Apelin inhibits cyst growth and improves kidney function in mice with polycystic kidney disease

This study demonstrates that diminished apelin levels are associated with autosomal dominant polycystic kidney disease (ADPKD) and that exogenous apelin receptor activation inhibits cyst growth and improves kidney function in ADPKD mice, suggesting the apelin receptor as a promising therapeutic target.

The Gist

The Big Picture: A Leaky Faucet and a Missing Brake

Imagine your kidneys are like a sophisticated water filtration plant. In a healthy person, the pipes (tubules) are the right size, and the water flows smoothly.

But in a disease called Polycystic Kidney Disease (PKD), something goes wrong. The pipes start to sprout massive, fluid-filled balloons called cysts. Think of these cysts as balloons inflating inside a crowded room. As they grow, they push against the healthy pipes, crushing them and eventually causing the whole filtration plant to shut down. This leads to kidney failure.

Currently, the only approved medicine (Tolvaptan) works by turning off the "water faucet" to stop the balloons from filling up. However, it has a major side effect: it makes patients pee constantly, leading to extreme thirst and dehydration. It's like fixing a leaky roof by opening a window in the middle of a blizzard—you stop the leak, but you freeze.

This new study asks: Is there a way to stop the balloons from growing without making people pee their pants?

The Discovery: The Missing "Apelin" Signal

The researchers discovered that people with PKD are missing a crucial chemical messenger in their blood called Apelin.

• The Analogy: Imagine the kidney cells have a "Stop" button. In healthy people, Apelin is the finger that presses this button. In PKD patients, that finger is missing or weak. Without it, the cells get confused and start multiplying uncontrollably, inflating those cyst balloons.
• The Finding: The study confirmed that even in young people with PKD whose kidneys still work fine, their Apelin levels are already dangerously low.

The Experiment: Testing the "Remote Control"

The team decided to see what would happen if they gave the kidneys a remote control (a drug) to press that "Stop" button for them. They tested two types of remotes:

1. Apelin: The natural peptide (the original remote).
2. Azelaprag: A small molecule drug (a generic, third-party remote).

They tested these on:

• Lab-grown kidney cells (in a petri dish).
• Mice that have a genetic version of PKD.

The Results: A Miracle Cure? (Almost)

Here is what happened when they pressed the "Stop" button:

1. The Natural Remote (Apelin) Worked Wonders:

• In the Lab: When they added Apelin to the growing cysts, the balloons stopped expanding.
• In the Mice: The treated mice had smaller kidneys, fewer cysts, and better kidney function (lower blood urea nitrogen) compared to untreated mice.
• The Mechanism: Apelin worked by turning down the internal "gas pedal" (a chemical called cAMP) that tells the cells to grow. It also turned off the "construction crew" (ERK signaling) that builds the cysts.

2. The Generic Remote (Azelaprag) Was a Mixed Bag:

• It worked well in the petri dish to stop cyst growth.
• But in the mice, it failed. The mice treated with Azelaprag didn't see the same kidney improvements.
• Why? The researchers suspect the drug gets metabolized (broken down) by the liver before it can reach the kidneys in high enough doses, or it might not trigger the exact same complex signals as the natural Apelin.

3. The Best Part: No "Pee Party" Side Effects!

• The current drug (Tolvaptan) works by blocking a different receptor, which causes the kidneys to dump water, leading to constant urination.
• The researchers were worried that Apelin might do the same thing. It didn't.
• The Analogy: Tolvaptan is like opening a floodgate to stop a leak. Apelin is like tightening the bolt on the pipe. The mice treated with Apelin kept their normal urine output and concentration. They didn't get dehydrated.

The "Secret Sauce": What's Happening Inside?

The researchers looked at the genetic "instruction manuals" inside the kidney cells. They found that Apelin didn't just stop growth; it actually re-wrote the instructions.

• It turned off genes that cause inflammation and scarring (fibrosis).
• It turned on genes that help the cells manage their energy and fats properly.
• Essentially, Apelin didn't just put a bandage on the problem; it helped the kidney cells return to a healthier, more normal state.

The Conclusion: A New Hope

This study suggests that Apelin is a promising new target for treating PKD.

• Why it matters: It stops the cysts from growing.
• Why it's better: It improves kidney function without causing the terrible side effect of constant urination.
• The Catch: The natural Apelin peptide breaks down too fast in the human body to be used as a pill. The small-molecule drug (Azelaprag) didn't work in the mice at the tested dose.

The Takeaway: The researchers are essentially saying, "We found the missing piece of the puzzle. Now, we need to invent a better, more stable 'remote control' (a new drug) that can deliver this Apelin signal to human kidneys without getting destroyed on the way."

If they can find that perfect drug, it could be a game-changer for the millions of people living with Polycystic Kidney Disease, offering a treatment that saves their kidneys without making them run to the bathroom every 10 minutes.

Technical Summary

1. Problem Statement

• Disease Context: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a leading genetic cause of end-stage renal disease (ESRD), characterized by the progressive growth of renal cysts that impair kidney function.
• Current Limitations: Tolvaptan, the only FDA-approved drug for ADPKD, acts as a V2 receptor antagonist to inhibit cAMP-driven cyst growth. However, it causes significant side effects, including polyuria (excessive urination), nocturia, polydipsia, and potential liver toxicity. Furthermore, it offers no direct cardiovascular benefit, despite cardiovascular disease being a major complication in ADPKD patients.
• The Gap: There is an urgent need for novel therapeutic targets that can inhibit cystogenesis without the diuretic side effects of V2R antagonists and potentially offer cardiovascular protection.
• Hypothesis: The authors hypothesized that diminished apelin signaling contributes to ADPKD pathogenesis. Since the apelin receptor (APJ) signals via inhibitory G-proteins ($G\alpha_i$) to reduce cAMP, exogenous activation of this receptor might inhibit cyst growth while avoiding the adverse diuretic effects associated with V2R blockade.

2. Methodology

The study employed a multi-faceted approach combining human clinical data, in vitro cell models, and in vivo mouse models.

• Human Clinical Analysis:

  • Cohort: Plasma samples from 100 ADPKD patients (children and young adults) and 20 age/sex-matched healthy controls from the Early PKD Observation Cohort (EPOC).
  • Measurements: Circulating apelin levels were measured via ELISA. Correlations were analyzed against eGFR, BUN, creatinine, and total kidney volume (TKV).
  • Transcriptomics: Microarray data from ADPKD kidneys were analyzed for expression of APLN (apelin), APLNR (receptor), and APELA (Elabela).

• In Vitro Experiments:

  • Cell Model: Primary human ADPKD renal epithelial cells.
  • Assay: 3D collagen-embedded microcyst assay. Cysts were induced with forskolin (cAMP agonist) and EGF.
  • Treatment: Cells were treated with the peptide agonist [Pyr1]apelin-13 and the small-molecule agonist Azelaprag (AMG986) to assess effects on cyst volume and number.

• In Vivo Mouse Models:

  • Model: Pkd1^RC/RC; Pkd2^+/- mice, an orthologous model of early-onset, rapidly progressive PKD.
  • Intervention: Mice were treated from postnatal day 22 to 48 with:
    • Vehicle (Control)
    • [Pyr1]apelin-13 (2 mg/kg, IP)
    • Azelaprag (2 mg/kg, IP)
    • Mozavaptan (V2R antagonist, positive control)
  • Endpoints: Kidney weight-to-body weight ratio (KW/BW), cystic index, Blood Urea Nitrogen (BUN), renal cAMP levels, ERK1/2 phosphorylation, and histological analysis (fibrosis, inflammation).
  • Diuresis Test: Wild-type mice were treated with agonists to measure urine volume and osmolality, comparing effects against Mozavaptan.

• Omics and Molecular Analysis:

  • RNA Sequencing: Bulk RNA-seq performed on kidneys from vehicle vs. apelin-treated mice to identify differentially expressed genes (DEGs).
  • Validation: qPCR and Western blotting for specific markers (e.g., Lcn2, Postn, Havcr1, pERK).

3. Key Results

• Human Biomarker Findings:

  • Circulating apelin levels were significantly reduced in ADPKD patients compared to healthy controls, even in patients with normal eGFR and renal function.
  • Apelin levels inversely correlated with disease severity markers (htTKV) but showed no correlation with current renal function (eGFR, BUN).
  • Transcriptomic analysis of human kidneys showed reduced APLNR (receptor) expression in large cysts and reduced APELA expression across all cyst sizes.

• In Vitro Cyst Growth:

  • Both [Pyr1]apelin-13 and Azelaprag caused a dose-dependent reduction in cyst volume in 3D human ADPKD cell cultures.
  • [Pyr1]apelin-13 reduced cyst volume but did not significantly reduce cyst number, whereas Azelaprag reduced both volume and number.

• In Vivo Therapeutic Efficacy (Mouse Model):

  • [Pyr1]apelin-13: Significantly reduced kidney weight-to-body weight ratio (KW/BW), cystic index, and cyst number. It also significantly lowered BUN (improving renal function).
  • Mozavaptan: Confirmed efficacy in reducing KW/BW, cystic index, and BUN.
  • Azelaprag: Surprisingly, while effective in vitro, Azelaprag failed to reduce KW/BW or cystic index in vivo at the tested dose, though it did reduce cyst number.

• Mechanistic Insights:

  • cAMP and ERK: Treatment with [Pyr1]apelin-13 and Mozavaptan significantly reduced renal cAMP levels and phosphorylated ERK1/2 (pERK) in PKD mice. Azelaprag did not reduce cAMP or pERK in vivo.
  • Gene Expression: Apelin treatment downregulated genes associated with cyst progression and inflammation (Lcn2/Ngal, Postn, Havcr1/Kim-1, Timp1, Mmp14) and upregulated genes involved in lipid metabolism (Acaa1b, Dio1).
  • Inflammation: Apelin treatment showed a trend toward reducing inflammatory markers (Il1β, Ccl2, Tnfα).

• Safety Profile (Diuresis):

  • Crucial Distinction: Unlike Mozavaptan, which caused significant diuresis (increased urine volume) and reduced urine osmolality, [Pyr1]apelin-13 did not induce diuresis or alter urine osmolality in wild-type mice at therapeutic doses.
  • Azelaprag showed a mild diuretic effect only at a higher dose (4 mg/kg), but not at the therapeutic dose used in PKD mice.

4. Key Contributions

1. Novel Biomarker: Identified circulating apelin as a potential early prognostic biomarker for ADPKD, noting its reduction occurs before detectable loss of renal function.
2. Therapeutic Validation: Demonstrated that exogenous apelin receptor activation effectively inhibits cyst growth and improves renal function in a severe orthologous mouse model of PKD.
3. Mechanistic Differentiation: Provided evidence that apelin-mediated cyst inhibition occurs via cAMP/ERK suppression but dissociates from the diuretic mechanism of V2R antagonists. This suggests apelin signaling may utilize distinct cAMP pools (e.g., primary cilia) or G-protein coupling (G$\alpha_q$ vs G$\alpha_i$) that spare water reabsorption pathways.
4. Transcriptomic Profiling: Mapped the gene expression changes induced by apelin, highlighting the downregulation of fibrosis and inflammation markers, suggesting a broader disease-modifying effect beyond simple volume reduction.

5. Significance and Implications

• Clinical Potential: The apelin receptor represents a promising novel therapeutic target for ADPKD. Unlike Tolvaptan, apelin agonists may offer cyst growth inhibition without the debilitating side effect of polyuria, potentially improving patient compliance and quality of life.
• Cardiovascular Benefit: Given the known vasodilatory and anti-hypertrophic effects of apelin on the heart, targeting this receptor could simultaneously address the high cardiovascular risk prevalent in ADPKD patients.
• Drug Development Challenges: The study highlights a discrepancy between the peptide agonist ([Pyr1]apelin-13) and the small molecule (Azelaprag). While the peptide worked well in vivo, the small molecule failed to show efficacy at the tested dose, possibly due to biodistribution issues (hepatic metabolism) or biased signaling (G$\alpha_i$ only vs. G$\alpha_i$/G$\alpha_q$). This underscores the need for developing new, kidney-targeted small-molecule apelin agonists.
• Future Directions: The authors propose that further studies are needed to optimize small-molecule agonists for ADPKD and to evaluate long-term efficacy in models that progress to renal failure.

Conclusion: The study establishes that apelin signaling is downregulated in ADPKD and that restoring this signaling via receptor activation inhibits cyst growth, reduces cAMP/ERK signaling, and improves kidney function without causing diuresis, positioning the apelin receptor as a safe and effective potential therapeutic avenue.