Endogenous markers of kidney function and renal drug clearance processes of filtration, secretion, and reabsorption

Abstract

The kidneys are responsible for maintaining physiologic homeostasis. The kidneys clear a variety of drugs and other substances through passive (filtration) and active processes that utilize transport proteins. Renal clearance is comprised of the processes of glomerular filtration, tubular secretion, and tubular reabsorption. Endogenous biomarkers, such as creatinine and cystatin C, are routinely used to estimate renal clearance. Understanding the contributing components of renal function and clearance, through the use of biomarkers, is necessary in elucidating the renal pharmacology of drugs and other substances. While exogenous markers of kidney function have been known for decades, several complexities have limited their usage. Several endogenous markers are being evaluated and hold promise to elucidate the individual components of kidney function that represent filtration, secretion, and reabsorption.

Introduction

The kidneys are responsible for maintaining water, electrolyte, and acid-base homeostasis. They are effective at removing metabolic waste products, xenobiotics (drugs and toxins) and maintaining physiologic osmolality (Fig. 1). Renal clearance is characterized as the composite of three processes – glomerular filtration, tubular secretion, and tubular reabsorption (Equation (1)) [1]. While filtration and secretion add substances to the urinary ultrafiltrate, reabsorption removes compounds from the ultrafiltrate. The expanded renal clearance equation includes a term for fraction reabsorbed to account for the negative contribution to clearance (Equation (2)).

$${\text{Cl}}_{\text{R}}={\text{Cl}}_{\text{filtration}}+{\text{Cl}}_{\text{secretion}}-{\text{Cl}}_{\text{reabsorption}}$$ Equation 1

$${\text{Cl}}_{\text{R}}=[\left(\text{Fu}*\text{GFR}\right)+\left[\text{Q}*\text{Fu}*{\text{Cl}}_{\text{i}}/\left[\text{Q}+\left(\text{Fu}*{\text{Cl}}_{\text{i}}\right)\right]\right]*\left(1-\text{Fr}\right)$$ Equation 2

where Cl_R is renal clearance, Fu is the fraction unbound, Q is the renal blood flow, Cl_i is the intrinsic renal clearance, and Fr is fraction of the compound reabsorbed from the tubule lumen.

Figure. 1.

The kidneys maintain physiologic osmolality and remove waste products, drugs, and toxins from the blood through the processes of filtration, secretion, and reabsorption. Compounds must fit within size and charge exclusion properties to be filtered across the glomerulus and into the urinary ultrafiltrate. Transporters in the proximal and distal convoluted tubules secrete compounds from the blood into the ultrafiltrate and reabsorb substances from the ultrafiltrate back into the blood. Adapted from “Kidney Reabsorption and Secretion”, by BioRender.com (2022).

While some small molecular weight drugs are excreted unchanged through the kidneys, metabolism leads to the addition of a functional group which increases the charge and molecular weight, requiring renal transporters for the urinary excretion of most drugs. Secretion and reabsorption of drugs in the kidneys are facilitated by transport proteins in the tubules, leading to unidirectional or bidirectional movement of both organic anions and cations (Fig. 2).

Figure. 2.

Representative renal transporters that are responsible for secretion and reabsorption in proximal tubule cells. Transporters on the basolateral membrane transport molecules from the blood into the proximal tubule cells. Apical transporters are responsible for the efflux of molecules from the proximal tubule cells into the urinary ultrafiltrate. Some transporters work bi-directionally and reabsorb certain molecules from the ultrafiltrate back into the blood. UT: urea transporter; OAT: organic anion transporter; MATE: multidrug and toxin extrusion protein; MRP: multidrug resistance-associated protein; P-gp: P-glycoprotein; URAT: urate transporter; SGLT: sodium-glucose co-transporter; OCT: organic cation transporter.

Glomerular filtration rate (GFR) and creatinine clearance (CrCl) are used for evaluating kidney function and deciding drug dosing requirements (Table 1) [2–4]. Similar to GFR and CrCl equations is the use of serum creatinine as the endogenous marker. Numerous publications have reported on the limitations of serum creatinine for determination of kidney function [5–8]. The use of creatinine to estimate GFR is limited secondary to several factors including muscle mass differences, age, sex, drugs, disease states, diets, and physical activity levels [5,6]. As creatinine is generally assumed to be 85% filtered and 15% secreted, it is primarily used to represent glomerular filtration. Equations to estimate GFR (referred to in the nephrology community as eGFR, representing an estimate) include age, sex, and serum creatinine (Table 1) [9]. Race was removed as a variable from the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula in 2021 following a scientific debate [10].

Table 1.

Equations for estimating kidney function using endogenous markers.

Name	Biomarkers	Equation				References
CKD-EPI Creatinine GFR Equation (2021)	Creatinine	GFR = 142 * (SCr/A)B * 0.9938Age * (1.012 if female)				[10]
		Male		Female
		SCr ≤0.9	A = 0.9 B = −0.302	SCr ≤0.7	A = 0.7 B = −0.241
		SCr >0.9	A = 0.9 B = −1.2	SCr >0.7	A = 0.7 B = −1.2
CKD-EPI Creatinine GFR Equation (2009)	Creatinine	GFR = A * (SCr/B)C * 0.993Age * (1.159 if Black)				[9]
		Male		Female
		SCr ≤0.9	A = 141 B = 0.9 C = −0.411	SCr ≤0.7	A = 144 B = 0.7 C = −0.329
		SCr >0.9	A = 141 B = 0.9 C = −1.209	SCr >0.7	A = 144 B = 0.7 C = −1.209
MDRD GFR Equation (2006)	Creatinine	GFR = 175 * (SCr)−1.154 * Age−0.203 * (1.212 if Black) * (0.742 if female)				[89]
CrCl GFR Equation	Creatinine	GFR ≈ CrCl = (UCr * V)/SCr				[90]
Cockcroft-Gault CrCl Equation (1976)	Creatinine	CrCl = (140 – Age) * (Weight) * (0.85 if female)/(72 * SCr)				[30]
CKD-EPI Cystatin C GFR Equation (2012)	Cystatin C	GFR = 133 * (Scys/A)B * 0.9938Age * (1.012 if female)				[37]
		Male		Female
		A = −0.499 B = 1		A = −0.499 B = 0.932
CKD-EPI Creatinine-Cystatin C GFR Equation (2021)	Creatinine, Cystatin C	GFR = 135 * (SCr/A)B * (Scys/C)D * 0.9961Age * (0.963 if female)				[10]
		Male:
				SCr ≤0.9	SCr >0.9
		Scys ≤0.8		A = 0.9 B = −0.144 C = 0.8 D = −0.323	A = 0.9 B = −0.544 C = 0.8 D = −0.323
		Scys >0.8		A = 0.9 B = −0.144 C = 0.8 D = −0.778	A = 0.9 B = −0.544 C = 0.8 D = −0.778
		Female:
				SCr ≤0.7	SCr >0.7
		Scys ≤0.8		A = 0.7 B = −0.219 C = 0.8 D = −0.323	A = 0.7 B = −0.544 C = 0.8 D = −0.323
		Scys >0.8		A = 0.7 B = −0.219 C = 0.8 D = −0.778	A = 0.7 B = −0.544 C = 0.8 D = −0.778
CKD-EPI Creatinine-Cystatin C GFR Equation (2012)	Creatinine, Cystatin C	GFR = A * (SCr/B)C * (Scys/0.8)D * 0.995Age * (1.08 if Black)				[37]
		Male:
				SCr ≤0.9	SCr >0.9
		Scys ≤0.8		A = 135 B = 0.9 C = −0.207 D = −0.375	A = 135 B = 0.9 C = −0.601 D = −0.375
		Scys >0.8		A = 135 B = 0.9 C = −0.207 D = −0.711	A = 135 B = 0.9 C = −0.601 D = −0.711
		Female:
				SCr ≤0.7	SCr >0.7
		Scys ≤0.8		A = 130 B = 0.7 C = −0.248 D = −0.375	A = 130 B = 0.7 C = −0.601 D = −0.375
		Scys >0.8		A = 130 B = 0.7 C = −0.248 D = −0.711	A = 130 B = 0.7 C = −0.601 D = −0.711
CKD-EPI B2M GFR Equation (2016)	B2M	GFR = 133 * B2M−0.852				[40]
CKD-EPI BTP GFR Equation (2016)	BTP	GFR = 55 * BTP−0.695 * 0.998Age * (0.899 if female)				[40]
CKD-EPI BTP-B2M GFR Equation (2016)	B2M, BTP	GFR = 96 * B2M−0.588 * BTP−0.278				[40]
CKiD Pediatric GFR Equation (2012)	Creatinine, Cystatin C, Urea	GFR = 39.8 * (Height/SCr)0.456 * (1.8/Scys)0.418 * (30/BUN)0.079 * (Height/1.4)0.179 * (1.076 if male)				[62]
Hippurate RPF Equation	Hippurate	RPF = (UH * V)/PH				[67]
Renal Clearance Equation	Secretory solutes	ClR = (UEx from 0 to 24 h)/(AUC Px from 0 to 24 h)				[71,75]
Net Renal Secretion Clearance Equation	Secretory solutes	Clsec = ClR − Fu * GFR				[74]
Fractional Excretion Equation	Secretory solutes	FEx = (Ux * PCr)/(Px * UCr)				[72]
Tubular Reabsorption Equation	Reabsorbed solutes	FEx = (Ux/Px)/(UCr/PCr)				[77]
Tubular Reabsorption of Phosphate Equation	Phosphate	TRP (%) = 1 − [(UP/PP) * (PCr/UCr)]				[88]

CKD-EPI: Chronic Kidney Disease Epidemiology Collaboration; MDRD: Modification of Diet in Renal Disease; CrCl: creatinine clearance; GFR: glomerular filtration rate; B2M: beta-2 microglobulin; BTP: beta-trace protein; CKiD: Chronic Kidney Disease in Children; RPF: renal plasma flow; SCr: serum creatinine; UCr: urinary creatinine; V: urine volume; SCys: serum cystatin C; BUN: blood urea nitrogen; U_H: urinary hippurate; P_H: plasma hippurate; Cl_R: renal clearance; UE_x: urinary excretion amount of solute x; AUC: area under the curve; P_x: plasma concentration of x; Cl_sec: net renal secretion clearance; Fu: fraction unbound; FE_x: fractional excretion of x; U_x: urine concentration of x; PCr: plasma creatinine; TRP: tubular reabsorption of phosphate; U_P: urinary phosphate; P_P: plasma phosphate.

There has been a renewed interest within the nephrology community to find improved markers of kidney function and more precisely differentiate the individual renal clearance components. There is increasing recognition of the limitations of the markers currently in use [3,4,11]. This review will briefly discuss exogenous markers of renal filtration and then highlight the most contemporary literature related to endogenous markers of glomerular filtration, tubular secretion, and tubular reabsorption (Table 2).

Table 2.

Endogenous markers of kidney function.

Biomarker	Matrix	Usage	Normal reference range	Comments
Creatinine	Serum	Glomerular filtration	M: 0.74–1.35 mg/dL F: 0.59–1.04 mg/dL	Most frequently used clinical marker to estimate GFR but heavily influenced by muscle mass; increases only after significant reductions in GFR
	Urine	Glomerular filtration and tubular secretion	M: 20–320 mg/dL F: 20–275 mg/dL	CrCl overestimates GFR due to tubular secretion of creatinine
Cystatin C	Serum	Glomerular filtration	0.62–1.15 mg/L	Outperforms SCr in calculating eGFR; less variation across populations than SCr
	Urine	Tubular damage	<0.1 mg/dL	Catabolized in proximal tubular cells, presence in urine indicates kidney injury
B2M	Plasma	Glomerular filtration	0.8–2.5 mg/L	Elevated concentrations associated with decreased kidney function
	Urine	Tubular reabsorption	<300 μg/L	Catabolized in proximal tubular cells, presence in urine indicates impaired tubular reabsorption; unstable in acidic urine
BTP	Serum	Glomerular filtration	M: 0.37–0.77 mg/L F: 0.40–0.70 mg/L	Elevated concentrations associated with reduced GFR
	Urine	Glomerular filtration	M: < 7.79 mg/L F: < 3.13 mg/L	Urinary excretion increases as GFR decreases
SDMA	Serum	Glomerular filtration	M: 61–136 μg/L F: 55–127 μg/L	Elevated concentrations associated with reduced GFR; consistent across species, often used in veterinary medicine
Albumin	Serum	Glomerular filtration	3.4–5.4 g/dL	Low concentrations associated with decreased eGFR and increased creatinine tubular secretion
	Urine	Glomerular filtration and tubular reabsorption	0.2–1.9 mg/dL	Not excreted by healthy kidneys, high specificity for renal injury
Urea	Serum	Glomerular filtration and tubular reabsorption	6–24 mg/dL	BUN increases as GFR decreases
	Urine	Glomerular filtration and tubular reabsorption	12–20 g/day	Low concentrations indicate kidney injury while high concentrations can indicate high protein intake
Hippurate	Urine: plasma ratio	Tubular secretion	P: < 0.5 mg/dL U: < 1070 mg/g Cr FE: > 3.19	Fractional excretion is decreased in patients with kidney injury
Cinnamoylglycine	Urine: plasma ratio	Tubular secretion	P: < 8.1 μg/L U: < 4 mg/g Cr FE: > 0.89	Fractional excretion is decreased in patients with kidney injury
NMN	Plasma	Tubular secretion	6–40 μg/L	Used to evaluate OCT2 and MATE1/2K secretory transporters in drug–drug interactions
	Urine	Tubular secretion	4.3–7.8 mg/day	Decreased excretion associated with impaired kidney function
A1M	Serum	Tubular reabsorption	20–42 mg/L	Elevated concentrations associated with decreased kidney function; concentrations correlated with creatinine concentrations
	Urine	Tubular reabsorption	<8 mg/L	Catabolized in proximal tubule cells, presence in urine indicates impaired tubular reabsorption
FABP1	Serum	Tubular reabsorption	9–17 μg/L	Concentration increases in parallel with worsening diabetic nephropathy
	Urine	Tubular reabsorption	1.7–9.3 μg/g Cr	Increased excretion associated with impaired tubular reabsorption
Phosphate	Plasma	Tubular reabsorption	3–4.5 mg/dL	Elevated concentrations associated with reduced kidney function
	Urine	Tubular reabsorption	0.4–1.31 g/day TRP >85%	TRP calculated using plasma and urine concentrations over 24 h

B2M: beta-2 microglobulin; BTP: beta-trace protein; SDMA: symmetric dimethylarginine; NMN: N¹-methylnicotinamide; A1M: alpha-1-microglobulin; FABP1: fatty acid binding protein 1; M: for males; F: for females; P: plasma; U: urine; Cr: creatinine; FE: fractional excretion relative to creatinine; TRP: tubular reabsorption of phosphate; CrCl: creatinine clearance; SCr: serum creatinine; BUN: blood urea nitrogen.

Exogenous markers of filtration

Exogenous markers that focus on filtration and effective renal plasma flow (eRPF) have been used for several decades and inulin and para-aminohippurate (PAH), respectively, are considered the gold standards [12,13]. There are well-established methods for using these markers [14–18]. Inulin is not routinely used clinically as it requires a continuous infusion, frequent timed serum and urine collections, and an assay that is not convenient for clinical practice [12]. PAH is unbound, has a high clearance, and undergoes both filtration and tubular secretion. At low plasma concentrations (10–20 mg/L), about 90% of PAH is cleared by the kidneys in a single pass [19]. To measure eRPF, PAH is administered as an intravenous infusion, to sustain a plasma concentration of 20 mg/L, and frequent timed plasma and urine samples are collected [16–19]. However, these studies are arduous due to the long infusion needed to achieve steady-state and the need for timed collections of plasma and urine [18]. High doses of PAH at plasma levels 400–600 mg/L, saturate tubular secretion of PAH and can be used to parse out the tubular secretion component [19,20].

Although inulin and PAH are considered gold standards for assessment of kidney function, they are expensive, invasive, and cumbersome procedures that are not conducive to routine use. The search for an ideal exogenous filtration marker should be centered on the characteristics of 1) excreted solely by the kidneys, 2) no protein binding, 3) no secretion or reabsorption, 4) easy to use, and 5) low distribution volume, indicating a relatively short redistribution phase and therefore a shorter test. Newer exogenous markers, such as iothalamate, iohexol, and radioactive compounds, have been evaluated. Common methods for measuring GFR are the urinary clearance of non-radioactive iothalamate and the plasma clearance of iohexol [12]. For these tests, the exogenous compound is administered as either a sub-cutaneous injection (for urinary clearance) or infusion (for plasma clearance) and timed blood and/or urine samples are collected over the clearance period [21]. Measuring plasma clearance is often advantageous in populations with bladder impairment as it does not require urine collection [22]. However, when calculating GFR by plasma clearance using a limited number of samples, an equation to correct for the absence of the early compartment must be used to account for the redistribution phase [23]. Iohexol is not metabolized or transported in the kidneys and is excreted predominantly by glomerular filtration, making it an excellent candidate marker of GFR [24]. Radioactive compounds such as I-125 iothalamate, chromium-51 labelled ethylene diamine tetra-acetic acid (⁵¹Cr-EDTA), and technetium-99 m diethylenetriaminepentaacetic acid (^99mTc-DPTA) can also be used for measuring GFR. Urinary and plasma clearance methods following a single injection accurately measure GFR and produce similar results [25,26]. The CKD-EPI 2009 and 2012 eGFR equations were validated using urinary non-radioactive iothalamate clearance while the CKD-EPI 2021 equations were validated using the plasma clearance of iohexol and ⁵¹Cr-EDTA (Table 1) [10,27].

Numerous limitations with exogenous assessment methods exist including long infusions to achieve equilibration, bias due to methodology or sample timing, and in individuals with low GFR, the elimination of the marker may not be fully elucidated during the sample collection period [12,13]. Given these limitations, there has been interest in evaluating endogenous compounds.

Endogenous markers of filtration

An ideal endogenous marker of GFR has been defined to have a constant rate of endogenous production, free passage through the glomerulus, no-to-limited protein binding, excretion exclusively by glomerular filtration, and measurement that is simple, accurate, and cost-effective [28]. This section will review conventional and contemporary endogenous markers of filtration and Table 2 provides concise data usages and normal ranges.

1. Creatinine

Serum creatinine (SCr) is the oldest and most commonly used endogenous marker to estimate GFR (Tables 1 and 2). Since creatinine undergoes filtration and secretion, patients with reduced kidney function can exhibit a reduction in creatinine filtration and an increase in the secretion contribution [29]. There can be confusion around the degree of kidney function with SCr, as observations of elevated concentrations may be delayed until after GFR has been reduced by over 50% [7,8]. Creatinine is a relatively insensitive endogenous biomarker for early detection of kidney injury.

Endogenous CrCl has traditionally been used to guide drug dosing and uses the Cockcroft–Gault equation (Table 1) [30]. CrCl can also be calculated by collecting urine and blood and using SCr and urinary creatinine concentrations (Table 1). CrCl can overestimate actual GFR due to the tubular secretion of creatinine [31].

2. Cystatin C

Cystatin C is a protein that is eliminated exclusively by glomerular filtration but subsequently undergoes catabolism in the lysosomes of tubular cells, resulting in limited appearance in the urine of healthy individuals [32,33]. Cystatin C in the blood is not a product of muscle mass and is instead produced by all nucleated cells, meaning its production is more uniform across populations than creatinine [34]. However, increased age, smoking, obesity, hyperthyroidism, and the use of corticosteroids are all associated with increased concentrations [34,35]. Many studies have demonstrated that eGFR determination using both SCr and serum cystatin C concentrations results in improved accuracy over eGFR determined from either SCr or serum cystatin C alone (Tables 1 and 2) [5,10,36,37].

3. Beta-2 microglobulin (B2M)

Beta-2 microglobulin (B2M) is generated by all nucleated cells in the body. B2M is freely filtered by the glomerulus and concentrations (plasma and urinary) can increase early after a kidney insult [38,39]. Studies have shown that the CKD-EPI B2M equation for estimating GFR, which is independent of age and sex, performs similarly to the CKD-EPI creatinine-cystatin C equation (Table 1) [40–42].

4. Beta-trace protein (BTP)

Beta-trace protein (BTP), also known as lipocalin-type-prostaglandin-d-synthase, is filtered by the glomerulus with limited tubular reabsorption [43]. Elevated serum and urine BTP concentrations have been associated with renal and cardiovascular diseases, as well as mortality [44]. However, the CKD-EPI BTP equation does not outperform either the CKD-EPI creatinine or cystatin C equations in estimating GFR (Table 1) [40,45].

5. Symmetric dimethylarginine (SDMA)

Symmetric dimethylarginine (SDMA) is the endogenous catabolic product of methylated arginine-containing proteins and is mainly excreted by the kidneys [46]. SDMA is not influenced by the non-renal factors that impact creatinine and/or cystatin C, such as diet, muscle mass, etc., and is only minimally influenced by obesity, gender, and age [47]. SDMA levels are increased in the plasma of patients with kidney disease and correlate with GFR in patients with CKD [48]. Additionally, the use of SDMA as a biomarker of renal function is consistent across species (human, cat, dog) and is often used in veterinary medicine [47,49–51].

6. Albumin

Albumin is filtered by the glomerulus and then largely reabsorbed in the tubules (~97%) [52]. The presence of albumin in the urine (albuminuria) signifies structural damage to the kidney glomerulus, due to diseases such as glomerulonephritis or diabetes mellitus. This damage results in the “filtering” of large molecular weight compounds. There can also be a reduced ability of the tubules to reabsorb albumin due to either saturation of reabsorption capacity or injury leading to decreases in the function of uptake pathways [53,54]. Urinary albumin excretion is used in CKD staging and is an independent risk factor of mortality [55]. After a stage of hyperfiltration (high GFR), individuals with diabetes or obesity develop albuminuria, and this is associated with the development of CKD, cardiovascular disease, and death [56]. Increased GFR is associated with albuminuria and an increased urinary albumin-to-creatinine ratio [57]. Decreased serum albumin (hypoalbuminemia) is associated with decreased eGFR [58].

7. Urea

Urea is produced by the liver as a product of protein and amino acid catabolism. In the kidneys, it is freely filtered from the blood and allows the kidneys to create hyperosmotic urine, helping to prevent the loss of water [12]. The amount of urea reabsorbed in the collecting ducts (~50%) is dependent on the permeability and the tubular concentration of urea, which are both regulated by antidiuretic hormone (ADH), also known as vasopressin. ADH synthesis in the hypothalamus is triggered by increases in blood osmolarity, such as an increase in sodium concentration, and acts in the kidneys to increase water reabsorption to prevent dehydration [59]. ADH renders the medullary collecting ducts highly permeable to urea by increasing phosphorylation and apical plasma-membrane accumulation of urea transporters A1 (UT-A1) and A3 (UT-A3) (Fig. 2) [60]. UT-A1 (apical) and UT-A3 (basolateral, apical after ADH stimulation) are found in the inner medullary collecting duct. Additionally, in the presence of ADH, water is avidly reabsorbed in the distal tubule and urea becomes highly concentrated, driving urea reabsorption. Alternatively, the absence of ADH results in decreased collecting duct permeability and high levels of water in the collecting duct, diluting the concentration of tubular urea and decreasing urea reabsorption. While blood osmolarity and volume are the main factors that affect ADH synthesis, angiotensin, pain, nausea, hypoglycemia, nicotine, and certain medications can also promote ADH secretion [59]. ADH secretion is inhibited by ethanol, explaining the increased diuresis and free water loss during intoxication [59].

Blood urea nitrogen (BUN) concentrations are ~46% of blood urea concentrations [61]. BUN has an inverse relationship with GFR. Previous reports have demonstrated the usefulness of eGFR calculated by equations based on SCr, BUN, height, gender, and cystatin C serum levels, particularly in children with CKD (Table 1) [62,63]. Since the serum and urinary concentrations of urea depend on ADH production, urine flow rate, diet, and urea cycle enzymes, it is a poor marker of GFR [7].

Endogenous markers of secretion

Tubular secretion is the primary mechanism of drug excretion through the kidneys (Fig. 1) [3,4]. Tubular secretion removes drugs from circulation, including those that are protein-bound, via transporter proteins on the basolateral membrane of the proximal tubule, while proteins on the apical membrane further contribute by moving drugs from the proximal tubule to the urine ultrafiltrate (Fig. 2). Transporters can be bidirectional depending on pH. Transporters can also facilitate the movement of drugs back into the blood from the urinary ultrafiltrate. Unlike glomerular filtration, tubular secretion is a saturable process and can be altered by competitive binding interactions between medications and other circulating substances [2]. An ideal endogenous marker of secretion should be excreted into the urine without degradation in the body and be measured easily, accurately, and cost-effectively. Despite the importance of tubular secretion, it is difficult to quantify using endogenous markers and hence there are no established user-friendly equations analogous to GFR or CrCl for filtration [2,4].

1. Creatinine

As mentioned previously, creatinine is actively secreted by the tubules in addition to being filtered by the glomerulus. CrCl calculations result in an overestimation of kidney filtration function since creatinine is 15% cleared through tubular secretion [31]. Studies often compare the measured renal clearance of a drug to the CrCl to predict whether the drug undergoes a significant secretory component [64]. For example, if the renal clearance of a drug is greater than CrCl, secretion is presumed to be a contributing process. Drugs that are substrates of organic cation transporters may compete with creatinine renal secretion and result in an underestimation of GFR [65]. It is important to differentiate whether a reduction in GFR or CrCl is due to a transporter competition or due to drug-induced kidney injury [64].

2. Hippurate

Hippurate, an organic anion, is a glycine conjugate of benzoic acid formed by gut bacterial metabolism and by mitochondria in the liver and kidneys [66]. The primary excretion route is through renal tubular secretion via OAT1 and OAT3, located on the tubular basolateral membrane (Fig. 2). Endogenous hippurate is cleared on a single pass through the kidneys (50–90% extraction) and the clearance, measured using a timed urine collection and a single plasma sample, can provide an estimate of an individual’s eRPF (Table 1) [67]. Hippurate urinary excretion decreases with decreased tubular secretion and accumulates in the plasma of patients with renal failure [68,69]. Individual variability in the gut production of hippurate, influenced by diet and microbiome composition, may limit its ability to estimate eRPF [66].

3. Cinnamoylglycine

Cinnamoylglycine is a gut-derived metabolite with a renal clearance greater than CrCl, suggestive of a secretion component [69]. Clearance of cinnamoylglycine is only moderately correlated with eGFR (0.40), confirming that it is primarily secreted rather than filtered [70]. Like hippurate, cinnamoylglycine accumulates in the plasma of patients with renal failure [69]. Multiple studies have used cinnamoylglycine excretion to estimate tubular secretion (Table 1) [71,72].

4. N¹-methylnicotinamide (NMN)

Nicotinamide undergoes liver metabolism to produce N¹-methylnicotinamide (NMN), a substrate of OCT2 and the multidrug and toxin extrusion proteins MATE1 and MATE2-K, the latter of which are transporters on the tubular apical membrane (Fig. 2). NMN is unbound and renal clearance is higher than GFR, implying significant excretion via tubular secretion [73]. NMN clearance has been evaluated as a potential endogenous biomarker of OCT2/MATE transporter secretory function in drug–drug interaction studies (Table 1) [73–75].

Endogenous markers of reabsorption

The tubules reabsorb most of the urinary ultrafiltrate, including water, sodium, and other nutrients (Fig. 1). Most reabsorption of the filtrate occurs in the proximal tubules, which reabsorb ~70% of the filtered load. The distal tubules are responsible for the finer regulation of the water, electrolyte, and hydrogen-ion balance. With damage to the glomerulus and tubules, more proteins undergo filtration through the leaky glomerulus and the reabsorption mechanisms in the tubules are oversaturated (and damaged), leading to the presence of proteins in the urine ultrafiltrate. Megalin, also known as low-density lipoprotein receptor-related protein 2 (LRP2), is an endocytic receptor that works in cooperation with the receptor cubilin to drive the reabsorption of nearly all filtered plasma proteins (Fig. 2) [76]. Certain compounds that are filtered out of the blood by the glomerulus and subsequently reabsorbed by the tubules, such as those discussed in this section, can be quantified in urine samples to estimate tubular reabsorption. For example, if a drug undergoes significant reabsorption after filtration, the renal clearance would be predicted to be lower than CrCl. Tubular reabsorption of drugs is calculated by dividing the urine to plasma ratio of the drug by the urine to plasma ratio of creatinine (Table 1) [77].

1. Alpha-1-microglobulin (A1M)

Alpha-1-microglobulin (A1M) is produced by hepatocytes and freely filtered by the glomerulus. About 99% of free A1M in the urinary ultrafiltrate is reabsorbed by megalin/cubilin-mediated endocytosis and subsequently catabolized in the tubules [32,78]. An increase in urinary A1M concentration indicates impaired tubular reabsorption and an increased risk of AKI, rapid CKD progression, and higher mortality [79–81].

2. Beta-2 microglobulin (B2M)

B2M is removed from circulation primarily by glomerular filtration and more than 99.9% of the protein is reabsorbed from the urinary ultrafiltrate and catabolized by lysosomes in the tubules [41]. Elevated urinary B2M is indicative of decreased tubular reabsorption [39].

3. Fatty acid binding protein 1 (FABP1)

Fatty acid binding protein 1 (FABP1), also known as liver-type FABP (LFABP), is expressed mainly in the liver and kidneys, and is freely filtered by the glomerulus and reabsorbed in the tubules by megalin/cubilin. Both serum and urinary FABP1 concentrations have been proposed as biomarkers for early detection of AKI [82,83]. Urinary FABP1 concentrations are associated with serum FABP1 concentrations and the inverse of protein reabsorption capacity [84]. Excretion of FABP1 increases in both kidney and liver injury, meaning it can be used as a biomarker of both reduced tubular reabsorption and liver function [84,85].

4. Phosphate

The electrolyte phosphate is formed when phosphorous is combined with oxygen. Phosphate is the most abundant intracellular anion in the body and is involved in energy production. Elevated plasma concentrations (hyper-phosphatemia) are associated with AKI, CKD, cardiovascular events, and mortality [86]. Phosphate levels are highly dependent on dietary intake so CKD patients with hyper-phosphatemia are prescribed phosphate-restrictive diets and phosphate binding drugs to reduce intestinal absorption of phosphate [87]. When phosphate intake is excessive, circulating levels of fibroblast growth factor 23 (FGF23) increase. FGF23 suppresses phosphate reabsorption in renal tubules, raising the tubular phosphate concentration until eventually, calcium phosphate crystals are formed which damage tubule cells and lead to interstitial fibrosis and nephron loss [87]. Urinary and plasma concentrations measured over 24 h can be used to calculate the percent tubular reabsorption of phosphate (Table 1) [88].

5. Albumin

Albumin is filtered by the glomerulus and then largely reabsorbed in the tubules (~97%; 71% in proximal convoluted tubule, 23% in loop of Henle and distal tubule, 3% in collecting duct) by megalin/cubilin-mediated endocytosis [52]. Albuminuria, or albumin in the urine, can signify tubular stress/dysfunction and decreased reabsorption.

6. Urea

Urea is freely filtered in the glomerulus and the amount of urea reabsorbed in the collecting ducts (~50%) is regulated by antidiuretic hormone (ADH) [12,59]. BUN levels increase as a result of enhanced tubular reabsorption [64].

Conclusions

While exogenous markers of kidney function have been used for decades, limitations related to lengthy, cumbersome protocols and extensive sample collection have limited their utility. In the era of personalized medicine and to circumvent the observed limitations, there is interest in using endogenous markers as tools to assess an individual’s kidney functional capacity. Several endogenous markers are being evaluated and hold promise to more fully elucidate the individual components of filtration, secretion, and reabsorption of kidney function and clearance.

Abbreviations:

⁵¹Cr-EDTA

chromium-51 labelled ethylene diamine tetra-acetic acid

^99mTc-DTPA

technetium-99 m diethylenetriaminepentaacetic acid

A1M

alpha-1-microglobulin

ADH

antidiuretic hormone

AKI

acute kidney injury

B2M

beta-2 microglobulin

BTP

beta-trace protein

BUN

blood urea nitrogen

CKD

chronic kidney disease

CKD-EPI

Chronic Kidney Disease Epidemiology Collaboration

CrCl

creatinine clearance

eGFR

estimated glomerular filtration rate

eRPF

effective renal plasma flow

FABP1

fatty acid binding protein 1

FGF23

fibroblast growth factor 23

GFR

glomerular filtration rate

LFABP

liver-type fatty acid binding protein

LRP2

low-density lipoprotein receptor-related protein 2

MATE

multidrug and toxin extrusion

NMN

N¹-methylnicotinamide

OAT

organic anion transporter

OCT

organic cation transporter

PAH

para-aminohippurate

SCr

serum creatinine

SDMA

symmetric dimethylarginine

UT

urea transporter