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Investigating Citric Acid Cycle metabolism in
Chronic Kidney Disease

A metabolic hypothesis involving CAC intermediates, nitrogen metabolism and acid-base balance.

The Origin

Everyday proteins we consume are synthesized and degraded into amino acids (AA) that contain nitrogen (N). Since amino acids can not be stored, they are catabolized in the liver, loosing their amino group (NH2) through deamination and forming ammonia (NH3). In the aqueous medium of the organism, at the physiological pH, ammonia occurs as ammonium ion (NH4+). Ammonium is converted to urea through the Urea Cycle in the liver.

In healthy people, urea is filtered in the renal glomeruli and mostly excreted in the urine. It corresponds to approximately 90-95% of the total nitrogen excretion. Alternately to renal excretion, expelling of urea and other low molecular weight nitrogen compounds is also carried out through sweat, feces, and as ammonium through the urine.

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Patients with Chronic Kidney Disease (CKD) experience a decreased filtration rate given the loss of kidney function. Therefore, urea, ammonium and other nitrogenous compounds can not be excreted in full or as necessary in relation to the amount of proteins consumed, which degrade into nitrogen-containing amino acids. Thus, CKD patients are prescribed low-protein diets which typically range from 0.6 to 0.8 grams of protein per kilo of weight per day.

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  • According to a diet restricted to 0.6 grams of protein per kilo of weight per day, a person of 70 kilos of weight would ingest 42 grams of protein, which would provide 6.72 grams of nitrogen (equivalent to 480 millimoles of nitrogen).

  • In the case of a diet of 0.8 grams of protein per kilo of weight per day, a person of 70 kilos of weight would ingest 56 grams of protein, which would provide 8.96 grams of nitrogen (equivalent to 640 millimoles of nitrogen).

Proteins

to AA

Urea Toxicity

Since patients with Chronic Kidney Disease suffer from a reduced glomerular filtration rate (GFR), their kidneys can not excrete sufficient urea, ammonium and other nitrogenous compounds. Thus, these toxic substances begin to accumulate in the blood up to a serious point causing uremia. Guidelines suggest that when the patient's GFR lowers to between 5 to 10 mL/min, the patient must begin with a renal replacement treatment (RRT), such as peritoneal dialysis (PD), hemodialysis (HD) or renal transplant (from living or deceased donor kidneys). At this point, classified as End-Stage Renal Disease (ESRD), quantity and quality of life become severely detrimental. Life-expectancy with dialysis is well known and documented, as well as the side-effects and life-threatening complications of the established RRT. 

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Retention of nitrogenous waste products is a characteristic feature of advanced CKD and contributes to the biochemical environment associated with uremia.

Elevated urea concentrations have increasingly been recognized as biologically active rather than merely a filtration marker. Experimental and clinical literature has associated urea and urea-derived carbamylation with oxidative stress, metabolic dysfunction and adverse cardiovascular outcomes in CKD.

  • “Higher BUN [blood urea nitrogen] levels were identified as a risk factor for kidney disease progression in patients with moderate to severe CKD, independent of eGFR” [1].

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  • “Urea can exert direct toxicity to various tissues, such as the intestinal epithelium, vascular walls, pancreatic β cells, and adipocytes, and indirect toxicity through carbamylation” [2].

urea molecule

Urea

  • “Urea slowly dissociates into cyanate, which is rapidly converted to isocyanate. […] Carbamylation has been recognized as a spontaneous post-translational modification of amino acids and proteins mediated by cyanate, which leads to biochemical alterations” [1].

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  • “Hypercarbamylation is present in all stages of CKD before and after the start of hemodialysis” [3].

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  • “Urea induces the production of reactive oxygen species (ROS) in adipocytes, leading to insulin resistance” [4].

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  • “The insulin-secreting defects associated with CKD arise from elevated circulating levels of urea that increase the islet protein O-GlcNacylation and impair glycolysis” [5​].

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  • Carbamylated proteins have been reported to be associated with general mortality and cardiovascular mortality in patients with End-Stage Renal Disease (ESRD) [3,6,7​​].

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  • "Carbamoylation has important effects on the immune system, atherosclerosis, lipid metabolism, and the progression of chronic kidney disease" [8].

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  • "​​As carbamoylated amino acids cannot be used for protein synthesis, carbamoylation might contribute heavily to amino acid deficiencies and, thus, to protein malnutrition" [8].

Our Approach

Our approach to Chronic Kidney Disease lives on the understanding of the Tricarboxylic Acid Cycle (TAC) -also known as Citric Acid Cycle (CAC) and Krebs Cycle- and its intertwined relationship with the Urea Cycle. The Krebs Cycle is a universal metabolic process that takes place not only in the human being, but in the three domains of life on earth: 1) domain eukarya, where animalia, plantae, fungi, and protista kingdoms are included; 2) domain archaea, and 3) domain bacteria.

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The Krebs Cycle should not be viewed as a closed circle, since several compounds enter and exit the cycle and intermediates of the cycle connect to other metabolic pathways. The Krebs Cycle is both catabolic and anabolic in nature, thus regarded as amphibolic.

The Citric Acid Cyle

a) Catabolic pathway, in which nutrients such as proteins via amino acids, carbohydrates via glucose, triacylglycerols via glycerol and fatty acids, end up as pyruvate before entering the cycle. By losing a carbon and generating CO2, pyruvate is converted to acetyl coenzyme A and when it binds with oxaloacetate it regenerates citrate to enter the Krebs Cycle, generating energy through the formation of ATP and various electron carriers like NAD and FAD.

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b) Anabolic pathway, in which certain intermediates of the Krebs Cycle are used for the biosynthesis of monomeric molecules. Thus, for glucose biosynthesis (gluconeogenesis), oxaloacetate is used, and for biosynthesis of fatty acids we use acetyl coenzyme A. For the biosynthesis of non-essential amino acids (glycine, L alanine, L asparagine, L aspartate, L cysteine, L glutamate, L glutamine, L proline, L serine and L tyrosine), oxaloacetate intermediate is used as the initiator of synthesis. Another cycle intermediate is alpha-ketoglutarate which serves as the initiator to form glutamate by reversible transamination. Then, by additional transamination forms glutamine, as well as other related amino acids.

These amino acid biosynthetic pathways, glucose and fatty acids, are pathways that extract intermediates from the cycle and are known as cataplerotic pathways or reactions, which hypothetically speaking, when extracting intermediates from the cycle would exhaust it. This is not the case because there are replacement pathways of these intermediates known as anaplerotic pathways, of which the most important is the catabolized by the enzyme pyruvate carboxylase that generates oxaloacetate from pyruvate (reaction 1). Another anaplerotic reaction for the replacement of cycle intermediates involves the direct conversion of phosphoenolpyruvate by the action of the enzyme phosphoenolpyruvate carboxylase to oxaloacetate (reaction 2). Another involves reversible transamination from aspartate to oxaloacetate (reaction 3). By the action of the malic enzyme or malate dehydrogenase on pyruvate, it catalyzes the reductive carboxylation thereof to generate malate (reaction 4). Finally, glutamate generates alpha ketoglutarate by reversible transamination (reaction 5). ​

New studies further prove the intertwined relationship between the Krebs Cycle and Chronic Kidney Disease, since metabolites from the cycle have been proven diminished in CKD patients, both diabetic and non-diabetic etiology.

  • Sharma et al performed a study that "used gas chromatography-mass spectrometry to quantify 94 urine metabolites in screening and validation cohorts of patients with diabetes mellitus (DM) and CKD(DM+CKD), in patients with DM without CKD (DM–CKD), and in healthy controls. Compared with levels in healthy controls, 13 metabolites were significantly reduced in the DM+CKD cohorts, and 12 of the 13 remained significant when compared with the DM–CKD cohort " [9].

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  • ​​Mitochondrial dysfunction is associated with kidney disease in non-diabetic and diabetic contexts [10].

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  • Hallan et al "combined metabolomics (GCMS) with kidney gene expression studies to identify metabolic pathways that are altered in adults with non-diabetic stage 3–4 CKD versus healthy adults. Urinary excretion rate of 27 metabolites and plasma concentration of 33 metabolites differed significantly in CKD patients versus controls [...] Pathway analysis revealed that the citric acid cycle was the most significantly affected, [supporting] the emerging view of CKD as a state of mitochondrial dysfunction" [11].

Mitochondrial dysfunction has emerged as an important component of CKD pathophysiology. Metabolomic and gene-expression studies have reported alterations in Krebs Cycle metabolites and related pathways in both diabetic and non-diabetic kidney disease.

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This observation led our research group to investigate whether supplying selected Krebs Cycle intermediates could represent a complementary metabolic approach in CKD.

Studied Composition

The composition evaluated in the published clinical study consisted of selected Krebs Cycle intermediates—citric, succinic, fumaric and malic acids—in combination with calcium carbonate, calcium lactate and sodium bicarbonate.

Proposed Mechanism

Citric, succinic, fumaric and malic acids, in the body’s aqueous environment, dissociate into their corresponding anions: citrate, succinate, fumarate, and malate. The proposed mechanism is based on replenishing these Krebs-Cycle intermediates and increasing substrate availability for pathways involving α-ketoglutarate and oxaloacetate. In turn, these two intermediates participate in transamination reactions and therefore provide a mechanistic hypothesis linking Krebs Cycle metabolism, nitrogen handling and non-essential amino-acid synthesis.

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The formulation evaluated clinically also included sodium bicarbonate and calcium salts to address acid-base balance and phosphorus binding, respectively.

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The exact contribution of each pathway to the clinical observations remains to be established.

mechanism of action krebs metabolites

Clinical Observations

In a retrospective observational study of 55 patients with stages 3b, 4 and 5 CKD, the following changes were observed during the evaluated period:

  1. increased eGFR​

  2. decreased serum creatinine

  3. decreased serum urea

  4. decreased serum phosphorus

  5. increased serum hemoglobin

  6. albumin remained within normal ranges

The cohort included a predominantly advanced CKD population: 52.7% Stage 5, 36.4% Stage 4 and 10.9% Stage 3b. Mean baseline eGFR was 16.73 mL/min/1.73 m² and mean follow-up was approximately 11 months. [13].

Published Clinical Evidence

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Hernández-Miramontes JA, Méndez-Durán A, Hernández-Villanueva JA. Tricarboxylic Cycle Intermediates in Combination with Calcium Phosphate Chelators and Sodium Bicarbonate Increases eGFR in Patients with Stages 3b, 4 and 5 CKD: A Retrospective Observational Study. Revista Colombiana de Nefrología. 2024;11(2):e778.

READ THE FULL PUBLICATION  > >

Study Design & Limitations

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The 2024 publication was a retrospective observational study involving 55 adult patients with stages 3b, 4 and 5 CKD who were not receiving renal replacement therapy.

The study did not include a concurrent control group. Because of its observational retrospective design and relatively limited sample size and follow-up, the findings should be interpreted as hypothesis-generating and require confirmation in prospective controlled clinical trials.

Interested in our research?

Contact our scientific team for additional clinical information, publications or collaboration opportunities.

Cited References

  1. Seki M. et al. (2019). Blood urea nitrogen is independently associated with renal outcomes in Japanese patients with stage 3–5 chronic kidney disease: a prospective observational study. BMC Nephrology. 20:115.

  2. Vanholder R, Gryp T, Glorieux G. (2018). Urea and chronic kidney disease: the comeback of the century?. Nephrol Dial Transplant. 33:4–12.

  3. Berg AH, Drechsler C, Wenger J, Buccafusca R, Hod T, Kalim S, et al. (2013). Carbamylation of serum albumin as a risk factor for mortality in patients with kidney failure. Sci Transl Med. 5:175ra29.

  4. D’Apolito M, Du X, Zong H, Catucci A, Maiuri L, Trivisano T, et al. (2010). Urea-induced ROS generation causes insulin resistance in mice with chronic renal failure. J Clin Invest. 120:203–13. 

  5. Koppe L, et al. (2016). Urea impairs β cell glycolysis and insulin secretion in chronic kidney disease. The Journal of Clinical Investigation. 126(9), 3598-3612.

  6. Drechsler C, Kalim S, Wenger JB, et al. (2015). Protein carbamylation is associated with heart failure and mortality in diabetic patients with ESRD. Kidney Int. 87:1201–8.

  7. Koeth RA, Kalantar-Zadeh K, Wang Z, Fu X, Tang WH, Hazen SL. Protein carbamylation predicts mortality in ESRD. (2013). J Am Soc Nephrol. 24:853–61.

  8. Delanghe S, et al. (2017). Mechanisms and consequences of carbamoylation. Nature Reviews Nephrology. 1-14.

  9. Sharma K., Karl B., Matthew A.V., et al. (2013). Metabolomics Reveals Signature of Mitochondrial Dysfunction in Diabetic Kidney Disease. J Am Soc Nephrol. 24: 1901–1912.

  10. Forbes MJ, Thorburn DR. (2018). Mitochondrial dysfunction in diabetic kidney disease. Nature Reviews Nephrology. (14) 291-312.

  11. Hallan S, Afkarian M, Zelnick LR, Kestenbaum B, Sharma S, et al. (2017). Metabolomics and gene expression analysis reveal down-regulation of the Citric Acid Cycle in non-diabetic CKD patients. EBioMedicine. (26) 68-77.

  12. Czibik G, Steeples V, Yavari A, et al. (2014). Metabolomics, Citric Acid Cycle intermediates in cardioprotection. Circ Cardiovasc Genet. (7) 711-719.

  13. Hernández-Miramontes JA, Méndez-Durán A, Hernández-Villanueva JA. (2024). Tricarboxylic cycle intermediates in combination with calcium phosphate chelators and sodium bicarbonate increase eGFR in patients with stages 3b, 4 and 5 CKD: a retrospective observational study. Rev Colomb Nefrol. 11(2):1-18. https://revistanefrologia.org/index.php/rcn/article/view/778/1115

  14. Chen YR, Yang Y, Wang SC, et al. (2013). Effectiveness of multidisciplinary care for chronic kidney disease in Taiwan: a 3-year prospective cohort study. Nephrol Dial Transplant. 28:671–682.

  15. Perkovic V, Jardine MJ, Neal B, et al. (2019). Canaglifozin and renal outcomes in type 2 diabetes and nephropathy. N Engl J Med. 380(24): 2295-2306. https://www.nejm.org/doi/full/10.1056/NEJMoa1811744

  16. Heerspink HJL, Stefánsson BV, Correa-Rotter R, et al. (2020). Dapaglifozin in patients with chronic kidney disease. N Engl J Med. 383(15):1436-1445. https://www.nejm.org/doi/full/10.1056/NEJMoa2024816

  17. Herrington WG, Staplin N, Wanner C, et al. (2023). Empaglifozin in patients with chronic kidney disease. N Engl J Med. 388(2):117-126. https://www.nejm.org/doi/full/10.1056/NEJMoa2204233

  18. Bakris GL, Agarwal R, Anker SD, et al. (2020). Effect of finerenone on chronic kidney disease outcomes in type 2 diabetes. N Engl J Med. 383(23):2219-2229. https://www.nejm.org/doi/full/10.1056/NEJMoa2025845

References
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