Livestock production is a major contributor to nitrogen pollution, accounting for roughly 23 percent of global nitrous oxide and 60 percent of global ammonia emissions. Much of this nitrogen originates in manure, where microbial urease rapidly converts urinary urea into ammonia. The amount of urea a cow excretes depends heavily on the crude protein content of her diet, because rumen microbes break dietary protein down into ammonia, which the liver then detoxifies into urea. That urea leaves the body in urine and feces, but also in milk, where its concentration—milk urea nitrogen—is widely used as a herd-level indicator of whether dietary protein is being fed at optimal levels.

The complication is that individual cows fed identical rations can differ markedly in milk urea concentration. Low milk urea has been proposed as a breeding goal precisely because it was assumed to predict lower urinary nitrogen losses and a smaller nitrogen footprint. Yet previous work from the same group showed that although cows with high milk urea tend to have lower renal clearance rates for urea, creatine, creatinine, and uric acid, their total urinary urea and nitrogen excretions are similar to those of low-milk-urea herdmates. In other words, selecting for low milk urea does not automatically deliver the environmental benefit it promises, and the physiological reasons behind the divergent clearance rates remained unresolved.

To probe those mechanisms, researchers at the Research Institute for Farm Animal Biology (FBN) in Dummerstorf, Germany, together with colleagues at the University of Greifswald and the University of Veterinary Medicine Vienna, recruited 18 non-pregnant, multiparous German Holstein cows in their second to fourth lactation. The animals were screened from commercial farms in Mecklenburg-Western Pomerania and paired into nine blocks, each containing one cow with consistently high milk urea (averaging 285.9 milligrams per liter) and one with consistently low milk urea (averaging 190.5 milligrams per liter). Milk yield was comparable between groups at roughly 32 kilograms per day, and body condition scores were matched, ensuring that differences in kidney function could not be attributed to differences in productivity or body reserves.

After a two-week adaptation to a standardized total mixed ration containing 15 percent crude protein, the cows moved into a climate-controlled tie-stall facility where they were fitted with jugular catheters and urine collection devices. Blood and acidified urine were sampled over four days, allowing the team to calculate renal clearance rates and reabsorption ratios for sodium, chloride, lactate, creatinine, and nearly every proteinogenic free amino acid. On the fifth day the animals were slaughtered, and tissue from the renal cortex and medulla was flash-frozen for proteomic analysis. Using high-resolution mass spectrometry on gel-separated proteins, the researchers identified 6,793 kidney proteins, of which 4,463 passed quality filtering for statistical comparison.

The clearance results were strikingly uneventful. Sodium, chloride, lactate, creatinine, and virtually all free amino acids were cleared and reabsorbed at statistically indistinguishable rates in the two groups, with reabsorption ratios hovering near or above 0.98 for most compounds—meaning the kidneys reclaim almost everything the blood delivers. The single exception was carnosine, a histidine-and-beta-alanine dipeptide released mainly from skeletal muscle. High-milk-urea cows carried significantly more carnosine in their plasma (19.5 versus 13.2 micromoles per liter) yet tended to clear it more slowly, a paradox that pointed toward a transport bottleneck rather than a filtration problem.

That bottleneck found a molecular explanation in the proteome. The peptide transporter SLC15A2, also known as PEPT2, which shuttles carnosine and other di- and tripeptides across tubular membranes, was expressed at lower levels in the kidneys of high-milk-urea cows. Because PEPT2 is a high-affinity but low-capacity transporter, its transport rate barely rises even when plasma carnosine climbs, so reduced expression combined with elevated circulating carnosine plausibly accounts for the impaired clearance. The authors also note that increased release of carnosine from muscle tissue could contribute to the higher plasma concentrations, a hypothesis they flag for future testing.

Several other solute carriers shifted between the groups without producing measurable functional consequences. High-milk-urea cows showed reduced abundance of transporters for neutral amino acids (SLC7A8), organic anions (SLC22A8), chloride (SLC12A9), choline (SLC44A1), glucose (SLC2A2), lactate (SLC16A7), and peptides (SLC15A2), alongside increased expression of the anion exchanger SLC4A1 and the citrate transporter SLC25A1. Yet plasma and urinary concentrations of the corresponding substrates were unchanged, and clearance and reabsorption ratios were identical. The researchers suggest that transporters distributed unevenly along the nephron—some residing in tissue regions not sampled—may compensate, and they caution that total protein abundance does not reveal how many transporters are actually active in the tubular membrane.

The metabolic rewiring ran deeper than transport. Pathway enrichment analysis showed that in high-milk-urea cows the proteasome, endocytosis, and reactive oxygen species pathways were upregulated, while thyroid hormone signaling, aminoacyl-tRNA biosynthesis, and several metabolic pathways were downregulated. Notably, arginase 1 (ARG1), the urea-cycle enzyme that converts arginine to ornithine and urea, was less abundant, and plasma citrulline was elevated while ornithine and arginine remained stable—together suggesting diminished local urea cycling and reduced ammonia detoxification by kidney cells. A tendency toward lower urinary glutamate, a key carrier of amine groups feeding the urea cycle, reinforced this interpretation.

The activated proteasomal system, reflected in increased 20S alpha subunits and 11S regulators, may serve a protective role: the proteasome degrades misfolded and oxidized proteins that would otherwise accumulate at the glomerular filtration barrier and clog it. The authors propose that in cows with lighter kidneys, heightened proteasomal and endocytic activity—fueled by upregulated glycolysis, glycine-serine-threonine metabolism, and oxidative phosphorylation—compensates for the reduced filtration surface to maintain filter capacity. Whether this increased burden could eventually exceed proteasomal capacity and trigger proteotoxic stress, and whether the accompanying reactive oxygen signaling is cause or consequence, remain open questions. The team also acknowledges the study’s limitations: proteomics captures total abundance rather than membrane localization or enzyme activity, and with only nine animals per group, statistical power was limited and multiple-testing corrections could not be applied. Larger populations and functional follow-up studies will be needed before the findings can inform breeding programs that currently use milk urea as a proxy for nitrogen efficiency.

Subject of Research: Renal clearance and kidney proteome differences in dairy cows with divergent milk urea concentrations

Article Title: Renal clearance rate, estimated renal reabsorption ratio, and kidney proteome of Holstein dairy cows with divergent milk urea concentrations

Article References: Koch, F., Albrecht, D., Görs, S., Müller-Kiedrowski, C. B. M., Wimmers, K., & Kuhla, B. (2026). Renal clearance rate, estimated renal reabsorption ratio, and kidney proteome of Holstein dairy cows with divergent milk urea concentrations. Frontiers in Molecular Biosciences, 13, 1819426. https://doi.org/10.3389/fmolb.2026.1819426

Image Credits: AI Generated

DOI: 10.3389/fmolb.2026.1819426

Keywords: dairy cows, milk urea nitrogen, kidney, renal clearance, proteomics, urea cycle, carnosine, PEPT2 transporter, nitrogen excretion, Holstein cattle, proteasome, amino acids