Uncovering New Indications And Potential Intervention Targets For Podocyte Diseases

This study is divided into two parts, clinical research and animal research. The clinical research part is a multicenter study that analyzed the positive rate of anti-Nephrin antibodies in patients with adult glomerular diseases (including MCD, FSGS, MN, immunoglobulin A nephropathy [IgAN], neutrophil cytoplasmic antibodies [ANCA]-related glomerulonephritis and lupus nephritis [LN]), children with idiopathic nephrotic syndrome (INS) and controls. It is worth noting that the data for adult and pediatric patients mainly come from Europe (Germany, Italy, France).

Click to Cistanche for kidney disease

The animal research part is to establish a relevant mouse model, specifically male wild-type BALB/c mice that received subcutaneous immunization with recombinant mouse Nephrin and complete Freund's adjuvant. The control group mice received complete Freund's adjuvant mixed with phosphate-buffered saline.

Of course, this study innovatively used immunoprecipitation to detect anti-Nephrin. Previous examination methods, such as Blot, ELISA, etc., cannot accurately quantify or quantify anti-Nephrin antibodies, but the immunoprecipitation method is different. Specifically, anti-nephrin autoantibody status was determined by immunoprecipitation using recombinant human nephrin external domain (amino acids A36 to L1052). Proteins were incubated with human serum or plasma overnight, and then protein G was added. Immunoprecipitates were collected, subjected to electrophoresis, blotted, and analyzed for nephrin using an anti-nephrin antibody (AF4269; R&D Systems). Samples were analyzed in a blinded manner, and disease diagnosis and clinical data were revealed only after anti-nephrin autoantibody status was determined. For a quantitative readout of anti-nephrin autoantibodies, a hybrid assay was established that included immunoprecipitation and immunoprecipitated nephrin quantification. Such an assay allows for the clearest and most unambiguous detection of anti-nephrin antibodies.

1 Baseline Information

A total of 656 participants were included in this study, including 357 adults with glomerular disease, 182 children with glomerular disease, and 117 controls (67 adults and 50 children). The specific diseases of the patients with glomerular diseases included MCD, FSGS, MN, immunoglobulin A nephropathy (IgAN), anti-neutrophil cytoplasmic antibody-associated nephritis (ANCA-GN), lupus nephritis (LN), and idiopathic nephrotic syndrome.

2 Distribution of anti-Nephrin antibodies in adult patients

Among adult patients, 46 (44%) of 105 MCD patients and 7 (9%) of 74 primary FSGS patients were found to be positive for anti-Nephrin autoantibodies, while almost no patients (n≤1) with other glomerular diseases were positive for anti-Nephrin autoantibodies. If MCD patients did not receive any immunosuppressive treatment, the positive rate of anti-Nephrin autoantibodies was as high as 69% (24/35), which was higher than that of MCD patients who received immunosuppressive treatment (36/59).

3 Child patients

In children with idiopathic nephrotic syndrome (INS), the overall proportion of positive anti-Nephrin autoantibodies was 52%. In the subgroup not receiving immunosuppressive therapy, the overall proportion of positive anti-Nephrin autoantibodies was as high as 90%, while in healthy children, anti-Nephrin autoantibodies were almost negative, with only one patient showing positive results. It is worth noting that since children rarely undergo renal biopsy, this data has strong clinical guidance significance.

4 Relationship between positive examination and clinical characteristics

Whether in adults or children, patients with positive anti-Nephrin antibodies have higher urine protein creatinine ratio (UPCR) or urine albumin creatinine ratio (UACR), while patients with negative anti-Nephrin antibodies usually have a UACR or UPCR of <0.3.

In addition, if the level of anti-Nephrin antibodies changes after the patient receives effective immunosuppressive therapy, the patient's UACR level will also change. For example, a case report of a patient showed that the patient's anti-nephrin antibody and proteinuria levels rebounded after receiving glucocorticoids and cyclosporine A treatment, while after receiving rituximab, the anti-nephrin antibody and UACR levels continued to decline and were close to 0. Of course, this is not an isolated case, and many patients have similar findings. Data analysis showed that there was a strong correlation between all measured anti-Nephrin autoantibody levels and urine albumin to creatinine ratio (r=0.64).

Summary of clinical studies

In general, anti-Nephrin autoantibodies are widely present in MCD adults and ISN children and are effective biomarkers for predicting proteinuria in patients, and the titer of anti-Nephrin autoantibodies can guide clinical medication. Therefore, anti-Nephrin autoantibodies are a new classification biomarker for podocyte disease and a new standard for pathological classification, which can be used for disease activity monitoring, prognosis prediction, and treatment guidance.

1 Renal biopsy results and clinical characteristics

Male wild-type BALB/c mice received subcutaneous immunization with recombinant mouse Nephrin and complete Freund's adjuvant (intervention group). The control group mice received complete Freund's adjuvant mixed with phosphate-buffered saline. The intervention group mice rapidly developed nephrotic syndrome after 3 weeks.

Renal biopsies of the two groups of mice showed that although there were no significant differences between the two groups in PAS staining examination, electron microscopy showed widespread effacement of podocyte foot processes and no electron-dense deposits in the intervention group mice, while there was no similar phenomenon in the control group. Specifically, immunogold labeling of mouse IgG in the intervention group mice showed that it was specifically localized to the slit diaphragm between the areas of effacement of podocyte foot processes.

In addition, the intervention group mice showed a redistribution of nephrin from the membrane to the cytoplasm, with mouse IgG positivity in a fine punctate pattern, partially colocalized with nephrin, and no immune cell infiltration, reduced podocyte number, or complement deposition.

2 Molecular Mechanism

To clarify whether anti-nephrin autoantibodies have a direct effect on nephrin signaling and downstream pathways, the researchers performed proteomic and phosphoproteomic analyses on glomeruli isolated from control mice. The results showed increased phosphorylation of Nphs1, a tyrosine residue corresponding to Y1176 of human nephrin.

This phosphorylation site is known to be involved in nephrin signaling, leading to actin assembly, cytoskeleton reorganization, and nephrin endocytosis. Proteomics showed that in the glomeruli of the intervention group mice, the levels of adaptor proteins such as ShcA (Shc1) and clathrin (Cltc) involved in endocytic nephrin transport increased, while the core slit diaphragm-related protein was significantly downregulated, which is an important cause of podocyte lesions. That is, anti-Nephrin autoantibodies induced nephrotic syndrome, Nephrin phosphorylation, and cell rearrangement at the proteomic level, leading to ultrastructural changes typical of MCD.

How Does Cistanche Treat Kidney Disease?

Cistanche is a traditional Chinese herbal medicine used for centuries to treat various health conditions, including kidney disease. It is derived from the dried stems of Cistanche deserticola, a plant native to the deserts of China and Mongolia. The main active components of cistanche are phenylethanoid glycosides, echinacoside, and acteoside, which have been found to have beneficial effects on kidney health.

Kidney disease, also known as renal disease, refers to a condition in which the kidneys are not functioning properly. This can result in a buildup of waste products and toxins in the body, leading to various symptoms and complications. Cistanche may help treat kidney disease ase through several mechanisms.

Firstly, cistanche has been found to have diuretic properties, meaning it can increase urine production and help eliminate waste products from the body. This can help relieve the burden on the kidneys and prevent the buildup of toxins. By promoting diuresis, cistanche may also help Reduce high blood pressure, a common complication of kidney disease.

Moreover, cistanche has been shown to have antioxidant effects. Oxidative stress, caused by an imbalance between the production of free radicals and the body's antioxidant defenses, plays a key role in the progression of kidney disease. ies help neutralize free radicals and reduce Oxidative stress, thereby protecting the kidneys from damage. The phenylethanoid glycosides found in cistanche have been particularly effective in scavenging free radicals and inhibiting lipid peroxidation.

Additionally, cistanche has been found to have anti-inflammatory effects. Inflammation is another key factor in the development and progression of kidney disease. Cistanche's anti-inflammatory properties help reduce the production of pro-inflammatory cytokines and inhibit the activation of mandatory pathways for inflammation, thus alleviating inflammation in the kidneys.

Furthermore, cistanche has been shown to have immunomodulatory effects. In kidney disease, the immune system can be dysregulated, leading to excessive inflammation and tissue damage. Cistanche helps regulate the immune response by modulating the production and activity of immune cells, such as T cells and macrophages. This immune regulation helps reduce inflammation and prevent further damage to the kidneys.

Moreover, cistanche has been found to improve renal function by promoting the regeneration of renal tubes with cells. Renal tubular epithelial cells play a crucial role in the filtration and reabsorption of waste products and electrolytes. In kidney disease, these cells can be damaged, leading to damaged renal function. Cistanche's ability to promote the regeneration of these cells helps restore proper renal function and improve overall kidney health.

In addition to these direct effects on the kidneys, cistanche has been found to have beneficial effects on other organs and systems in the body. This holistic approach to health is particularly important in kidney disease, as the condition often affects multiple organs and systems. che has been shown to have protective effects on the liver, heart, and blood vessels, which are commonly affected by kidney disease. By promoting the health of these organs, cistanche helps improve overall kidney function and prevent further complications.

In conclusion, cistanche is a traditional Chinese herbal medicine used for centuries to treat kidney disease. Its active components have diuretic, antioxidant, anti-inflammatory, immunomodulatory, and regenerative effects, which help improve renal function and protect the kidneys from further damage. , cistanche has beneficial effects on other organs and systems, making it a holistic approach to treating kidney disease.