Takamitsu Saigusa

Collecting duct cells that lack normal cilia have mislocalized vasopressin-2 receptors

Polycystic kidney disease (PKD) is a ciliopathy characterized by renal cysts and hypertension. These changes are presumably due to altered fluid and electrolyte transport in the collecting duct (CD). This is the site where vasopressin (AVP) stimulates vasopressin-2 receptor (V2R)-mediated aquaporin-2 (AQP2) insertion into the apical membrane. Since cysts frequently occur in the CD, we studied V2R and AQP2 trafficking and function in CD cell lines with stunted and normal cilia [cilia (-), cilia (+)] derived from the orpk mouse (hypomorph of the Tg737/Ift88 gene). Interestingly, only cilia (-) cells grown on culture dishes formed domes after apical AVP treatment. This observation led to our hypothesis that V2R mislocalizes to the apical membrane in the absence of a full-length cilium. Immunofluorescence indicated that AQP2 localizes to cilia and in a subapical compartment in cilia (+) cells, but AQP2 levels were elevated in both apical and basolateral membranes in cilia (-) cells after apical AVP treatment. Western blot analysis revealed V2R and glycosylated AQP2 in biotinylated apical membranes of cilia (-) but not in cilia (+) cells. In addition, apical V2R was functional upon apical desmopressin (DDAVP) treatment by demonstrating increased cAMP, water transport, and benzamil-sensitive equivalent short-circuit current (I(sc)) in cilia (-) cells but not in cilia (+) cells. Moreover, pretreatment with a PKA inhibitor abolished DDAVP stimulation of I(sc) in cilia (-) cells. Thus we propose that structural or functional loss of cilia leads to abnormal trafficking of AQP2/V2R leading to enhanced salt and water absorption. Whether such apical localization contributes to enhanced fluid retention and hypertension in PKD remains to be determined.

Molecular Pathways and Therapies in Autosomal-Dominant Polycystic Kidney Disease

Autosomal-dominant polycystic kidney disease (ADPKD) is the most prevalent inherited renal disease, characterized by multiple cysts that can eventually lead to kidney failure. Studies investigating the role of primary cilia and polycystins have significantly advanced our understanding of the pathogenesis of PKD. This review will present clinical and basic aspects of ADPKD, review current concepts of PKD pathogenesis, evaluate potential therapeutic targets, and highlight challenges for future clinical studies.

Activation of the intrarenal renin‐angiotensin‐system in murine polycystic kidney disease

The mechanism for early hypertension in polycystic kidney disease (PKD) has not been elucidated. One potential pathway that may contribute to the elevation in blood pressure in PKD is the activation of the intrarenal renin‐angiotensin‐system (RAS). For example, it has been shown that kidney cyst and cystic fluid contains renin, angiotensin II (AngII), and angiotensinogen (Agt). Numerous studies suggest that ciliary dysfunction plays an important role in PKD pathogenesis. However, it is unknown whether the primary cilium affects the intrarenal RAS in PKD. The purpose of this study was to determine whether loss of cilia or polycystin 1 (PC1) increases intrarenal RAS in mouse model of PKD. Adult Ift88 and Pkd1 conditional floxed allele mice with or without cre were administered tamoxifen to induce global knockout of the gene. Three months after tamoxifen injection, kidney tissues were examined by histology, immunofluorescence, western blot, and mRNA to assess intrarenal RAS components. SV40 immortalized collecting duct cell lines from hypomorphic Ift88 mouse were used to assess intrarenal RAS components in collecting duct cells. Mice without cilia and PC1 demonstrated increased kidney cyst formation, systolic blood pressure, prorenin, and kidney and urinary angiotensinogen levels. Interestingly immunofluorescence study of the kidney revealed that the prorenin receptor was localized to the basolateral membrane of principal cells in cilia (−) but not in cilia (+) kidneys. Collecting duct cAMP responses to AngII administration was greater in cilia (−) vs. cilia (+) cells indicating enhanced intrarenal RAS activity in the absence of cilia. These data suggest that in the absence of cilia or PC1, there is an upregulation of intrarenal RAS components and activity, which may contribute to elevated blood pressure in PKD.

Suppressing angiotensinogen synthesis attenuates kidney cyst formation in a Pkd1 mouse model

Activation of the intrarenal renin angiotensin system (RAS) is believed to play an important role in the development of hypertension and cystogenesis in autosomal dominant polycystic kidney disease (ADPKD). Results of clinical studies testing RAS inhibitors in slowing the progression of cystic disease in ADPKD are inconclusive, and we hypothesized that current RAS inhibitors do not adequately suppress intrarenal RAS. For this study, we compared a novel Gen 2 antisense oligonucleotide (ASO) that inhibits angiotensinogen (Agt) synthesis to lisinopril in adult conditional Pkd1 systemic‐knockout mice, a model of ADPKD. Six weeks after Pkd1 global gene knockout, the mice were treated with Agt‐ASO (66 mg/kg/wk), lisinopril (100 mg/kg/d), PBS (control), or scrambled ASO (66 mg/kg/wk) for 10 wk, followed by tissue collection. Agt ASO resulted in significant reduction in plasma, liver, and kidney Agt, and increased kidney renin compared with control treatments. Kidneys from Agt‐ASO‐treated mice were not as enlarged and showed reduced cystic volume compared with lisinopril or control treatments. Blood pressure was better controlled with lisinopril than with Agt‐ASO. Agt‐ASO suppressed cell proliferation in both cystic and noncystic cells compared with lisinopril and control treatments. However, Agt‐ASO did not reduce cell proliferation in liver, which indicates that Agt‐ASO targets cell signaling pathways that specifically suppresses cystogenesis in the kidney. These data suggest that Agt‐ASO effectively attenuates intrarenal RAS and therefore can be a novel and effective agent for treating ADPKD.—Saigusa, T., Dang, Y., Mullick, A.E., Yeh, S.T., Zile, M.R., Baicu, C. F., Bell, P. D. Suppressing angiotensinogen synthesis attenuates kidney cyst formation in a Pkd1 mouse model. FASEB J. 30, 370‐379 (2016). www.fasebj.org

Reciprocal effect of angiotensin II in collecting duct renin synthesis

activation of the renin-angiotensin system (RAS) plays a central role in the regulation of hypertension and sodium homeostasis. Angiotensin II (ANG II) is the most potent biologically active peptide that possesses pleiotropic functions to increase renal vascular resistance, cardiac contractility,