Abstract
Kidney injury in hypertension and diabetes entails, among in other structures, damage in a key cell of the glomerular filtration barrier, the podocyte. Podocytes are polarized and highly differentiated cells in which vesicular transport, partly driven by Rab GTPases, is a relevant process. The aim of the present study was to analyze Rab GTPases of the Rab-Rabphilin system in human immortalized podocytes and the impact of high glucose and angiotensin II. Furthermore, alterations of the system in urine cell pellets from patients with hypertension and diabetes were studied. Apoptosis was analyzed in podocytes, and mRNA level quantification, Western blot analysis, and immunofluorescence were developed to quantify podocyte-specific molecules and Rab-Rabphilin components (Rab3A, Rab27A, and Rabphilin3A). Quantitative RT-PCR was performed on urinary cell pellet from patients. The results showed that differentiated cells had reduced protein levels of the Rab-rabphillin system compared with undifferentiated cells. After glucose overload and angiotensin II treatment, apoptosis was increased and podocyte-specific proteins were reduced. Rab3A and Rab27A protein levels were increased under glucose overload, and Rabphilin3A decreased. Furthermore, this system exhibited higher levels under stress conditions in a manner of angiotensin II dose and time treatment. Immunofluorescence imaging indicated different expression patterns of podocyte markers and Rab27A under treatments. Finally, Rab3A and Rab27A were increased in patient urine pellets and showed a direct relationship with albuminuria. Collectively, these results suggest that the Rab-Rabphilin system could be involved in the alterations observed in injured podocytes and that a mechanism may be activated to reduce damage through the vesicular transport enhancement directed by this system.
Highlights
Kidney injury, characterized by a progressive decrease in glomerular filtration rate and/or an increase in urinary albumin* O
In vitro studies have revealed that exposure to glucose and angiotensin II (ANG II) induces alteration of specific proteins and activation of intracellular pathways leading to podocyte injury [35, 56]
We observed that synaptopodin and nephrin mRNA levels were higher in differentiated podocytes (4.96-fold increase, P Ͻ 0.001, and 2.12-fold increase, P Ͻ 0.05, respectively), WT-1 levels were lower (2.58-fold decrease, P Ͻ 0.001), and CD2associated protein (CD2AP) showed no significant changes compared with the undifferentiated state (Fig. 1B)
Summary
Kidney injury, characterized by a progressive decrease in glomerular filtration rate and/or an increase in urinary albumin* O. Due to the complexity of their structure and function, the mechanisms involved in their injury are still not fully understood, but their involvement in the pathophysiology of renal diseases has been previously described [41, 46, 68]. In this regard, in vitro studies have revealed that exposure to glucose and angiotensin II (ANG II) induces alteration of specific proteins and activation of intracellular pathways leading to podocyte injury [35, 56]
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