group. However, both LFA and HFA treatment improved nephrin protein expression (Fig. 4b).

Fig. 4
figure 4

The mRNA and protein expression levels of nephrin in renal tissues and the foot processes of podocytes under transmission electron microscopy (TEM) among control, HHcy, HHcy + LFA and HHcy + HFA rats. a Relative mRNA levels of nephrin by qRT-PCR analysis in renal tissues, *P < 0.05 vs control, #P < 0.05 vs HHcy (n = 8). b Representative immunostaining for nephrin in the glomeruli from different groups of rats (original magnification, ×400) and morphological changes in the foot processes of podocytes under TEM

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Changes in podocytes observed by TEM

TEM experiments were conducted to evaluate the ultrastructural changes in podocytes. As shown in Fig. 4b, the glomerular basement membrane of the control group (hypertensive) was thickened, and partial fusion of podocyte foot processes was observed. However, compared with the control group, the brush-like structure of the podocyte foot process disappeared and was widely fused in the HHcy group, while the ultrastructure of the podocyte was significantly improved in the HHcy + LFA and HHcy + HFA groups, and this effect was greater with HFA.

TUNEL assay

As shown in Fig. 5a, b, the number of TUNEL-positive cells in kidney tissue was much higher in the HHcy group (3.88 ± 0.17) than in the control group (2.09 ± 0.15). The number of TUNEL-positive podocytes was significantly decreased after LFA and HFA treatment (2.31 ± 0.18 in HHcy + LFA and 2.03 ± 0.16 in HHcy + HFA rats) (p < 0.05) compared with the HHcy group, and there was no significant difference between the FA doses. The TUNEL assay results suggest that HHcy increased podocyte apoptosis in SHRs and that LFA alleviated HHcy-induced apoptosis.

Fig. 5
figure 5

Podocyte apoptosis by TUNEL assay among control, HHcy, HHcy + LFA and HHcy + HFA rats. a Representative images of podocyte apoptosis in the glomeruli from different groups of rats (original magnification, ×400), where the nuclei are stained brown in TUNEL-positive cells. b The number of TUNEL-positive cells/glomeruli. *P < 0.05 vs control, #P < 0.05 vs HHcy (n = 8)

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Discussion

In the present study, we demonstrated that lowering the plasma Hcy level by LFA treatment attenuates HHcy-induced glomerular injury in SHRs. Consistent with a previous report, elevated plasma Hcy was associated with glomerular damage in Dahl salt-sensitive hypertensive rats [26]. In the present study, considering the high incidence of HHcy in China, SHRs were injected intraperitoneally with DL-Hcy for 12 weeks to create an animal model for investigating the mechanism of the effect of HHcy on renal damage in hypertension, which has high clinical relevance. Previous research showed that enalapril–FA therapy slows the progression of CKD in patients with hypertension with mild-to-moderate CKD to a greater degree than enalapril alone [27]. However, the FA dose used for HHcy intervention varies significantly across previous studies [20, 22, 28], and different doses of FA have produced inconsistent results. To clarify the effects of different doses of FA on Hcy and related renal damage, we established LFA and HFA groups. Our results showed that both doses significantly reduced the plasma Hcy level, with no significant difference between the two groups. The results of this study are consistent with the results of Tamadon et al. [29], which showed that LFA can effectively reduce plasma Hcy levels. In addition, our results showed that Hcy had no effect on the BP of SHRs, suggesting that Hcy-associated renal damage may be independent of BP. We therefore turned our attention to the mechanism underlying the effects of Hcy and FA on renal damage in hypertension.

Research has shown that HHcy is associated with changes in the structure and function of cerebral blood vessels, and oxidative stress appears to play a major role in mediating these changes [29]. Yi et al. [30] found that Hcy activates NADH/Nox via ceramide-stimulated Rac GTPase activity in rat mesangial cells. A previous study has shown that excessive activation of Nox is an important cause of excessive reactive oxygen species production in cells [31]. In the current study, HHcy induced a decrease in serum SOD and an increase in serum MDA levels, and these effects were counteracted by FA treatment. We also observed that NOX2 and NOX4 mRNA and protein levels in kidney tissue were significantly increased in the HHcy group compared with the control group, and this effect was also counteracted by FA treatment. The above results indicate that hypertensive rats with HHcy are in a state of oxidative stress and that LFA and HFA can ameliorate this condition by reducing Hcy. In addition, although there was no significant difference in the plasma Hcy level between the HHcy + HFA and HHcy + LFA groups, NOX2 expression was lower in the HHcy + HFA group. This suggested that FA may play an antioxidant role independent of Hcy by inhibiting NOX2 expression. FA has been reported to exert an independent antioxidant effect by inhibiting Nox-mediated superoxide anion production in human kidney proximal tubular cells [32], and this effect has also been demonstrated in the heart and liver.

UACR is an important warning sign of early renal damage, especially glomerular filtration barrier damage. We found that UACR significantly increased and that GFR significantly decreased in the HHcy group. Furthermore, compared with the control group, the glomerular capillary loops in the HHcy group were significantly wrinkled and thickened, the glomerular extracellular matrix and the GSI were increased, and the podocyte foot process was widely fused, which was consistent with functional alterations in the kidney. These changes in glomerular function and structure were associated with a significantly decreased level of nephrin, which is an important podocyte slit diaphragm molecule that plays a critical role in maintaining the glomerular filtration barrier [33, 34]. In SHRs supplemented with LFA and HFA, nephrin expression was increased, and glomerular structure and function were significantly improved. Compared with the HHcy + LFA group, the expression of nephrin in the HHcy + HFA group was slightly higher, and podocyte ultrastructure, UACR and GFR were slightly improved. This may have been related to the decrease in NOX2 and the independent antioxidant effect of FA, but there were no significant differences in the above parameters between the two groups. Recent research [35] has also demonstrated that HHcy decreases nephrin expression in gp91+/+ (NOX2) mice but not gp91−/− mice. Considering the relevant literature, the results of this study showed that Hcy increased oxidative stress and downregulated nephrin expression, aggravating the damage of podocytes and glomerular structure and renal function in hypertensive rats, and LFA improved renal damage by reducing Hcy and playing an antioxidant role.

Nephrin is not only a structural molecule of the slit diaphragm but also an important signal transduction molecule that affects podocyte survival [36]. Huber et al. found that phosphorylation of intracellular nephrin can initiate PI3K/AKT-dependent signal transduction in glomerular podocytes and inhibit purinomycin-induced podocyte apoptosis [37]. Our data also suggest that HHcy reduces the expression of nephrin in podocytes by enhancing oxidative stress, which leads to podocyte apoptosis, and that LFA may increase nephrin expression and inhibit podocyte apoptosis by reducing Hcy and exerting antioxidant effects. In addition, Huber et al. also found that tyrosine residue phosphorylation in the intracellular segment of nephrin can further activate the downstream p38MAPK signaling pathway and enhance the activity of the transcription factor AP-1 [38], which, as a key transcription factor regulating cell survival and death pathways, is involved in the regulation of cell apoptosis [39]. Therefore, HHcy reduces the expression of nephrin and induces apoptosis of podocytes, which may involve the PI3K/AKT and p38MAPK signaling pathways mediated by nephrin.

In conclusion, based on a hypertensive rat model with HHcy, the current study established that HHcy synergistically increased glomerular function and morphological damage in hypertensive rats. These pathogenic effects of Hcy were independent of BP and were related to increased oxidative stress and downregulated nephrin expression, ultimately leading to podocyte apoptosis. By reducing plasma Hcy levels and exerting antioxidant effects, LFA delayed the development of HHcy-induced glomerular damage associated with hypertension, indicating the potential efficacy of LFA in treating renal damage in hypertensive patients with HHcy. However, the signaling mechanism of HHcy-induced podocyte injury remains to be further studied.