Vitamin D and Hydroxychloroquine Reduce Renal Injury and Ki67 Expression in a Rat Model of IgA Nephropathy via TLR4
IgA nephropathy (IgAN) is a significant health concern in China, where approximately 30% of patients experience poor outcomes, and the condition tends to develop in younger individuals. Alarmingly, 40% of IgAN patients progress to end-stage renal disease (ESRD) within 20 years of diagnosis. The pathogenesis of IgAN remains poorly understood, and current treatments, which typically involve glucocorticoids and immune inhibitors like cyclophosphamide and mycophenolate mofetil, are not fully effective. Therefore, there is an urgent need for a better understanding of the disease mechanisms and the development of more effective therapeutic strategies.
Two agents that have shown potential in protecting the kidneys are Vitamin D (VitD) and hydroxychloroquine (HCQ). VitD is a fat-soluble vitamin essential for maintaining calcium and phosphorus levels in the blood. It also has protective effects on the cardiovascular system and kidneys. Previous studies have demonstrated that VitD, in combination with tacrolimus, can reduce mesangial cell proliferation, glomerular basement membrane thickening, and inflammatory cell infiltration in the kidneys. HCQ, an antimalarial drug, is known to regulate immune responses by inhibiting inflammatory signaling pathways. It is commonly used in the treatment of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis.
In this study, we investigated the therapeutic effects of VitD and HCQ, both individually and in combination, on renal histology in a rat model of IgAN. The goal was to identify potential improvements in treatment protocols for this condition.
A total of 40 specific pathogen-free male Wistar rats, with an average body weight of 150 ± 10 grams, were used in the study. The rats were randomly divided into five groups, each containing eight animals: (1) Control, (2) IgAN, (3) IgAN + HCQ, (4) IgAN + VitD, and (5) IgAN + HCQ + VitD. The rats were housed under controlled lighting (12-hour light/dark cycle) and temperature (22–26°C) conditions, with free access to food and water. All experimental procedures were approved by the Animal Care Committee of China Medical University (Approval No. 2018046).
To establish the IgAN model, rats in the four experimental groups received 400 mg/kg/day of bovine serum albumin (BSA) for eight weeks, with a one-day interval between gavages. Additionally, they received weekly subcutaneous injections of castor oil (0.3 mL) and carbon tetrachloride (CCl4, 0.1 mL) for nine weeks. Lipopolysaccharides (LPS, 0.05 mg) were injected via the caudal vein in the 6th and 9th weeks. The control group received distilled water (4 mL/kg) every other day for eight weeks, along with weekly subcutaneous injections of normal saline (0.4 mL) for nine weeks. Normal saline (0.2 mL) was also administered via the caudal vein in the 6th and 9th weeks.
At the end of the 9th week, one rat from each group was randomly selected for kidney analysis. The kidneys were removed and divided into two sections: one portion was frozen at -80°C, while the other was embedded in paraffin for histological examination. Immunofluorescence staining revealed significant green fluorescence deposition in the IgAN group, indicating the presence of IgA. Hematoxylin-eosin (HE) staining showed that the degree of glomerular atrophy was significantly higher in the IgAN group compared to the control group, confirming the successful establishment of the IgAN model.
Starting from the 10th week, the specific treatments for each group were administered. The control and IgAN groups received distilled water (4 mL/kg/day) via gavage. The IgAN + HCQ group was treated with HCQ at a dose of 54 mg/kg/day, while the IgAN + VitD group received VitD at 0.2 mg/kg/day. The IgAN + HCQ + VitD group received both HCQ and VitD at the same doses. Treatments were administered daily for eight weeks. At the end of the 17th week, the renal cortex of the rats was preserved for further analysis.
Histological examination revealed atrophic glomeruli in the IgAN groups. However, pathological manifestations were significantly improved in the three drug treatment groups. The glomeruli in the HCQ and HCQ + VitD groups showed mild atrophy, with the degree of atrophy being slightly less in the HCQ + VitD group compared to the VitD group. The maximal reduction in glomerular atrophy was observed in the HCQ + VitD group, although the kidneys in this group did not fully return to normal compared to the control group.
IgA deposition intensity was significantly lower in the three drug treatment groups compared to the IgAN group (577.293 ± 168.973 ng/L). The HCQ + VitD combination therapy resulted in the lowest IgA deposition intensity (27.337 ± 15.998 ng/L), significantly lower than that observed in the HCQ (136.750 ± 54.373 ng/L) and VitD (71.338 ± 12.793 ng/L) groups. However, none of the treatments completely reduced IgA deposition to the levels observed in the control group (0 ng/L).
The ratio of fibrosis area was significantly increased in the IgAN group (2.468 ± 0.312%) compared to the control group (0.624 ± 0.173%). The fibrosis area ratio was significantly reduced in the HCQ (1.494 ± 0.315%) and HCQ + VitD (0.888 ± 0.208%) groups compared to the IgAN group. The HCQ + VitD group showed a significantly lower fibrosis area ratio compared to the HCQ group.
Ki67 expression, a marker of cell proliferation, was significantly increased in the IgAN group (0.603 ± 0.134%) compared to the control group (0.143 ± 0.018%). The Ki67 expression rate was significantly reduced in the three drug treatment groups, with the HCQ + VitD group showing the lowest expression rate (0.290 ± 0.073%), significantly lower than that in the HCQ (0.370 ± 0.087%) and VitD (0.450 ± 0.078%) groups.
TLR4 expression, which plays a role in inflammatory responses, was significantly increased in the IgAN group (0.754 ± 0.166%) compared to the control group (0.098 ± 0.023%). The TLR4 expression rate was significantly reduced in the three drug treatment groups, with the HCQ + VitD group showing the lowest expression rate (0.322 ± 0.247%), significantly lower than that in the HCQ (0.299 ± 0.073%) and VitD (0.310 ± 0.039%) groups. However, TLR4 expression in the treatment groups remained significantly higher than in the control group.
The results of this study demonstrate that the IgAN model rats exhibited glomerular atrophy, crescent formation, excessive mesangial cell proliferation, and significant IgA deposition in the mesangial region. The increased expression of Ki67 and TLR4 in the IgAN group suggests that these markers play a role in the disease’s progression. Ki67, a nuclear antigen expressed during cell proliferation, is commonly used to detect the proliferation of normal and malignant cells. TLR4, which regulates pro-inflammatory cytokines and chemokines through MyD88-dependent and independent pathways, has been implicated in promoting inflammation and fibrosis in IgAN.
VitD, in addition to its role in maintaining bone health, has anti-inflammatory properties that can protect the kidneys. In IgAN patients, the addition of active VitD to valsartan treatment has been shown to reduce urinary protein excretion, particularly in patients with moderate proteinuria who are intolerant to glucocorticoids or immunosuppressants. HCQ has also been shown to reduce proteinuria in IgAN patients receiving conventional immunosuppressive therapy.
The combined treatment of VitD and HCQ in this study resulted in the most significant improvements in renal histology. Rats in the HCQ + VitD group showed minimal glomerular atrophy, the lowest IgA deposition, and significantly reduced Ki67 and TLR4 expression compared to the other treatment groups. These findings suggest that the combined treatment may reduce inflammatory responses through TLR4 inhibition, thereby delaying the progression of IgAN.
In conclusion, this study demonstrates that the combination of VitD and HCQ has protective effects on the kidneys in a rat model of IgAN, primarily through the inhibition of TLR4 expression. These findings have important clinical implications, as they suggest that this combined treatment may be beneficial for patients with IgAN. Further long-term studies are needed to evaluate the quantitative effects of these treatments on kidney glomerular injury.
doi.org/10.1097/CM9.0000000000001618
Was this helpful?
0 / 0