Β1 Integrin Blockade Prevents Podocyte Injury in Experimental Models of Minimal Change Disease

Minimal change disease is the most type cause of nephrotic syndrome in children, which is characterized by proteinuria, body swelling, high cholesterol and loss of protein in the blood.

• ~85% of children with nephrotic syndrome have minimal change disease.
• No. 1 trigger of relapse of nephrotic syndrome in this population are infections.

Steroids initially induce remission in most patients with minimal change disease but almost all children will experience multiple relapses. Complications include:

• Acute kidney injury
• Infection
• Blood clots
• Progression to kidney failure

The disorder is believed to be caused by circulating factors that attack and injure podocytes, the specialized epithelial cells that cover the outer surfaces of glomerular capillaries in the kidney. There is poor understanding of the signaling pathways that lead to their injury.

Minimal change disease is directly associated with:

• Extensive foot process effacement (FPE)
• Synaptopodin loss
• Nephrin deactivation
• Nephrin loss and/or mislocalization
• Activation of the focal adhesion kinase

In experimental models, a reduction in nephrin activation has been observed in minimal change disease, suggesting other signaling pathways are involved in podocyte focal adhesion kinase activation in the disorder.

A study led by Gabriel Cara-Fuentes, MD, PhD, assistant professor of Nephrology-Pediatrics in the Department of Pediatric Nephrology at Children’s Hospital Colorado and other researchers, including Colin Bauer, Federica Piani, MD, Gabriela Garcia, MD, Petter Bjornstad, MD, and Richard Johnson, MD, hypothesized Beta1 (b1) integrin is the upstream signal for podocyte focal adhesion kinase activation in experimental models of minimal change disease-like injury. They combined experimental models and cell culture studies to test their hypothesis.

Methods: inducing minimal change disease using b1 integrin inhibitors

Researchers used lipopolysaccharide (LPS) and minimal change disease sera to induce minimal change disease-like changes in vivo and in cultured human podocytes.

Experimental models

Study authors looked for morphological and molecular changes in podocytes through functional studies using specific b1 integrin inhibitors in vivo and in vitro. Methods included:

• Histological analysis
  • Immunostaining
  • Transmission electron microscopy
  • Immunogold electron microscopy
• Western blotting
• Immunofluorescence
  • Human podocytes stimulation
  • b1 1 integrin blocking assays

For human studies, enzyme-linked immunoassay (ELISA) was used to measure serum LPS levels in:

• 35 children diagnosed with minimal change disease or idiopathic nephrotic syndrome (presumed minimal change disease) at time of disease onset or during relapse
• 18 healthy control children (no known history of glomerular disease or proteinuria)

Results: podocyte b1 integrin may be a molecular target for circulating factors

LPS replicates some key features of human minimal change disease

Study authors evaluated if the LPS model of podocyte injury replicates some key features of human minimal change disease.

When using light microscopy, compared to control models, the LPS-injected experimental models developed both:

• Transient albuminuria
• Transient albuminuria with normal appearing glomeruli

When using immunofluorescence, compared to control models, the LPS-injected experimental models experienced:

• Decreased synaptopodin
• Preservation of total nephrin expression during injury

Study authors performed dual immunofluorescence for phosphorylated nephrin and synaptopodin. At the peak of proteinuria, compared to controls, nephrin phosphorylation was:

• Increased
• Mislocalized

Notable finding:

Nephrin exhibited a peak of phosphorylation and its lineal pattern recovery coincided with proteinuria recovery.

ELISA was used to measured LPS levels to assess the potential relevance of the model to human minimal change disease. Compared to controls, serum LPS was significantly higher in minimal change disease/ idiopathic nephrotic syndrome.

Podocyte b1 integrin signaling mediates focal adhesion kinase activation and podocyte injury in vivo

Researchers found podocyte b1 integrin and focal adhesion kinase are activated following LPS.

To determine if b1 integrin activation was an important upstream mediator of focal adhesion kinase phosphorylation and podocyte injury in this model, researchers administered the blocking antibody HMb1 to b1 integrin into LPS and control murine models.

When only HMb1 was injected, baseline proteinuria in murine controls did not change.

Compared to controls, pre-treatment with a single dose of HMb1 followed by single dose of LPS 20 hours later:

• Improved proteinuria
• Prevented focal adhesion kinase phosphorylation
• Preserved podocyte health and shape

MCD sera mediate podocyte injury via b1 integrin signaling in vitro

Functional studies testing whether b1 integrin is a target of minimum change disease sera circulating factors and a mediator of podocyte injury found:

• Sera from relapsed children caused actin rearrangement in cultured human podocytes
• This change was negated by pre-emptive incubation with Mab13

Discussion and conclusion: role of podocyte b1 integrin in minimal change disease

• Podocyte b1 integrin signaling:
• Mediates podocyte injury in a traditional animal model of minimal change disease-like injury
• Replicates some key features of human minimal change disease in cultured human podocytes
• Podocyte b1 integrin activation and downstream signaling involving focal adhesion kinase phosphorylation is an important causal pathway for podocyte injury in vivo
• Support for study hypothesis that podocyte b1 integrin may be a molecular target for circulating factors in minimal change disease
• First study to show LPS levels are high in serum from children with minimal change disease/ steroid sensitive nephrotic syndrome during relapse
• Circulating factors may directly activate podocyte integrins and downstream pathways initiating podocyte shape changes

Proposed role of the podocyte b1 integrin-focal adhesion kinase axis in minimal change disease

Infections are the most common trigger for relapse of nephrotic syndrome in children with steroid sensitive nephrotic syndrome and minimal change disease.

Given their findings, study authors proposed:

• Microbial products like LPS can target podocytes, leading to cytoskeletal changes and proteinuria via activation of the b1 integrin/focal adhesion kinase axis
• Microbial products, with other molecules that can activate the axis, could be initial triggers leading to podocyte injury in minimal change disease
• Additional insults may be needed to initiate, aggravate and/or perpetuate podocyte injury and proteinuria in minimal change disease

Study authors concluded:

• Podocyte b1 integrin blockade improved podocyte injury in vivo and in vitro.
• The identification of molecules or mechanisms involved in podocyte b1 integrin activation could provide insights into the processes triggering minimal change disease and may help develop targeted therapies.