Evaluation of pancreatic β-cell functions is an important issue for precise management of diabetes. C-peptide, the molecule produced at an equimolar concentration by proinsulin cleavage, has been used as a superior biomarker over insulin in ordinary clinical settings because C-peptide has a longer half-life, higher serum concentration, and less variable peripheral clearance than insulin (1,2). Furthermore, C-peptide is principally cleared by the kidneys, with 5–10% being excreted unchanged in urine, unlike insulin (3). Thus, the C-peptide concentration in urine collected for a day is a useful marker in evaluation of β-cell functions throughout a day.

However, we experienced a patient in whom the urine C-peptide concentration appeared to be increased substantially upon administration of a neprilysin inhibitor. On 24 November 2021, a 47-year-old female patient with diabetes was admitted to our hospital for better glycemic control. Her fasting plasma glucose and HbA1c levels on the admission day were 172 mg/dL and 9.4%, respectively, with the use of oral semaglutide. She also had uncontrolled hypertension. Thus, sacubitril valsartan sodium hydrate or a dual angiotensin receptor–neprilysin inhibitor (ARNi) was administered from day 8 of admission. To evaluate pancreatic β-cell functions, urinary C-peptide excretion for a day was measured on day 14 of admission, which revealed inconceivably high urinary C-peptide excretion (1,079.4 μg/day despite a normal serum C-peptide concentration [4.62 ng/mL] as measured by electrochemiluminescence immunoassay). As described above, the patient was taking the ARNi for treatment of hypertension. Neprilysin hydrolyzes various peptide hormones and is mainly expressed in proximal convoluted tubules (3). Thus, we speculated that the nephritic inhibitor was the cause of such high urinary C-peptide excretion. To evaluate this idea, urine C-peptide–to–creatinine ratios were remeasured at 16 days after discharge from our hospital or 14 days after withdrawal of the drug because of hypotensive symptoms probably caused by the drug. This strengthened our idea, as the urine C-peptide–to–creatinine ratio had decreased within the normal range after the withdrawal (from 735.6 to 61.0 μg/g Cre).

Additionally, to confirm whether the findings in this patient could be generalized, we next investigated whether such discrepancies between urine C-peptide–to–creatinine ratio and serum C-peptide concentration were found in other subjects with diabetes. Among patients with diabetes who attended our hospital from 1 January 2021 to 31 December 2021, five more patients were found to be taking the ARNi and had measurements of urine C-peptide–to–creatinine ratio and serum C-peptide concentration. In these patients, the urine C-peptide–to–creatinine ratios were also inconceivably high (mean [range] 442.1 μg/g Cre [198.2–744.6]) despite normal serum C-peptide concentrations (5.58 ng/mL [3.79–7.57]) (Fig. 1). In addition, none of the patients reported here had severe kidney dysfunction or estimated glomerular filtration rate <30 mL/min/1.73 m2 (lowest, 35.7).

Figure 1

Discrepant C-peptide concentrations between serum and urine of each patient. Serum and urine C-peptide concentrations of each patient are plotted separately and connected with lines. The urine C-peptide concentration after withdrawal of the ARNi of the index patient is separately plotted as “off,” and those observed in patients treated with the ARNi are shown as “on.”

Figure 1

Discrepant C-peptide concentrations between serum and urine of each patient. Serum and urine C-peptide concentrations of each patient are plotted separately and connected with lines. The urine C-peptide concentration after withdrawal of the ARNi of the index patient is separately plotted as “off,” and those observed in patients treated with the ARNi are shown as “on.”

Close modal

Together, these findings clearly indicated that the neprilysin inhibitor increased urine excretion of C-peptide to a disproportionally high level relative to the serum C-peptide concentration. Although kidneys are a major site for C-peptide clearance, the detailed mechanism and related enzymes have not been well elucidated to date. Neprilysin is a membrane zinc-dependent metallopeptidase that hydrolyzes various substrates, including a range of peptides up to 40–50 amino acids in length, such as atrial natriuretic peptide family, substance P, bradykinin, somatostatin, glucagon, glucagon-like peptide 1, and insulin B chain (4,5). To date, although a growing number of peptides are now considered such substrates, C-peptide has not been well evaluated. The findings observed here indicate that C-peptide is a substrate of neprilysin. Therefore, neprilysin may hydrolyze C-peptide in the kidneys, and neprilysin inhibition attenuates this process and increases urine C-peptide excretion. Furthermore, although neprilysin is expressed abundantly in the kidney brush border membrane, its effects on the urine concentration of any peptide have not been well evaluated. Therefore, this study appears to warrant further analyses to examine the role of neprilysin in C-peptide metabolism.

Here, we report that neprilysin inhibition may substantially increase the urinary C-peptide excretion that has been thought to be less varied than insulin depending on condition, including drugs taken. Therefore, evaluation of the urinary C-peptide excretion must include careful consideration of drugs taken, especially neprilysin inhibitors or ARNis; otherwise, pancreatic β-cell functions may be overestimated. Further, the results may open a new research field for evaluation of the mechanism involved in the urine C-peptide increment.

Acknowledgments. The authors thank Mitchell Arico from Edanz for editing a draft of the manuscript.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. Y.N. and M.D. wrote the manuscript. Y.N., S.M., M.Y., J.T., E.S., and K.Matsum. contributed to case curation and data acquisition. Y.N. and M.D. contributed to data interpretation. All authors reviewed and edited the manuscript and approved the final version of the manuscript. M.D. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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