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Prevailing strategies directing early-phase drug discovery heavily rely on equilibrium-based metrics such as affinity, which overlooks the kinetic process of a drug molecule interacting with its target. Herein, we developed a number of vasopressin V2 receptor (V2R) antagonists with divergent binding affinities and kinetics for autosomal dominant polycystic kidney disease (ADPKD). Surprisingly, the residence time of the V2R antagonists, but not their affinity, was correlated with the efficacy in both ex vivo and in vivo models of ADPKD. We envision that the kinetics-directed drug candidate selection and development may have general applicability for ADPKD and other therapeutic areas as well.

Cyst formation and enlargement in autosomal dominant kidney disease (ADPKD) is mainly driven by aberrantly increased cytosolic cAMP in renal tubule epithelial cells. Because the vasopressin V2 receptor (V2R) regulates intracellular cAMP levels in kidneys, a series of benzodiazepine derivatives were developed targeting the V2R. Among these derivatives, compound 25 exhibited potent binding affinity to the V2R (K i = 9.0 ± 1.5 nM) and efficacious cAMP inhibition (IC50 = 9.2 ± 3.0 nM). This led to the suppression of cyst formation and growth in both an MDCK cell model and an embryonic kidney cyst model. Further advancing compound 25 in a murine model of ADPKD demonstrated a significantly improved in vivo efficacy compared with the reference compound tolvaptan. Overall, compound 25 holds therapeutic potential for the treatment of ADPKD.

Background: The cAMP-PKA signaling pathway and TGF-β1-dependent fibrosis pathways are of particular importance in ADPKD progression, but the cross-talk between these pathways remains unclear. Therefore, we used an MDCK-cell model and embryonic kidney-cyst model to study the regulatory role of cAMP-PKA signaling in the TGF-β1 induced fibrotic process. Method and Results: Pkd1^flox/flox; Ksp-Cre and Pkd1^+/+; Ksp-Cre mice were used as an in vivo model. Increased kidney volume, renal cysts formation and up-regulation of the fibrosis-related proteins TGF-β1, connective tissue growth factor (CTGF), and fibronectin (FN) can be observed in Pkd1^flox/flox; Ksp-Cre mice. In an embryonic kidneys-cyst model, TGF-β1, FN and collagen type I were highly expressed. Western blotting revealed the obviously up-regulation of TGF-β1, CTGF, FN and collagen type I expression following forskolin treatment in MDCK cells. Selective PKA inhibition with H89 may partially reversed the above effects. Pretreatment with the TGF-β RI kinase inhibitor VI SB431542 suppressed the increased expression of CTGF, FN and collagen type I caused by forskolin. Our data also indicate that forskolin inhibited TGF-β-induced ERK1/2 phosphorylation and FN up-regulation. ERK inhibition useing PD98059 significantly inhibited the expression of CTGF, FN and collagen type I caused by TGF-β1. Conclusions: The cAMP-PKA signaling pathway can directly promote the production of TGF-β1 and/or TGF-β1-dependent fibrogenetic molecules in MDCK cells and embryonic kidney cysts, but when TGF-β1 and its downstream pathways were highly expressed in MDCK cells, cAMP-PKA had a significantly negative effect on TGF-β1 induced p-ERK1/2 and FN expression.

Background/Aims: A sodium-glucose co-transporter-2 inhibitor dapagliflozin is widely used for lowering blood glucose and its usage is limited in type 2 diabetes mellitus patients with moderate renal impairment. As its effect on kidney function is discrepant and complicated, the aim of this study is to determine the effect of dapagliflozin on the progression of diabetic nephropathy and related mechanisms. Methods: Twelve-week-old male C₅₇BL/6 wild-type and db/db mice were treated with vehicle or 1 mg/kg dapagliflozin for 12 weeks. Body weight, blood glucose, insulin tolerance, glucose tolerance, pyruvate tolerance and 24-hour urine were measured every 4 weeks. At 24 weeks of age, renal function was evaluated by blood urea nitrogen level, creatinine clearance, urine output, urinary albumin excretion, Periodic Acid-Schiff staining, Masson’s trichrome staining and electron microscopy. Changes in insulin signaling and gluconeogenic key regulatory enzymes were detected using Western blot analysis. Results: Dapagliflozin did not alleviate but instead aggravated diabetic nephropathy manifesting as increased levels of microalbuminuria, blood urea nitrogen, and glomerular and tubular damage in db/db mice. Despite adequate glycemic control by dapagliflozin, urinary glucose excretion increased after administration before 24 weeks of age and was likely associated with renal impairment. Increased urinary glucose excretion was mainly derived from the disturbance of glucose homeostasis with elevated hepatic and renal gluconeogenesis induced by dapagliflozin. Although it had no effect on insulin sensitivity and glucose tolerance, dapagliflozin further induced the expression of gluconeogenic key rate-limiting enzymes through increasing the expression levels of FoxO1 in the kidney and liver. Conclusion: These experimental results indicate that dapagliflozin aggravates diabetes mellitus-induced kidney injury, mostly through increasing gluconeogenesis.