Kidney Week

Abstract: SA-PO303

The CaSR Signals Through CDC42, MKK6, and p38 to Inhibit SP1, a Repressor of Claudin-14 Expression

Session Information

• Fluid and Electrolytes: Basic - II
  November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Lee, Justin, University of Alberta, Edmonton, Alberta, Canada

Justin Lee,

• Alzamil, Jawad Fahad, University of Alberta, Edmonton, Alberta, Canada

Jawad Fahad Alzamil,

• Rehman, Saba, University of Alberta, Edmonton, Alberta, Canada

Saba Rehman,

• Alexander, R. Todd, University of Alberta, Edmonton, Alberta, Canada

R. Todd Alexander,

Background

Urinary calcium (Ca²⁺) excretion increases in direct response to elevated plasma [Ca²⁺] , independent of hormonal signalling, by attenuating paracellular Ca²⁺ reabsorption from the thick ascending limb (TAL). This occurs via sensing plasma [Ca²⁺] by the calcium sensing receptor (CaSR) expressed in the basolateral membrane of the TAL. This increases expression of the tight junction protein claudin-14, which blocks paracellular Ca²⁺ reabsorption. This pathway is inappropriately activated in some kidney stone formers, causing their disease. However, the signalling pathway between CaSR activation and increased claudin-14 transcription is unknown.

Methods

We identified the renal CLDN14 transcript variant regulated by CaSR activation via quantitative PCR, using specific primers to the different variants on cDNA isolated from kidneys of mice treated with cinacalcet. We cloned the promoter region of this gene into a luciferase reported construct. This region also contained elements responsive to cinacalcet. We used this tool to delineate the signalling pathway downstream of CaSR activation.

Results

The region 1500 bp 5’ to the 1^st transcript variant when transfected in HEK293 cells contained promoter activity. Further, this region displayed more than double reporter activity in the presence of the CaSR and cinacalcet, but not in the absence of the CaSR. Increasing extracellular [Ca²⁺] similarly increased reporter activity in the presence, but not the absence, of the CaSR. A prior microarray found increased renal MKK6 and CLDN14 expression in mice treated with cinacalcet. However, expression of MKK6 reduced CLDN14 reporter activity after cinacalcet treatment. MKK6 can signal through JNK or p38 MAPK. Inhibition of p38 but not JNK enhanced the cinacalcet mediated increase in CLDN14 reporter activity. Sp1, a known downstream effecter of p38, was down regulated by CaSR activation, and co-expression attenuated the effect on the reporter construct. Upstream activation of MKK6 is through CDC42, as dominant-negative CDC42 attenuated reporter activity.

Conclusion

CaSR activation increases CLDN14 transcription via signalling through CDC42, MKK6 and p38 to attenuate the expression of SP1 a repressor of CLDN14.

Funding

• Government Support - Non-U.S.