Activation of the Renin–Angiotensin System Disrupts the Cytoskeletal Architecture of Human Urine-Derived Podocytes

Abstract

High blood pressure is one of the major public health problems that causes severe disorders in several tissues including the human kidney. One of the most important signaling pathways associated with the regulation of blood pressure is the renin–angiotensin system (RAS), with its main mediator angiotensin II (ANGII). Elevated levels of circulating and intracellular ANGII and aldosterone lead to pro-fibrotic, -inflammatory, and -hypertrophic milieu that causes remodeling and dysfunction in cardiovascular and renal tissues. Furthermore, ANGII has been recognized as a major risk factor for the induction of apoptosis in podocytes, ultimately leading to chronic kidney disease (CKD). In the past, disease modeling of kidney-associated diseases was extremely difficult, as the derivation of kidney originated cells is very challenging. Here we describe a differentiation protocol for reproducible differentiation of sine oculis homeobox homolog 2 (SIX2)-positive urine-derived renal progenitor cells (UdRPCs) into podocytes bearing typical cellular processes. The UdRPCs-derived podocytes show the activation of the renin–angiotensin system by being responsive to ANGII stimulation. Our data reveal the ANGII-dependent downregulation of nephrin (NPHS1) and synaptopodin (SYNPO), resulting in the disruption of the podocyte cytoskeletal architecture, as shown by immunofluorescence-based detection of α-Actinin. Furthermore, we show that the cytoskeletal disruption is mainly mediated through angiotensin II receptor type 1 (AGTR1) signaling and can be rescued by AGTR1 inhibition with the selective, competitive angiotensin II receptor type 1 antagonist, losartan. In the present manuscript we confirm and propose UdRPCs differentiated to podocytes as a unique cell type useful for studying nephrogenesis and associated diseases. Furthermore, the responsiveness of UdRPCs-derived podocytes to ANGII implies potential applications in nephrotoxicity studies and drug screening.

Derivation of the immortalized cell line-UM51-PrePodo-hTERT and its responsiveness to Angiotensin II and activation of RAAS

“…We recently reported a detailed protocol for the differentiation of SIX2-positive urine derived renal progenitor cells (UdRPCs) into mature podocytes [26]. We applied this protocol to differentiate the UM51-PrePodo-hTERT cell line into podocytes.…”

“…These results confirm the differentiation capacity of UM51-PrePodo-hTERT into mature podocytes-based on both protein and mRNA expression of NPHS1, SYNPO and WT1. Interestingly, by employing our recently published podocyte differentiation protocol [26], the proliferation capacity of UM51-PrePodo-hTERT was diminished and a significant upregulation of SYNPO expression was observed. SYNPO has been recognized as a key marker of differentiated post-mitotic podocytes [15, 17, 18], which cannot be detected in undifferentiated or dedifferentiated cells [19, 20].…”

“…Finally, we evaluated the responsiveness of UM51-PrePodo-hTERT to Angiotensin II (ANGII) which is a key mediator of the renin-angiotensin-system (RAS) [1, 26]. Elevated levels of ANGII have been identified as a main risk factor for the initiation and progression of chronic kidney disease (CKD).…”

“…The cell line UM51-PrePodo was derived from the urine of a 51-year-old male of African origin. The cells were cultured, and differentiation was induced as described in [26]. Immortalization of the cells was achieved by lipofection of the pCDNA-3xHA-hTERT plasmid with Xfect (Takara BIO INC, Kusatsu, prefecture Shiga, Japan).…”

“…We recently showed that Angiotensin II (ANGII) treatment in UdRPC differentiated podocytes trigger disruption of the cytoskeleton, resulting in the inhibition of podocyte spreading and subsequent loss of foot processes as observed by a round and condensed morphology [26]. To evaluate the effect of ANGII on the differentiated podocytes derived from the UM51-Pre-Podo-hTERT cell line, we treated the cells with 100 µM ANGII for 24 h and with a combination of 100 µM ANGII and 1 µM of the selective, competitive angiotensin II receptor type 1 antagonist Losartan for 24 h. After 24 h, dynamic changes in morphology and a significant down-regulation of α-ACTININ expression could be observed in cells treated with 100 µM ANGII by immunofluorescence-based detection (fig.…”