Kidney fibrosis is the final common pathway of progressive kidney diseases, the underlying mechanisms of which are not fully understood. The purpose of the current study is to investigate a role of Piezo1, a mechanosensitive nonselective cation channel, in kidney fibrosis. In human fibrotic kidneys, Piezo1 protein expression was markedly upregulated. The abundance of Piezo1 protein in kidneys of mice with unilateral ureter obstruction (UUO) or with folic acid treatment was significantly increased. Inhibition of Piezo1 with nonspecific inhibitor GsMTx4 markedly ameliorated UUO- or folic acid–induced kidney fibrosis. Mechanical stretch, compression, or stiffness induced Piezo1 activation and profibrotic responses in human HK2 cells and primary cultured mouse proximal tubular cells (mPTCs), which were greatly prevented by inhibition or silence of Piezo1. TGF-β ₁ induced increased Piezo1 expression and profibrotic phenotypic alterations in HK2 cells and mPTCs, which were again markedly prevented by inhibition of Piezo1. Activation of Piezo1 by Yoda1, a Piezo1 agonist, caused calcium influx and profibrotic responses in HK2 cells and induced calcium-dependent protease calpain2 activation, followed by adhesion complex protein talin1 cleavage and upregulation of integrin β ₁ . Also, Yoda1 promoted the link between ECM and integrin β ₁ . In conclusion, Piezo1 is involved in the progression of kidney fibrosis and profibrotic alterations in renal proximal tubular cells, likely through activating calcium/calpain2/integrin β ₁ pathway.

Previously, we demonstrated that 24 h of bilateral ureteral obstruction (BUO) and short-term release of BUO was associated with a decrease in the expression of aquaporin-2 (AQP2), polyuria, and a reduced urinary concentrating capacity (10). The purposes of the present study were to examine whether BUO and the long-term release of BUO (BUO-R) for 3, 14, and 30 days were associated with changes in the expression of renal AQP1, AQP2, and AQP3 and whether such changes were associated with parallel changes in urinary output and urinary concentrating capacity. Rats ( n = 4–7 in each group) were kept in metabolic cages for measurements of urinary output. Kidneys were removed to determine the expression levels of AQP1, AQP2, and AQP3 by semiquantitative immunoblotting. AQP2 was downregulated after 24 h of BUO (42 ± 3%). Downregulation of AQP2 persisted 3 (43 ± 14%; P < 0.01) and 15 days after BUO-R (48 ± 11%; P < 0.01) but was normalized 30 days after BUO-R. AQP3 showed a similar pattern. Moreover, AQP1 was downregulated in response to BUO (65 ± 7%) and remained downregulated 3 days after BUO-R (41 ± 5%), 14 days after BUO-R (57 ± 8%), and 30 days after BUO-R (59 ± 5%). BUO-R resulted in a significant polyuria that gradually decreased, although it remained significant at day 30. Urinary concentrating capacity remained significantly impaired when determined 3, 14, and 30 days after BUO-R in response to a 24-h period of thirst (1,712 ± 270 vs. 2,880 ± 91 mosmol/kgH₂O at day 30, P < 0.05). In conclusion, the expression of AQP1, AQP2, and AQP3 were long-term downregulated after BUO-R, suggesting that dysregulation of aquaporins located at the proximal tubule, thin descending limb of the loop of Henle, and the collecting duct may contribute to the long-term polyuria and impairment of urinary concentrating capacity associated with obstructive nephropathy.

Bilateral ureteral obstruction (BUO) is associated with marked changes in the expression of renal aquaporins (AQPs) and sodium transport proteins. To examine the role of prostaglandin in this response, we investigated whether 24-h BUO changed the expression of cyclooxygenases (COX-1 and -2) in the kidney and tested the effect of the selective COX-2 inhibitor parecoxib (5 mg·kg⁻¹·day⁻¹via osmotic minipumps) on AQPs and sodium transport. Sham and BUO kidneys were analyzed by semiquantitative immunoblotting, and a subset of kidneys was perfusion fixed for immunocytochemistry. BUO caused a significant 14-fold induction of inner medullary COX-2 (14.40 ± 1.8 vs. 1.0 ± 0.4, n = 6; P < 0.0001) and a reduction in medullary tissue osmolality, whereas COX-1 did not change. Immunohistochemistry confirmed increased COX-2 labeling associated with medullary interstitial cells. COX isoforms did not change in cortex/outer medulla after 24-h BUO. In BUO kidneys, inner medullary AQP2 expression was reduced, and this decrease was prevented by parecoxib. In the inner stripe of outer medulla, the type 3 Na⁺/H⁺exchanger (NHE3) and apical Na⁺-K⁺-2Cl⁻cotransporter (BSC-1) were significantly reduced by BUO, and this decrease was significantly attenuated by parecoxib. Immunohistochemistry for AQP2, NHE3, and BSC-1 confirmed the effect of parecoxib. Parecoxib had no significant effect on the Na-K-ATPase α₁-subunit, type II Na-P_icotransporter, or AQP3. In conclusion, acute BUO leads to marked upregulation of COX-2 in inner medulla and selective COX-2 inhibition prevents dysregulation of AQP2, BSC-1, and NHE3 in response to BUO. These data indicate that COX-2 may be an important factor contributing to the impaired renal water and sodium handling in response to BUO.

The expression of aquaporin-2 (AQP2) is decreased in rats with bilateral ureteral obstruction (BUO) and unilateral ureteral obstruction (UUO). Therefore, the expression of additional renal aquaporins (AQP1–4) and phosphorylated AQP2 (p-AQP2), known to play a role in urinary concentration, was examined in a Wistar rat model with 24 h of UUO. In obstructed kidneys, immunoblotting revealed a significant decrease in the expression of inner medullary AQP2 to 42 ± 4, p-AQP2 to 23 ± 5, AQP3 to 19 ± 6, AQP4 to 11 ± 5, and AQP1 to 64 ± 8% of sham levels. AQP1 expression located in the proximal tubule decreased to 74 ± 4% of sham levels ( P < 0.05). Immunocytochemistry confirmed the downregulation of AQP3, AQP4, and p-AQP2. In contralateral nonobstructed kidneys, immunoblotting also revealed significant reductions of AQP1 in the inner medulla, outer medulla, and cortex, whereas expression of AQP2, AQP3, AQP4, and p-AQP2 was unchanged. Furthermore, we collected the urine from both obstructed and nonobstructed kidneys for 2 h, respectively, after 24 h of UUO. Urine collection from obstructed kidneys during 2 h after release of UUO revealed a significant reduction in urine osmolality and solute-free water reabsorption (T^cH₂O). Moreover, an increase in urine production and T^cH₂O was observed in contralateral kidneys. To examine whether vasopressin-independent mechanisms are involved in AQP2 regulation, vasopressin-deficient Brattleboro (BB) rats with 24 h of UUO were examined. Immunoblotting revealed downregulation of AQP2, p-AQP2, AQP3, and AQP1 in obstructed kidneys and downregulation of p-AQP2 and AQP1 in nonobstructed kidneys. In conclusion, 1) UUO is associated with severe downregulation of AQP2, AQP3, AQP4, and AQP1; thus all of these AQPs may play important roles in the impaired urinary concentrating capacity in the obstructed kidney; 2) the reduced levels of AQP1 in the nonobstructed kidney may contribute to the compensatory increase in urine production; and 3) downregulation of AQPs in BB rats supports the view that vasopressin-independent pathways may be involved in AQP2 and AQP3 regulation in the obstructed kidney.