Kidney Week

Abstract: SA-PO629

A Novel CLC-Kb Pore Mutation Disrupts Glycosylation and Associates with Distal Tubular Remodeling

Session Information

• Genetic Kidney Diseases: Models, Mechanisms, and Therapies
  October 26, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1202 Genetic Diseases of the Kidneys: Non-Cystic

Authors

• Lo, Robin H., Stanford University School of Medicine, Stanford, California, United States

Robin H. Lo, MD

• Sharma, Yogita, Stanford University School of Medicine, Stanford, California, United States

Yogita Sharma, PhD

• Tomilin, Victor N., The University of Texas Health Science Center at Houston John P and Katherine G McGovern Medical School, Houston, Texas, United States

Victor N. Tomilin, PhD

• Deremo, Holly A., Stanford University School of Medicine, Stanford, California, United States

Holly A. Deremo, MPH

• Dong, Wuxing, Stanford University School of Medicine, Stanford, California, United States

Wuxing Dong

• Charu, Vivek, Stanford University School of Medicine, Stanford, California, United States

Vivek Charu, MD, PhD

• Kambham, Neeraja, Stanford University School of Medicine, Stanford, California, United States

Neeraja Kambham, MD

• Mutig, Kerim, Charite Universitatsmedizin Berlin, Berlin, Germany

Kerim Mutig, DrMed

• Pochynyuk, Oleh, The University of Texas Health Science Center at Houston John P and Katherine G McGovern Medical School, Houston, Texas, United States

Oleh Pochynyuk, PhD

• Bhalla, Vivek, Stanford University School of Medicine, Stanford, California, United States

Vivek Bhalla, MD, FASN

Background

Bartter’s and Gitelman’s syndromes are inherited, monogenic autosomal recessive tubulopathies resulting in defects of renal electrolyte handling with a propensity for chronic kidney disease. One type of Bartter’s syndrome involves inactivating mutations in CLCNKB, a gene encoding the chloride channel CLC-Kb expressed on the basolateral membranes of the thick ascending limb and the distal tubule. Here, we identified a family with a mixed Bartter’s/Gitelman’s phenotype and early-onset kidney failure and employing a candidate gene approach, discovered a novel mutation (ntG499T, G167C) in exon 6 of CLCNKB in the index patient.

Methods

The mutation was identified via Sanger and whole exome sequencing. Wild type and mutant CLCNKB plasmid constructs were generated and transfected in vitro for electrophysiological analysis via patch-clamp experiments. Wild type and mutant CLC-Kb N-link glycosylation patterns were analyzed by Endo H or PNGase F digestion and localization by cell surface biotinylation or subcellular fractionation. Nephrectomy samples from the index patient and healthy controls were used for histology, morphometry, and qualitative immunofluorescence.

Results

Compared to wild type CLC-Kb, the G167C mutant conducted less current. The G167C mutation also caused impaired N-linked CLC-Kb glycosylation. By targeted mutagenesis of potential CLC-Kb glycosylation sites, we demonstrated that complex N-linked glycosylation at Asn-364 glycosylation is required for proper channel function but not for its surface expression. Moreover, using other known CLC-Kb mutations, we deduced that lack of glycine rather than gain of cysteine is responsible for the findings. Morphologic evaluation of kidney biopsies revealed basolateral localization of mutant G167C CLC-Kb in cortical distal tubular epithelia. However, we detected attenuated expression of distal sodium transport proteins, changes in abundance of distal tubule segments, and hypokalemia-associated intracellular condensates from the index patient compared to control nephrectomy specimens.

Conclusion

We demonstrate a novel regulatory mechanism of CLC-Kb activity by glycosylation and demonstrate nephron remodeling caused by mutant CLC-Kb, with implications for renal electrolyte handling, blood pressure control, and kidney disease.

Funding

• NIDDK Support