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The natriuretic peptide (NP) family—comprising atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), D-type natriuretic peptide (DNP), and urodilatin (UNP)—plays an indispensable role in the regulation of hypertension and cardiovascular function. Unlike ANP and BNP—whose core function is the promotion of sodium excretion—CNP does not participate in sodium elimination; instead, it primarily functions as a local autocrine or paracrine signaling molecule to regulate chondrocyte differentiation and promote longitudinal bone growth, induce vascular smooth muscle relaxation to lower blood pressure, inhibit neuronal excitability, and facilitate oocyte maturation.

The receptors for the various NPs are classified into three types: NP Receptor A (NPRA/NPR1), NP Receptor B (NPRB/NPR2), and NP Receptor C (NPRC/NPR3). The CNP-NPR2 signaling system constitutes a pivotal pathway for regulating longitudinal bone growth, cardiovascular homeostasis, and neurological and reproductive functions; a loss of function within this system directly results in chondrodysplasia, rendering the activation of NPR2 (e.g., through CNP analogs) an effective therapeutic strategy for treating this condition. Acting as a local paracrine signal, this pathway functionally complements the endocrine signaling of ANP and BNP, thereby collectively maintaining physiological homeostasis. Furthermore, the specific catalytic activity of NPR2 regarding cGMP presents a novel therapeutic target for the treatment of diseases such as cardiovascular fibrosis.

The HEK293 Human NPR2 Effector Reporter Cell Model—effectively simulates the signal transduction process of NPR2 *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the HEK293 Human NPR2 Effector Reporter Cell  Model

Figure 2. Dose Response of CNP(1-22), human TFA in NPR2 Effector Reporter Cell(C6).

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed  HEK293 Human NPR2 Effector Reporter Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.