YI QI QING RE GAO ALLEVIATES PODOCYTE CYTOSKELETON DYSFUNCTION VIA TUBULE-PODOCYTE CROSSTALK MEDIATED BY RENAL TUBULAR SIRT1

The damage of podocyte cytoskeleton in the glomerulus is a key link in the formation of proteinuria, and the damage to renal tubular epithelial cells (TEC) is an important driving force for disease progression. Studies have shown that Sirt1 in TEC can regulate NMN metabolism and repair podocyte cytoskeleton damage through tubular-podocyte crosstalk. This study is based on the interaction between glomeruli and renal tubules, treating the renal tubular and glomeruli as an organic whole, and exploring the intervention effect of traditional Chinese medicine formula YiQi QingRe Gao (YQQRG) in the crosstalk between glomeruli and tubules. YQQRG has been used in clinical practice for over 20 years to treat proteinuria, with significant clinical efficacy. This experiment used an ADR nephropathy model to observe the effects of YQQRG on glomeruli and tubules, and explored possible mechanism.

In vivo studies were conducted to construct conditional Sirt1 knockout mice of renal tubular epithelial cells, induced by Adriamycin (ADR) nephropathy model. After intervention with Yiqi Qingre Gao, biochemical indicators, 24-hour urine protein quantification levels, expression of fluorescently labeled β 2-microglobulin, renal pathological damage and ultrastructural changes, distribution and expression levels of Sirt1 and Cortactin in the glomerulus, expression of tubular crosstalk junction molecule NMN in renal tissue, and expression levels of cytoskeletal proteins were detected in each group of mice. In vitro studies were conducted by co-culturing proximal renal tubular epithelial cells (HK-2) and conditionally immortalized mouse podocyte lines. Sirt1 siRNA and recombinant plasmid were transfected into HK-2 cells, and Yiqi Qingre Gao containing serum was used for intervention. Observation of the movement, migration, and adhesion abilities of podocytes after treatment, the NMN content in the culture medium, the arrangement of cytoskeletal actin microfilaments, the distribution and expression levels of Sirt1 and Cortactin in podocytes, and their interactions, as well as the expression of podocyte cytoskeletal proteins were detected in each group of cells.

YQQRG can significantly reduce proteinuria levels, increase serum albumin, and improve renal function in SIRT1 knockout mice after modeling. It can reduce β 2-microglobulin in urine, and the repairing effect appeared earlier than the alleviation of proteinuria. It can also alleviate foot process fusion and reduce renal pathological damage. The in vitro experimental results showed that YQQRG can alleviate podocyte injury and apoptosis after modeling, reduce podocyte movement and migration, and repair cytoskeletal damage. Both in vivo and in vitro experiments have shown that YQQRG can upregulate the expression of Sirt1 and Cortactin in renal tissue and podocytes, and increase the expression of NMN, a tube ball crosstalk junction molecule, both in renal tissue and cell supernatant. YQQRG restored the normal expression of glomerular cytoskeleton proteins Actin and Synaptopodin, as well as Slit diaphragm protein Nephrin and Podocin.

Yiqi Qingre Gao can alleviate podocyte damage caused by ADR both in vivo and in vitro, and the mechanism may be through activating Sirt1 renal tubular epithelial cells, regulating NMN metabolism changes, and repairing cytoskeletal damage. This study elucidates the crosstalk between renal tubular epithelial cells and podocytes, providing scientific evidence for the use of Yiqi Qingre to reduce damage to renal tubular epithelial cells and podocytes, and to lower proteinuria. YQQRG may be a promising natural drug for treating kidney diseases.