Carfilzomib-Induced Hypertension Is Mediated By Ion Channel Dysregulation in the Kidneys; The Potent Role of AMP-Activated Kinase α

Introduction: Carfilzomib (Cfz), an irreversible proteasome inhibitor (PI), is an approved agent against relapsed/refractory multiple myeloma (R/R MM). Cfz is associated with high incidence of cardiovascular adverse effects. Hypertension stands as the most frequent cardiovascular complication of Cfz. Even though thrombotic microangiopathy (TMA) is inculpated of Cfz's hypertensive phenotype, its exact pathophysiology is still elusive. In our previous work, we showed that Cfz establishes cardiotoxicity in vivo in a Protein Phosphatase 2A (PP2A)-AMP-activated kinase α (AMPKα)-dependent manner (Efentakis P et al. Blood. 2019;133(7):710-723) but does not lead to a permanent vascular deficit, indicating that hypertension is not vascular derived [Efentakis P et al. IJMS 2020;21(15):E5185]. Taking under consideration that renal homeostasis plays an important role in blood pressure regulation we sought to (i) characterize the dose-dependent manifestation of Cfz-induced hypertension; (ii) investigate the molecular signaling of Cfz in the kidneys by proteomic and immunoblotting analyses and (iii) study the renal ion channels regulation.

Methods: Forty C57Bl/6 mice (12-14 weeks of age) were randomly assigned to: (i) Acute Protocol: a. Control [Normal Saline (N/S) 0.9%] and b. Cfz (8mg/kg) for two days and (ii) Sub-acute Protocol: a) Control (N/S 0.9%,) and b) Cfz (8mg/kg) for seven days. Intraperitoneal administration of N/S 0.9% and Cfz was performed at two consecutive and on alternate days for the acute and sub-acute protocols respectively. At baseline and endpoint of the experiments, systolic (SBP) and diastolic blood pressure (DBP) were measured, and subsequently mice were sacrificed for the collection of blood and renal samples. Blood samples were collected in citrate buffer for hematological/coagulation profiling [prothrombin time (PT) and INR estimation] and for cleaved Von Willebrand Factor (cl. VWF) immunoblotting assessment as well as for blood testing of white blood cells (WBCs) and platelets. Renal samples underwent histological proteomic and molecular analyses.

Results: SBP and DBP were found to be elevated in Cfz group only in the sub-acute protocol compared to control (SBP: 78.5±2.0 vs 68.2±0.7, p<0.01 and DBP: 104.1±2.3 vs 97.4±2.4, p<0.05, respectively). Neutrophil count in the whole blood was elevated both in the acute and sub-acute protocols, while platelet count was decreased in the acute protocol and restored after sub-acute Cfz administration. Mice presented a thrombotic phenotype characterized by increased PT and INR and decreased cl. VWF after acute Cfz treatment, and a hemorrhagic phenotype with restored cl. VWF after sub-acute Cfz administration. However, the presence of TMA was not confirmed by histological evaluation of the kidneys in any of the protocols. Nonetheless histological evaluation of the kidneys revealed inflammation of the perirenal adipose tissue only in the sub-acute protocol. Proteomic analysis presented that proteasome regulation, metabolism and transport of organic anion processes are affected by Cfz administration. Molecular analysis of the renal tissue showed that Cfz induced an increase in the inducible nitric oxide synthase (iNOS) and microtubule-associated proteins 1A/1B light chain 3B (LC3-B) expression and a decreased AMPKα phosphorylation. Moreover, RT-PCR analysis of the renal samples revealed that collecting duct ion channels epithelial Na⁺ channel (ENaC), Na⁺/K⁺/ATPase and urea transporter 1 (UTA-1) mRNA levels were increased in Cfz group.

Conclusion: Sub-acute Cfz treatment establishes a renal-derived hypertensive phenotype and a circulating inflammatory phenotype as indicated by the increased neutrophil and WBCs count in the blood. TMA does not seem to be implicated with the observed phenotype in vivo as investigated molecularly and histologically. Cfz-induced dephosphorylation of AMPKα and the subsequent dysregulation of the collecting duct renal ion channel homeostasis is found to be responsible for the observed effect. Therefore, Cfz seems to induce hypertension by a dysregulation of water-ion re-absorbance leading to increased cardiac preload. The latter comes in agreement with clinical data showing that some Cfz-treated patients present with fluid retention, which can be attributed to the aforementioned renal effects of the drug.