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Abstract: TH-PO862

Tubular and Injury Marker Expression Across Nephron Subsegments in Diabetic Nephropathy

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Barwinska, Daria, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Cheng, Yinghua, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Ferkowicz, Michael J., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Winfree, Seth, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Collins, Kimberly S., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Dunn, Ken, Indiana University, Indianapolis, Indiana, United States
  • Kelly, Katherine J., Indiana University, Indianapolis, Indiana, United States
  • Sutton, Timothy A., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Rovin, Brad H., Ohio State University Wexner Medical Center, Columbus, Ohio, United States
  • Parikh, Samir V., Ohio State University Medical Center, Columbus, Ohio, United States
  • Dagher, Pierre C., Indiana University, Indianapolis, Indiana, United States
  • El-Achkar, Tarek M., Indiana University, Indianapolis, Indiana, United States
  • Eadon, Michael T., Indiana University Division of Nephrology, Indianapolis, Indiana, United States
Background

Laser Microdissection (LMD) allows precise separation of nephron structures under immunofluorescence (IF). Several markers have been identified in the literature that are associated with specific subsegments of a nephron. Here, we examine the expression of known tubular and injury markers in kidney tissue of diabetic nephropathy (DN) biopsies and healthy nephrectomies.

Methods

Kidney biopsies from subjects with DN as well as a wedge biopsy from a healthy donor kidney were obtained. Tissue was preserved in OCT for 4 years at -80° C and sectioned into 12 µm thick sections on LMD PPS-membrane slides. Each slide was subjected to a rapid staining protocol, using DAPI, FITC-Phalloidin and Na/K channel antibody. Each slide underwent identical antibody staining and LMD, followed by RNA isolation, and sequencing. Transcriptomics data were obtained using RNAseq methodology on Illumina platform, and analyzed using R Studio.

Results

This study revealed that LMD methodology successfully allows for accurate identification of specific sub segments of a nephron, as shown by presence of markers associated with those structures and the absence of markers associated with other regions: NPHS1 and NPHS2 for the glomerulus, CUBN and SLC34A1 for the S1 and S2 proximal tubule, LRP2 for the S3 proximal tubule, and AQP2 and SCCN1G for the collecting duct. In addition, the transcriptomic analysis revealed differential expression of tubular injury markers like LCN2 (NGAL) expressed in the S1 and S2 subsegment. In contrast, HAVCR1 (KIM1) and TIMP2 expression was increased in the S3 subsegment.

Conclusion

LMD is an important tool that allows for accurate identification and isolation of tubular subsegments within renal tissue and subsequent RNA analysis. An increased expression of injury markers associated with different tubules was observed in the diseased kidney tissue samples. This suggests that specific regions of the nephron may respond to injury in different ways, which may further contribute to the pathogenesis and progression of DN.

Funding

  • NIDDK Support