Kidney Week

Abstract: PUB076

Hematuria Rescues Urothelium

Session Information

• Publication Only

Category: Anemia and Iron Metabolism

• 200 Anemia and Iron Metabolism

Authors

• Barasch, Jonathan M., Columbia University, New York, New York, United States

Jonathan M. Barasch, MD, PhD

• Xu, Katherine, Columbia University, New York, New York, United States

Katherine Xu, PhD

• Shen, Tian, Columbia University, New York, New York, United States

Tian Shen, PhD

• Ghotra, Aryan S., Columbia University, New York, New York, United States

Aryan S. Ghotra

• Nesanir, Kivanc, University of Richmond, Richmond, Virginia, United States

Kivanc Nesanir

• Sturley, Rachel, Columbia University, New York, New York, United States

Rachel Sturley

Background

Hematuria is a frightening complication of urinary diseases. Hematuria may be pathogenic because heme carries lipophilic Fe²⁺ which damages cells. In addition, iron is limiting for bacterial growth, but bacteria express heme transporters and siderophore receptors. Hence, hematuria may accelerate UTI and tissue damage.

Methods

We loaded the urinary system with ferric iron or with heme by phenylhydrazine (PHZ) hemolysis. UTI was titrated in male-females (20uL of 10⁷ UTI89 CFU) and tissue damage monitored by CK20⁺ superficial shedding versus preserved CK5⁺ basal cells. RNA was captured by (1) physical isolation of urothelia from bladder mucosa (2) and by 4-thiouracil pulse labeling of nascent RNA in vivo in Upk2Cre;Uprt^f/f urothelia. RNAscope probed anatomical expression of induced genes.

Results

UTI induced detachment and shedding of superficial cells (6-12hrs post infection). Ferric iron worsened and ferric iron deficiency suppressed bacterial growth and shedding. In contrast, hematuria did not affect bacterial growth but surprisingly prevented shedding (n=75 mice). Retention of superficial cells and suppression of erythema were grossly visibile even after bacterial invasion.

Urothelial RNA demonstrated that pyroptosis genes mediate heme sensitive cell death. Indeed, GsdmD KO mice suppressed urothelial shedding and accordingly hematuria suppressed GsdmD RNA expression. Upstream regulators of GsdmD, such as Nlrp3 and cleaved casp1 and 11 proteins were depressed by hematuria in superficial and intermediate cells. Pyroptosis associated cytokines, chemokines, neutrophil chemoattractants, interleukins and inflammatory cell death regulators were quantitatively downregulated. In accordance, neutrophil invasion of the infected bladder was suppressed.

Heme is captured in urothelia by HRG1, which in turn transports heme into the cytoplasm where upregulated BMAL, NPAS2, and HMOX1 metabolize heme to Carbon Monoxide (detectable with our Palladium-based fluorescence quenched probes) and iron (detectable by Perls and Turnbull's staining). However, HRG1 KO and HMOX KO still demonstrated rescue by hematuria. Rather, multiple TLR components mediate heme’s activity since KO prevented urothelial rescue.

Conclusion

Hematuria, a frightening sign of urological disease, is surprisingly anti-inflammatory and cell protective in UTI-Cystitis.

Funding

• NIDDK Support