Mechanism Of Proteinuria in Kidney Injury And Non-immune Intervention

On June 9, Professor Lin Hongli, vice president of the Nephrology Branch of the Chinese Medical Doctor Association, director of the Nephrology Institute of Dalian Medical University, and director of the Nephrology Department of the First Affiliated Hospital of Dalian Medical University, attended the Nephrology Branch of Hebei Medical Association, Hebei Province At the 2023 academic conference of the Nephrology Physician Branch of the Physician Association, the "Renal Injury Mechanism of Proteinuria and Non-immune Intervention" was introduced.

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At the beginning of the report, Professor Lin Hongli pointed out that persistent proteinuria is an important factor leading to the progression of chronic kidney disease (CKD) to end-stage renal disease. A study divided patients with the same baseline estimated glomerular filtration rate into three groups: massive proteinuria, no proteinuria, and a small amount of proteinuria. The lower the value, the better the patient's renal function outcome. Therefore, reducing proteinuria may delay the progression of CKD to end-stage renal disease.

The relationship between glomeruli and proteinuria: "Three highs" in glomeruli cause proteinuria

Immune factors can mediate the production of proteinuria. The glomerular filtration barrier is a key structure that blocks protein components in the plasma. When proteinuria occurs, it means that the glomerular filtration membrane and renal interstitium have been damaged. Hyperperfusion, high pressure, and high filtration (“three highs”) in the glomerulus are non-immune factors that produce proteinuria. At present, the drugs that are widely used clinically to reduce proteinuria and delay the progression of kidney disease are renin-angiotensin system inhibitors, and their important pharmacological effects are to reduce the "three highs" in the glomeruli.

Professor Lin Hongli introduced that the direct biological effects of the "three highs" in the glomerulus on the endothelial cells and podocytes of the glomerular filtration membrane, as well as the intracellular signal transduction have not yet been clarified. Previous studies of the effects of podocyte mechanics by stretching them on the elastic membrane can only simulate stretching forces. The hydrodynamics inside the glomerulus are very complicated, including not only stretching force but also longitudinal shear force and transverse shear force. Therefore, her department began to study microfluidic chips in 2010, using microfluidic chip technology to build a glomerular model with a filtering function. 

In the chip, the upper channel is used to simulate the glomerular capillary, the inlet connection channel is used to simulate the afferent arteriole, the middle cell culture chamber is used to simulate the capillary network, the sample outlet connection channel is used to simulate the efferent arteriole and the porous polymer in the middle layer is used to simulate the glomerular arteriole. Glomerular endothelial cells and podocytes were cultured on the upper and lower sides of the ester membrane to simulate the main components of the glomerular filtration function. 

Through a series of studies on the glomerular model, it was found that: 

(1) The "three high" mechanical factors in the glomerulus caused the redistribution and expression of the cytoskeleton protein F-actin in glomerular endothelial cells and podocytes to decrease, indicating that the cytoskeleton was affected by the damage. 

(2) The "three high" mechanical factors in the glomerulus damage the junction protein CD-31 between glomerular endothelial cells, resulting in proteinuria leakage. 

(3) The "three high" mechanical factors in the glomerulus damage the SD protein podocin in the glomerular podocytes. In addition, the study also found that high perfusion can increase the filtration rate of albumin and globulin, and the increase of albumin is more significant. Therefore, the "three highs" in the glomerulus have a great impact on the glomerular endothelial cells and podocytes, and the treatment with renin-angiotensin system inhibitors is very important.

After introducing the influence of the "three highs" in the glomeruli and the main treatment methods, Professor Lin Hongli emphasized that the "three highs" in the glomeruli cause changes in hemodynamic factors, leading to changes in the structure and function of the glomerular filtration barrier, proteinuria. 

Therefore, care should be taken to avoid factors that cause "three highs" in the glomeruli, such as high-protein diets. Professor Lin Hongli introduced that when patients with nephrotic syndrome have hypoproteinemia and the water load is too heavy, they often use an intravenous infusion of albumin and use of diuretics to reduce edema. This method can relieve edema in the initial stage of treatment, but it will Aggravate the "three highs" in the glomerulus and cause damage to the glomerular filtration barrier.

Based on this, her team designed a single-center, prospective, randomized controlled trial. Low-protein diet + keto-acid therapy is used for patients with CKD stage 1-2 primary glomerular disease with hormone resistance and proteinuria as the main manifestation, and to study whether it has additional renal protection compared with a normal protein diet. The results showed that a low-protein diet combined with compound α-keto acid significantly reduced proteinuria in patients with primary glomerular disease in the CKD1-2 stage; benefit, and plays a role in regulating blood lipids; it is still safe to use in CKD patients with nephrotic syndrome level proteinuria.

The relationship between renal tubules and proteinuria: proteinuria acts on renal tubules, causing renal interstitial fibrosis

Regarding the kidney injury caused by proteinuria acting on renal tubules, Professor Lin Hongli pointed out that one of the main effects of proteinuria on the kidney is that proteinuria acts on renal tubules, causing renal interstitial fibrosis and promoting the progression of CKD. However, the mechanism of proteinuria-induced renal interstitial damage is not yet fully understood, and there is a lack of strategies to effectively intervene in proteinuria-induced renal interstitial fibrosis. Therefore, it is of great significance to elucidate the mechanism of proteinuria-induced renal interstitial fibrosis and seek a new effective therapeutic target.

Therefore, she led the team to use microfluidic chip technology to construct a bionic "tubule-interstitial" chip model and use the "tubule-interstitial" chip to reproduce the process of renal tubular epithelial cell-mesenchymal cell transdifferentiation in vitro migration process. Constructed a high-throughput bionic renal tubular chip, using a healthy human serum to simulate proteinuria, successfully induced renal tubular epithelial cell-mesenchymal cell transdifferentiation, and found that the protein load was proportional to the degree of transdifferentiation; inactivated healthy human serum (complement component inactivation) will not induce renal tubular epithelial cell-mesenchymal cell transdifferentiation. After discovering this point, exogenous C3a was added to inactivated serum, and it was found that exogenous C3a could induce renal tubular epithelial cell-mesenchymal cell transdifferentiation in inactivated serum.

Interaction of renal tubules with pericytes: promoting renal interstitial fibrosis

Interaction between renal tubules and pericytes also plays an important role in the progression of renal interstitial fibrosis. When renal tubules are stimulated by injury, they will transform into secretory cells, secrete cytokines and act on pericytes, forming a "pro-fibrotic" microenvironment. Professor Lin Hongli introduced that the Nephrology Department of the First Affiliated Hospital of Dalian Medical University pays close attention to pericytes, and the research on them is relatively early. 

It is the first unit in China to isolate renal interstitial pericytes. However, in the process of proteinuria, the paracrine mechanism between renal tubules and blood vessels and the detailed interaction between cells is not clear. Therefore, using microfluidic chip technology, a high-throughput "tubule-interstitial-microvessel" bionic microfluidic chip integrated with 8 units was further constructed. Then, based on the "tubule-interstitium-microvessel" bionic chip, a pathological model of proteinuria was constructed and a series of studies were conducted.

 The results showed that: 

(1) Serum protein stimulation can induce renal tubular epithelial cell transdifferentiation. 

(2) With the prolongation of serum stimulation time, the number and distance of renal tubular epithelial cells migrating into Matrigel gradually increased, and serum stimulation caused renal tubular epithelial cells to migrate. 

(3) As the serum-stimulated renal tubular epithelial cells prolonged, the proportion of renal tubular epithelial cells undergoing apoptosis increased, and serum stimulation caused renal tubular epithelial cells to undergo apoptosis. 

(4) Compared with the normal control group, after serum-stimulated renal tubular epithelial cells for 24 h and 48 h, the number and distance of endothelial cells and pericytes migrating to Matrigel were significantly increased. The distances were significantly higher than those of endothelial cells. Thus, serum stimulates renal tubular epithelial cells, causing the migration of vascular endothelial cells and pericytes to the interstitial zone. 

(5) Serum protein load in renal tubules can induce peritubular vascular endothelial cells and pericytes to transdifferentiate. 

(6) Serum stimulation of renal tubular epithelial cells caused an increase in the proportion of vascular endothelial cells and pericytes to undergo apoptosis.

Finally, Professor Lin Hongli said that based on this study, her team found that down-regulating FUT8 can inhibit the core fucosylation level of key receptors on vascular endothelial cells and pericytes, block the paracrine network, and inhibit downstream pathways Activated to protect peri tubercular vascular injury and renal interstitial fibrosis caused by protein load.