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. 2015 Aug 18;7(10):1285–1306. doi: 10.15252/emmm.201505444

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© 2015 The Authors. Published under the terms of the CC BY 4.0 license

This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

PMC Copyright notice

CUL3^WT^/Δ403–459 mice recapitulate PHAII electrolyte imbalances due to over-activation of the renal WNK4-SPAK pathway

Western blot of whole kidney lysates from mice culled after a minimum 4-h fast. Following exsanguination after surgery, mouse tissues were rapidly harvested and stored, samples were then homogenised, clarified and quantified prior to SDS–PAGE. Immunodetection with the antibodies shown highlights elevated signalling through the WNK kinase pathway in CUL3^WT^/Δ403–459 versus CUL3^WT mice. The lowest panel is an anti-CUL3 immunoprecipitation of the kidney lysate samples, whereby the CUL3 antibody was cross-linked to Protein G sepharose and used to affinity purify CUL3^WT and CUL3^Δ403–459 from kidney lysates. The samples were incubated together overnight to allow deneddylation of CUL3 proteins to occur and maximise CUL3 binding to the anti-CUL3 resin. Samples were thoroughly washed and eluted from the resin prior to SDS–PAGE and immunodetection for CUL3. The IP highlights that CUL3^Δ403–459 is indeed present within the kidney lysate.

Fresh frozen kidney tissues from transplant patient and donor cadavers were homogenised, clarified and quantified prior to SDS–PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human kidneys.

Plasma aldosterone after a minimum 4-h fast was calculated by HTRF (homogeneous time-resolved fluorescence) aldosterone assay. The average aldosterone level per mouse was calculated from duplicate samples run in parallel on the assay. Blood was rapidly harvested in heparin-coated plasma extraction tubes following exsanguination after surgery, and samples were snap-frozen for storage. A 58% increase in aldosterone was detected in CUL3^WT^/Δ403–459 versus CUL3^WT mice (*P = 0.0245). Two-tailed unpaired Student’s t-test; data are mean ± SEM.

Arterial blood biochemistries after a minimum 4-h fast. Under anaesthesia, the right carotid artery was cannulated to minimise atmospheric exposure of samples collected for iSTAT blood gas and electrolyte measurements. CUL3^WT^/Δ403–459 mice present with abnormal electrolyte homoeostasis compared to CUL3^WT mice, exhibiting hyperkalaemia (***P = 0.0004) and hyperchloraemia (***P = 9.5 × 10⁻⁵) with a compensated metabolic acidosis (P = 0.7766), marked by a decrease in bicarbonate () (***P = 3.4 × 10⁻⁵), base excess (BE) (***P = 9.1 × 10⁻⁵) and partial pressure of carbon dioxide (pCO₂) (***P = 0.0038). Two-tailed unpaired Student’s t-test; data are mean ± SEM.

Source data are available online for this figure.

Inline graphic — CUL3^WT^/Δ403–459 mice recapitulate PHAII electrolyte imbalances due to over-activation of the renal WNK4-SPAK pathway

Western blot of whole kidney lysates from mice culled after a minimum 4-h fast. Following exsanguination after surgery, mouse tissues were rapidly harvested and stored, samples were then homogenised, clarified and quantified prior to SDS–PAGE. Immunodetection with the antibodies shown highlights elevated signalling through the WNK kinase pathway in CUL3^WT^/Δ403–459 versus CUL3^WT mice. The lowest panel is an anti-CUL3 immunoprecipitation of the kidney lysate samples, whereby the CUL3 antibody was cross-linked to Protein G sepharose and used to affinity purify CUL3^WT and CUL3^Δ403–459 from kidney lysates. The samples were incubated together overnight to allow deneddylation of CUL3 proteins to occur and maximise CUL3 binding to the anti-CUL3 resin. Samples were thoroughly washed and eluted from the resin prior to SDS–PAGE and immunodetection for CUL3. The IP highlights that CUL3^Δ403–459 is indeed present within the kidney lysate.

Fresh frozen kidney tissues from transplant patient and donor cadavers were homogenised, clarified and quantified prior to SDS–PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human kidneys.

Plasma aldosterone after a minimum 4-h fast was calculated by HTRF (homogeneous time-resolved fluorescence) aldosterone assay. The average aldosterone level per mouse was calculated from duplicate samples run in parallel on the assay. Blood was rapidly harvested in heparin-coated plasma extraction tubes following exsanguination after surgery, and samples were snap-frozen for storage. A 58% increase in aldosterone was detected in CUL3^WT^/Δ403–459 versus CUL3^WT mice (*P = 0.0245). Two-tailed unpaired Student’s t-test; data are mean ± SEM.

Arterial blood biochemistries after a minimum 4-h fast. Under anaesthesia, the right carotid artery was cannulated to minimise atmospheric exposure of samples collected for iSTAT blood gas and electrolyte measurements. CUL3^WT^/Δ403–459 mice present with abnormal electrolyte homoeostasis compared to CUL3^WT mice, exhibiting hyperkalaemia (***P = 0.0004) and hyperchloraemia (***P = 9.5 × 10⁻⁵) with a compensated metabolic acidosis (P = 0.7766), marked by a decrease in bicarbonate () (***P = 3.4 × 10⁻⁵), base excess (BE) (***P = 9.1 × 10⁻⁵) and partial pressure of carbon dioxide (pCO₂) (***P = 0.0038). Two-tailed unpaired Student’s t-test; data are mean ± SEM.

Source data are available online for this figure.