Kidney Week

Abstract: TH-PO575

Retinol Binding Protein 4 Is a Marker for Proteinuria in Humans and Zebrafish

Session Information

• Glomerular Diseases: Omics, Biomarkers, and Tools
  October 24, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology

Authors

• Haak, Jan René, Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

Jan René Haak, MSc

• Kolb, Cathiana, Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

Cathiana Kolb

• Pollinger, Lena, Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

Lena Pollinger, MSc

• Grosch, Melanie, Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

Melanie Grosch, PhD

• Drummond, Iain A., Mount Desert Island Biological Laboratory, Salsbury Cove, Maine, United States

Iain A. Drummond, PhD

• Haller, Hermann, Mount Desert Island Biological Laboratory, Salsbury Cove, Maine, United States

Hermann Haller, MD

• Hildebrandt, Friedhelm, Division of Nephrology, Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts, United States

Friedhelm Hildebrandt, MD

• Schiffer, Mario, Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

Mario Schiffer, MD, DrMed, MBA

• Rinschen, Markus M., Aarhus Universitet Institut for Biomedicin, Aarhus, Midtjylland, Denmark

Markus M. Rinschen, MD

• Jobst-Schwan, Tilman, Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

Tilman Jobst-Schwan, DrMed, MD

Background

A clinical hallmark of glomerular kidney diseases is proteinuria. In humans, proteinuria is described as the urinary albumin-creatine-ratio. The zebrafish is a widely used model in kidney research. However, up to date proteinuria detection in zebrafish requires transgenic lines, because the zebrafish does not possess an albumin orthologue. Although allowing for proteinuria-related kidney research in zebrafish, these transgenic lines may possess altered physiology and lack direct translational potential to human health.

Methods

We performed proteomics on fish water samples of zebrafish larvae with a described glomerular damage phenotype (magi2a^cl604). We designed and 3D-printed a custom dot blot plate to subject water samples of zebrafish larvae individually housed for 24 hours on a PVDF membrane. Assessment of proteinuria was performed by detecting Rbp4 on the membrane. To induce glomerular damage, different transgenic lines, a chemical induction model and somatic CRISPR knock-out models were used. Additionally, a fluorescent eye assay using the transgenic line Tg(lfab:VDBP-eGFP) was performed to compare the performance of the novel assay to a standard zebrafish proteinuria assay.

Results

Using a proteomics approach, we found high abundance of rbp4 in fish water of edema fish compared to control fish. It has been shown before that RBP4 is proteinuria marker in humans as well. Based on that, we developed a dot blot-based assay where fish water is used to quantify proteinuria in individually housed zebrafish larvae by immunodetection of Rbp4. Using four different established models to induce proteinuria in zebrafish, we validated the dot blot-based assay.

Conclusion

We developed a novel proteinuria assay in zebrafish based on the endogenous orthologue of a known human proteinuria marker. This assay allows for individual proteinuria assessment, independent of transgenic zebrafish lines and with a higher physiological relevance. Thereby, we established a novel translational assay that is useful for drug discovery as it is scalable and suitable for high-throughput procedures.

Funding

• Other NIH Support