Several molecules involved in kidney function, signaling, and structure have been evaluated as potential markers for CKD. The biomarkers currently used in clinical practice for the diagnosis and prognosis of CKD are markers of loss of kidney function. The most widely used are the eGFR, serum creatinine, blood urea nitrogen, and albuminuria or proteinuria. These markers indicate impaired renal function but have no disease specificity, and detectable changes in their concentration only appear after the biological changes in the organ causing the functional impairment.

The most important new biomarkers for kidney function include cystatin C [8], a small protein filtered and metabolized after tubular absorption, and the beta-trace protein [9], a lipocalin glycoprotein, which is a sensitive marker of glomerular filtration. According to the MMKD study, both are good markers of CKD progression; however, cystatin C seems to be more sensitive. Uric acid has also emerged as a novel and potentially modifiable risk factor for the development and progression of CKD; however, it is not currently clear whether hyperuricemia plays a causative role in CKD progression or is merely a biomarker of reduced kidney function [10].

Markers of renal tubular injury are not used routinely to describe kidney function and little is known about the risk of cardiovascular events and death associated with these biomarkers. Neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), N-acetyl-β-D-glucosaminidase (NAG), liver-type fatty acid-binding protein (L-FABP), tenascin, and the tissue inhibitor of metalloproteinase 1 (TIMP-1) have emerged as potential biomarkers of tubulointerstitial injury.

According to the CRIC study [11], involving 3,386 participants, urine levels of NGAL were associated independently with future ischemic atherosclerotic events, but not with heart failure events or deaths. The Multi-Ethnic Study of Atherosclerosis (MESA), involving 686 participants, evaluated both NGAL and KIM-1 as markers of tubular injury and found that urinary KIM-1 level was associated with future risk of kidney disease independent of albuminuria and that both biomarkers are a promising tool for identifying persons at risk of CKD [12].

KIM-1 is a transmembrane tubular protein that is not found in healthy people, emerging therefore as a potential biomarker of kidney disease. It has been reported that KIM-1 is upregulated in patients with renal cell carcinoma, urate nephropathy, acute and chronic tubular injury, allograft nephropathy, and acute kidney injury after cardiac surgery. Actually, KIM-1 has been also reported as an independent predictor of graft loss in renal transplant recipients [13] and as a good marker to monitor potentially nephrotoxic therapies.

NAG is a lysosomal enzyme that is present in proximal tubular cells and is widely used to evaluate tubular renal function. NAG has a high molecular weight, which does not permit its filtration through the glomerular basal membrane and is rapidly cleared from the circulation by the liver. Thus, urinary NAG originates primarily from the proximal tubule, and increased urinary excretion is a consequence of renal tubular cell breakdown; its urinary excretion is, therefore, almost constant with minimal diurnal changes. The urinary NAG values should be expressed as a ratio to urinary creatinine concentration, as this relationship shows less variability than the urinary enzyme excretions related to volume or time.

NGAL is another biomarker of renal tubular injury that belongs to the superfamily of lipocalins, a group of iron-carrying proteins [13]. It was firstly reported to be produced by neutrophils, but it is also expressed in other tissues, such as kidney, liver, epithelial cells, and vascular cells in atherosclerotic plaques [14]. When tubular injury occurs, the expression of NGAL rises and is rapidly secreted; urine and plasma concentrations increase proportionally to severity and duration of renal injury and its concentration rapidly decreases with attenuation of renal injury.

There are different commercially available ELISA kits to evaluate NGAL. As it was found that NGAL is ultrafiltered and absorbed by polysulfone membranes, the NGAL blood levels may be reduced in patients under therapeutic hemodialysis using such type of membranes for the hemodialysis procedure; a moderate to severe inflammatory condition, coexisting with kidney injury, may be also a confounding factor in NGAL measurement, as the activation of neutrophils will contribute to increase the NGAL blood levels.

Several clinical studies and systematic reviews proposed NGAL as a reliable diagnostic and prognostic biomarker for kidney injury. A multicenter analysis of pooled data [15], including 2,322 critically ill children and adults, evaluated the prognostic value of acute kidney injury detected by NGAL and found that about 20 % of the patients presented an early increase in the concentration of NGAL, in the absence of diagnostic increases in serum creatinine. The increase in NGAL levels, usually, occurs 24–72 h before the increase in creatinine to diagnosis values. Thus, NGAL evaluation seems to be a useful early biomarker of kidney injury. Moreover, this study group showed that NGAL is also a useful prognostic biomarker, as patients with increased NGAL were at greater risk of adverse outcomes, including death, renal replacement therapy, and hospitalization, both in the presence and absence of an increase in serum creatinine levels. However, future prospective studies are needed to confirm histopathological agreement of NGAL with tubular injury, already shown in animal models; further studies are also needed to test whether NGAL-based early diagnosis of kidney injury leads to more successful and prompt therapeutic intervention and to improve the outcome of the patients. Indeed, in the absence of an early detection of kidney injury, the therapeutic intervention could only be delivered late in the course of AKI, with worsening of the disease and of its outcome.

L-FABP is a newly emerging biomarker that has antioxidant properties and is expressed in the cytoplasm of human renal proximal tubules. Its expression is upregulated and its urinary excretion is increased by various stressors, including urinary protein, hyperglycemia, tubular ischemia, toxins, and salt-sensitive hypertension, which lead to the progression of kidney disease [16]. Urinary L-FABP levels accurately reflect the degree of tubulointerstitial damage and are strongly correlated with the prognosis of CKD patients [17]. Concerning AKI, urinary L-FABP seems to be able to detect kidney injury before an increase in serum creatinine occurs. Urinary L-FABP may be also useful for the early detection of diabetic nephropathy, the leading cause of CKD. In a longitudinal study, a higher level of urinary L-FABP was found to be a risk factor for the progression of diabetic nephropathy. A recent single-center, prospective observational study reported that urinary L-FABP may be also a useful predictor of adverse long-term outcomes in kidney transplant patients [18].

Tenascin and TIMP-1 have also been proposed as biomarkers of tubulointerstitial injury; however, there are still few studies supporting their value. Tenascin has emerged as an important extracellular matrix, playing an important role in nephrogenesis and in several pathological processes in glomerulus and tubulointerstitial renal cells. TIMP-1 is a physiological inhibitor of matrix-degrading enzymes, namely, collagenase, gelatinase, and stromelysin. Patients with CKD present elevated serum and urinary levels of both tenascin and TIMP-1; however, the urinary levels did not correlate with the degree of proteinuria [19].

These biomarkers of tubulointerstitial injury may be used as earlier markers of AKI, as markers of the underlying predominant kidney injury in CKD, as well as markers of progression and outcome.

Proteinuria is widely used as a marker of glomerular function. More recently, new biomarkers of kidney podocyte injury, including urinary nephrin, podocin, and podocalyxin, have emerged as biomarkers of glomerular injury.

Podocytes are differentiated epithelial cells covering the outer surface of the glomerular capillaries, presenting interdigitating foot processes and forming narrow slits to provide a pathway for glomerular filtration. They act as a size and charge barrier to anionic proteins, due to the presence of podocalyxin, a negatively charged apical membrane protein. The injury of podocytes may lead to a reduced foot process and proteinuria, while the detachment of podocytes from the glomerular basement membrane leads to progression of glomerular diseases. Podocyte injury might be induced by angiotensin II, hyperglycemia, oxidative stress, infections, deposition of antigen-antibody complexes, mechanical stretch, and drugs. In these conditions, urinary levels of nephrin, podocin, and podocalyxin are increased and, therefore, may be used in a noninvasive way, as useful markers of glomerular function. Moreover, the evaluation of the number of urinary podocytes can provide real-time data about the number of podocytes detached in a certain period of time; thus, urinary podocyte count may be also used as a marker of ongoing glomerular injuries. According to Hanamura et al. [20] a decrease in the number of glomerular podocytes is associated with glomerulosclerosis, decline in renal function, and impaired selectivity of proteinuria, suggesting a causative relationship between detachment and loss of podocytes and progression of renal dysfunction. Urinary podocyte excretion was associated with proteinuria and active histological lesions. These biomarkers are promising and more specific of glomerular injury; however, further studies are needed to assess their values in clinical practice.

Asymmetric dimethylarginine (ADMA) is a naturally occurring amino acid found in tissues and cells, acting as an endogenous inhibitor of the nitric oxide synthase, impairing the ability of nitric oxide for vasodilation, and is excreted in urine. Several studies have suggested that plasma concentration of ADMA provides a marker of risk for endothelial dysfunction and cardiovascular disease. ADMA blood levels are increased in patients with CKD (stages 1–5 with or without proteinuria) [21] and seem to provide a biomarker for CKD progression [22]; furthermore, it has been associated with CVD complications [23]. Thus, ADMA concentration has been proposed as a biomarker for endothelial dysfunction and for increased risk of cardiovascular mortality and morbidity, as well as a prognostic marker for the loss of renal function.

A recent prospective controlled 1-year follow-up study [24] on the effects of ADMA and other variables related to ADMA metabolism on the progression of kidney dysfunction, in 181 patients with CKD stages 3–5, showed that elevated ADMA was a strong predictor of progression only for patients with eGFR between 25 and 40 mL/min/1.73 m² (the borderline of CKD stages 3–4).

To evaluate ADMA blood levels, enzyme-linked immunosorbent assays have been mostly used; however, a recent study showed that these measurements overestimated ADMA levels in eGFR < 30 mL/min, as compared to the gold-standard liquid chromatography-electrospray tandem mass spectrometry.

CRP is a member of the family of pentraxins, which are small pentameric innate immunity effector proteins that are absent or weakly expressed in healthy conditions. Its synthesis by hepatocytes is induced by proinflammatory cytokines, such as IL-1, IL-6, and TNF-α. The plasmatic levels of CRP are widely used by clinicians to assess infectious or noninfectious types of systemic inflammation, and nephrologists recognize CRP levels as a useful biomarker for the outcome of CKD and ESKD patients. Several studies, such as the Jackson Heart study and the CARE (Cholesterol and Recurrent Events) trial, showed that plasma concentrations of CRP were associated with the presence of CKD and with a higher rate of kidney function decline, respectively [1].

CRP values show time-dependent variability, as they might be influenced by intercurrent clinical events, including comorbidities, infections, and changes in blood volume. Therefore, the motorization of CRP value is especially important when using CRP as a biomarker for the evolution of the disease and for the outcome of the patient. The persistence of high CRP values (from 3 to 6 months) marks a worst survival in CKD and in ESKD patients.

The prevalence of the utilization of CRP measurement varies across countries and dialysis facilities; however, according to the Dialysis Outcomes and Practice Patterns Study (DOPPS) III, there has been an increasing use of CRP measurement in nearly all countries [35]. This study hypothesized that the evaluation of CRP as a marker of underlying infection/inflammation in dialysis facilities could lead to lower adverse outcomes for the dialyzed patients. It was found that the measurement of CRP in the majority of patients within a dialysis facility (vs. less than 50 % of patients) was significantly related to a lower cardiovascular mortality, for facilities measuring CRP in at least 50 % of its patients. This finding strongly suggests that a more prevalent measurement of CRP may influence patient outcomes. Indeed, the use of a CRP screening for inflammation would trigger the evaluation of the possible underlying causes, when a rise in CRP value occurs, allowing a rapid clinical intervention and, therefore, an effective improvement in patient outcome. Moreover, this study confirmed that higher CRP values are associated with all-cause mortality and showed that this relationship with mortality was independent of the correlation with other common inflammatory markers.

Of all the acute-phase proteins and plasma biomarkers of inflammation, CRP is the most widely used. CRP is rapidly synthesized in hepatocytes, following an inflammatory stimulus by IL-6 during the acute phase of inflammation. Il-6 is a proinflammatory cytokine released by a variety of activated cell types, which has multiple cell targets and regulates the hepatic acute-phase response, promoting the synthesis of positive acute-phase reactants and inhibiting that of negative acute-phase reactants. IL-6 also controls several homeostatic functions including glucose metabolism, the hypothalamic-pituitary-adrenal axis, affecting mood, fatigue and depression, and hematopoiesis. A systemic increase in IL-6 causes hyperthermia and leads to a general loss of activity and appetite.