39 Biomarkers of renal and hepatic failure

In the last few years, major advances have been achieved in the understanding of the molecular and pathophysiological mechanisms which underlie the complex interactions between the heart and the kidney, as well as between the heart and the liver. According to these new insights, new biomarkers have been proposed for better evaluating and monitoring patients affected by cardiovascular diseases. In addition, some biomarkers should be used as risk factors and for an early identification and treatment of these severe diseases. This chapter reviews the most important biomarkers for evaluating the ‘cardiorenal syndrome’, in particular, the measurement of serum creatinine and its use for calculating the glomerular filtration rate which, with the new and more efficient equation, namely Chronic Kidney Disease Epidemiology Collaboration, still remains the most widely used biomarker. The role of newer biomarkers will be explored. The measurement of cystatin C, representing additional information, particularly in paediatric age groups and in the early phase of kidney disease, plays an increasing role. Neutrophil gelatinase-associated lipocalin is a recently developed and very promising new biomarker for the diagnosis of acute kidney injury, while the well-known albumin/creatinine ratio has been re-evaluated as a simple and useful tool for an early identification of kidney disease. Regarding liver diseases, a growing body of evidence demonstrates the usefulness of non-invasive makers of hepatic fibrosis that may avoid the need for a liver biopsy in most patients. A promising field of research is represented by the role of non-alcoholic fatty liver disease in the pathogenesis of cardiovascular disease.

The term ‘cardiorenal syndrome’ (CRS) [1, 2] can be generally defined as bidirectional disorders of the heart and kidney, whereby an acute or chronic dysfunction in one organ may induce an acute or chronic dysfunction in the other organ.

The pathophysiological mechanism [3] seems to be primary, via the circulatory system (haemodynamic factors), or secondary to underlying endogenous humoral or exogenous factors that are associated with disease of either organ, or a combination of both.

This interaction can occur in normal organs (acute dysfunction) or diseased organs (acute or chronic dysfunction), and in one or both organs, or a combination. The cause and temporality of this interaction, in terms of kidney damage and subsequent clinical deterioration, are unpredictable.

In order to include the vast array of interrelated derangements and to stress the bidirectional nature of the heart–kidney interactions, the proposed classification includes five subtypes, the etymology of which reflects the primary and secondary pathology, the time frame, and the simultaneous cardiac and renal co-dysfunction secondary to systemic disease [4], in particular:

Quantitation of the overall renal function is based on the assumption that all functioning nephrons are performing normally and that a decline in renal function is due to a loss of functioning nephrons quantitatively related to the decline. Thus, in nearly all types of renal disease, an impaired function is attributed to a diminished number of nephrons, rather than to a compromised function of individual nephrons. Because glomerular filtration is the initiating phase of all nephron functions, a quantitative or qualitative assessment of filtration generally provide the most useful indices to assess the severity and progress of kidney damage.

The biochemical markers that have been proved to be the most practical and useful to diagnose and monitor impaired kidney function are the following.

Creatinine is the most widely used endogenous marker of the glomerular filtration rate (GFR), produced at fairly constant rates and freely filtered at the glomerulus; a distal tubular secretion may account, however, for up to 10–40%. As a GFR marker, it is convenient and cheap to measure, but the concentrations are affected by age, sex, exercise, certain drugs, muscle mass, nutritional status, and meal intake. Plasma creatinine remains within the reference interval, until significant renal function has been lost, showing an unsatisfactory sensitivity in the early diagnosis of kidney insufficiency.

The most relevant limitation in the clinical sensitivity of creatinine lies in the evaluation of the circulating concentration on the basis of a general reference interval; creatinine concentration shows, in fact, a high variability between subjects, making the adoption of a general reference interval not adequate to evaluate small changes [5]. The knowledge of the biological variability and usefulness of the reference change value (RCV) represents a fundamental tool in clinical chemistry [6]. However, because of all the described limitations, it is recommended that plasma creatinine measurement alone is not used to assess kidney function.

Because creatinine is endogenously produced and released into body fluids at a constant rate, its clearance has been proposed as an indicator of the GFR. Historically, creatinine clearance has been seen as more sensitive for the detection of renal dysfunction than the measurement of plasma creatinine, but it requires a timed urine collection which introduces its own inaccuracies. Furthermore, in adults, the intraindividual day-to-day creatinine clearance may exceed 25%, and this further devalues the use of creatinine clearance as a measure of the GFR. Creatinine clearance and the GFR are not equivalent; as the kidney function declines, creatinine clearance becomes significantly higher, due to the preserved tubular secretion of creatinine, and may be twice the true GFR when the GFR is severely reduced. The best overall measure of kidney function is the GFR measured as the urinary or plasma clearance of an ideal filtration marker, such as inulin, or of alternative exogenous markers such as iothalamate or ethylenediamine tetraacetic acid (EDTA) [7].

More than 25 different formulae have been derived that estimate the GFR, using plasma creatinine corrected for some, or all, of gender, body size, race, and age. Indeed, the National Kidney Foundation has recommended, in its last recently published guideline [8], the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine equation that uses the same four variables, as well as the known Modified Diet in Renal Disease (MDRD) study equation, but shows a high accuracy, particularly at high GFRs.

The improvement in accuracy is attributable to a substantial decrease in systematic differences between the measured GFR (mGFR) and the eGFR, especially for people with an eGFR of >60 mL/min/1.73 m². In particular, there was an improvement in bias in subgroups at low risk of chronic kidney disease (CKD), for which an underestimation of the mGFR may have led to an overestimation of the CKD prevalence, including those aged younger than 65 years, women, and whites. For eGFR up to >90 mL/min/1.73 m², bias is negligible: unbiased GFR estimations in groups at increased risk of CKD, such as the elderly, blacks, patients with diabetes, organ transplant recipients, and the overweight and obese, have important implications in public health and clinical care [9].

On the basis of eGFR values obtained with the CKD-EPI equation, five GFR categories have been defined:

Cystatin C, a 13 kDa endogenous cysteine proteinase inhibitor, is a member of a family of proteins having an important role in the intracellular catabolism of several peptides and proteins [10]. It is synthesized at a constant rate by all nucleated cells in the body. With regard to renal function, due to the free filtration of cystatin C by the glomerulus, its complete reabsorption, and its catabolism without secretion in the renal tubule, the serum cystatin C circulating concentration has been thought to depend almost exclusively on the GFR [11, 12]. Cystatin C represents an alternative blood biomarker of kidney function [13, 14], and several GFR-estimating equations have been developed in the past [15–18]. The influence of body composition and other non-renal factors on cystatin C serum concentrations seems less than for creatinine, and cystatin C correlates with true GFR more accurately than creatinine [19]. An international certified cystatin C primary reference material (ERM^®-DA471/IFCC) was prepared and released in 2010 by the International Federation for Clinical Chemists (IFCC) [20], and a standardized assay of cystatin C traceable to that reference material is available. Recently, the CKD-EPI developed new equations, based on standardized cystatin C and cystatin C in combination with standardized creatinine [21]. Cystatin C measurement is recommended when a confirmation of CKD is required for patients with a creatinine-based eGFR of 45–59 mL/min/1.73 m² and no albuminuria; in these patients, a cystatin C-based eGFR of <60 mL/min/1.73 m² confirms a diagnosis of CKD [8]. Cystatin C-based eGFRs seem more powerful predictors of patients’ clinical outcomes than those based on creatinine [22]. The prognostic advantage of cystatin C is more evident among patients showing a GFR of >45 mL/min/1.73 m². Moreover, measuring cystatin C, in addition to creatinine, can improve the accuracy of GFR estimation and CKD classification [8].

Given that the kidney function is tightly correlated with cardiovascular risk, due to the strong impact of renal dysfunction on impaired survival and cardiovascular disease progression, cystatin C might prove a useful predictor of cardiovascular risk (see Figure 39.1). Several studies have shown cystatin C to be a powerful risk marker for adverse cardiovascular events in different cohorts of subjects. In a population of elderly ambulatory patients without a previous history of coronary artery or cerebrovascular disease, higher cystatin C concentrations at baseline were significantly associated with increased rates of all-cause and cardiovascular mortality. Moreover, patients having the highest cystatin C values showed higher rates of MI and stroke (median follow-up: 7.4 years) [23]. The association of cystatin C levels with mortality has been confirmed in a population of older subjects [24, 25]. In elderly patients without renal insufficiency at baseline, cystatin C, but not creatinine, was significantly associated with stroke, MI, heart failure, and all-cause and cardiovascular mortality, resulting as a better predictor of cardiovascular events and death than creatinine [26].

In patients with stable coronary artery disease (CAD), high cystatin C concentrations were associated with an increased risk of death, cardiovascular events, and hospitalization (follow-up: 3 years) [27, 28], both in patients with and without kidney insufficiency at baseline [27]. In patients without CKD, higher cystatin C levels were correlated with a higher rate of stroke, myocardial infarction (MI), and angina pectoris [29]. In ACS patients, including both ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI), cystatin C levels predicted the subsequent incidence of death and MI [30–32]. Several studies have highlighted the significant association between cystatin C concentrations and heart failure incidence and outcomes. In particular, it was shown that cystatin C, but not creatinine, was a predictor of heart failure, independently of other risk factors [26, 33]. High concentrations of cystatin C levels were related to left ventricle (LV) hypertrophy and diastolic dysfunction in patients with no previous cardiac failure [34–36]. In patients with heart failure, cystatin C increases with the severity of the disease, as defined by the New York Heart Association (NYHA) classification [37]. Several studies documented cystatin C to be an independent predictor of mortality in heart failure patients [38–43]. The association between higher cystatin C levels and increased rates of cardiovascular diseases (CVDs) and/or adverse outcomes has been reported in different cohorts of subjects without CKD or with normal kidney function [26, 29, 44, 45]. In CKD patients, higher cystatin C values significantly predicted mortality and progression to renal transplantation or dialysis [46]. In populations of older adults, cystatin C predicted CKD or a renal function decline, associated with an increased risk of all-cause and cardiovascular death [26, 47, 48].

In summary, cystatin C is a useful blood biomarker of kidney function that accurately reflects the renal GFR in various populations of healthy and diseased subjects. In the CRS, cystatin C is an interesting biomarker in the evaluation of kidney function, as well as in the risk stratification of patients with cardiac failure or CVD, reflecting even a modest impairment in the renal function not detected by traditional routine markers such as creatinine.

Proteinuria refers to the presence of increased amounts of protein in the urine. It may reflect an abnormal loss of serum proteins being attributable to: (1) an increased permeability of the glomeruli to large-molecular-weight proteins (glomerular proteinuria or albuminuria); (2) an incomplete tubular reabsorption of usually filtered low-molecular-weight proteins (tubular proteinuria); and (3) an increased serum concentration of low-molecular-weight proteins (overproduction proteinuria, e.g. immunoglobulin light chains). Similarly, proteinuria may reflect an abnormal loss of proteins from the kidney (renal tubular damage causing a leak of tubular cell constituents), as well as from the lower urinary tract. Albuminuria, tubular proteinuria, and the presence of renal tubular cell constituents in urine samples are pathognomonic of kidney damage [8]. Moreover, proteinuria appears to play a significant role in the pathogenesis of disease progression of CKD [49]. The recently published KDIGO clinical practice guideline for the evaluation and management of CKD recommends, as diagnostic criteria for CKD: a GFR threshold of <60 mL/min/1.73 m² or the detection of kidney injury (albuminuria, urine sediment abnormalities, electrolyte and other abnormalities due to tubular disorders, abnormalities detected by histology, structural abnormalities detected by imaging, a history of kidney transplantation). A CKD classification, based on cause, GFR, and albuminuria, is promoted (see Chapter 68). As a matter of fact, albumin is the main component of urinary protein in most kidney diseases, and a strong graded relationship exists between urinary albumin concentrations and the risk of kidney disease and/or CVD [8].

Albuminuria refers to the presence of an abnormal loss of albumin in the urine. Albumin is the most abundant protein measured in serum, contributing significantly to maintain a normal serum oncotic pressure. Despite the high concentration of albumin in serum, only small quantities of this protein appear in the urine of normal subjects, due to the size and charge selectivity of the glomerular filtration barrier, in addition to the reabsorption of filtered albumin by the renal tubuli [50]. Even if albumin is one of the serum proteins measured in the urine of normal subjects, it is a common, but not uniform, finding in CKD. Albuminuria is the earliest biomarker of glomerular diseases, appearing generally before a reduction in the GFR (e.g. diabetic glomerulosclerosis). On the other hand, it is also a biomarker of hypertensive nephrosclerosis, in which it may rise after a reduction of the GFR. Albuminuria is also associated with an underlying hypertension, obesity, and vascular disease, in which the underlying kidney disease is not overt [8]. Albuminuria, as well as proteinuria, is expressed as the urinary loss rate: AER (albumin excretion rate) and PER (protein excretion rate), respectively. Three main albuminuria categories have been identified, on the basis of daily urinary albumin loss, generally detected as the albumin/creatinine ratio (ACR): A1 (normal to mildly increased) <30 mg/24 hours or <30 mg/g; A2 (moderately increased) 30–300 mg/24 hours or 30–300 mg/g; A3 (severely increased) >300 mg/24 hours or >300 mg/g [8]. To indicate CKD, the clinical decision threshold for the urinary AER in the timed urine sample is ≥30 mg/24 hours sustained for >3 months, a value approximately equivalent to an ACR measured in a random urine sample of ≥30 mg/g [8]. These values are >3 times the normal levels found in young adults (AER 10 mg/24 hours, ACR 10 mg/g) and are associated with an increased risk of CKD complications, as well as with a subsequent risk of all-cause mortality, cardiovascular mortality, AKI, kidney failure, and CKD progression in the general population, as well as in subjects with cardiovascular risk factors [51–53]. Different studies showed evidence that urine samples collected at the first morning void provide lower albuminuria values than the random urine sample collected throughout the day (urinary AER is normally higher during the day, due to physical exercise and/or body posture). Moreover, albuminuria levels measured in the first morning void urine sample correlate better with results obtained from a 24-hour collected urine sample than with results obtained from a random urine sample [54–56]. According to these observations, a sequential testing is recommended, following up an increased ACR obtained from a random urine sample with testing on a first morning void urine sample. If a more accurate estimate of albuminuria (or proteinuria) is required, it is recommended to measure AER (or PER) in a timed urine sample [8]. Albuminuria is a well-established diagnostic and prognostic biomarker in diabetic nephropathy, and it is associated with glomerulonephritis, hypertension, hyperlipidaemia, obesity, smoking, the metabolic syndrome, and a previous history of stroke or MI [57–59]. Across every categories of eGFR, higher levels of albuminuria define an increased risk of death, CVD, and kidney disease progression [60]. Due to its additional predictive ability, above and beyond the eGFR, albuminuria has been proposed as an additional biomarker to classify the CKD stages [60]. Supporting data are lacking to definitively recommend albuminuria reduction as a surrogate clinical target [61]. Albumin represents a major urinary protein in many severe glomerular injuries, being, for example, the recommended marker for early diabetic nephropathy. However, the determination of albuminuria, rather than total proteinuria, may underdiagnose kidney disease associated with multiple myeloma, in which filtered light chains may be the main protein in the urine sample [50]. Indeed, if significant non-albumin proteinuria is suspected, analytical assays to measure specific urinary proteins (e.g. monoclonal heavy or light chains, α₁-microglobulin) should be used [8].

In summary, the AER is strongly recognized as a convenient, cheap, and helpful tool in routine clinical practice to assess the onset, as well as the progression, of renal disease. Albuminuria or proteinuria adds significantly to the risk stratification of subjects with, or at risk of, CKD.

NGAL, also known as lipocalin 2 or siderocalin, is a small molecule (178 amino acids) that belongs to the superfamily of lipocalins, which are proteins specialized in binding and transporting small hydrophobic molecules. It is expressed by neutrophils and various human tissues, with the loop of Henle and the collecting ducts as main production sites in the kidney [62], and may have an important role not only in defending against bacterial strains depending on siderophores through iron sequestration [63], but also in kidney development, as a growth factor, in the renal regeneration and repair after ischaemic injury, and in kidney protection [64]. Human NGAL was identified and isolated from secondary granules of neutrophils and is detectable in serum and urine in three main forms: a 25 kDa monomer, a 45 kDa disulfide-linked homodimer, or a 135 kDa heterodimer where the protein is covalently bonded to matrix metalloproteinase-9 (MMP-9). According to recent experimental data, the monomer may be predominantly released by tubular cells, whereas the homodimer may be synthesized by neutrophils [65]. This aspect seems to be relevant in the configuration and performance of the measurement assays [66, 67]. The expression of NGAL rises 1000-fold in humans, in response to renal tubular injury, and it appears so rapidly in the urine and serum that it is useful as an early biomarker of renal failure. Therefore, from a clinical point of view, the use of the NGAL assay is particularly useful for the diagnosis and prognosis of patients suffering from AKI, including patients with CRS related to heart failure [68].

AKI represents a serious and frequent clinical complication among hospitalized patients [69] that has been shown to correlate with adverse patient outcome (see Chapter 68). Serum creatinine, as well as BUN or other urinary markers of kidney injury, reflects the functional alteration when a significant amount of renal function has been lost, thus limiting their usefulness in the early detection of AKI [70]. Consequently, more reliable biomarkers are necessary, given a rapid and accurate identification of the early phase of AKI is a major challenge in clinical laboratories; an ideal biomarker should be non-invasive to measure, using urine or blood, rapid and inexpensive, automated on assay platforms allowing to report results with a turnaround time adequate for clinical needs. From the clinical point of view, it should be associated with a known mechanism of renal injury, sensitive to establish an early diagnosis, and able to identify tubular damage and to differentiate AKI from CKD [70].

In recently published guidelines from the Acute Dialysis Qualitative Initiative (ADQI), among several biomarkers proposed for an accurate and early detection of AKI, only NGAL and cystatin C have been proposed as novel biomarkers, with the potential to be integrated into clinical practice [4]

Several recent studies in homogeneous (adult and paediatric cardiac surgery) [71], as well as heterogeneous (intensive care and ED) [72], populations have further demonstrated the utility of NGAL measurement for the early diagnosis of AKI, and for the prediction of the severity of the acute disease and the need for RRT.

A recently published multicentre pooled analysis of prospective studies carried out on critically ill children and adults with CRS [73] demonstrates that approximately 20% of patients display an early increase in NGAL concentrations, without any increase in creatinine concentrations, during monitoring. In this subgroup of patients, a significant increase in the rate of adverse clinical events, such as mortality, dialysis requirement, and ICU and overall hospital stay, has been observed, suggesting that early NGAL measurements may allow to identify patients with subclinical AKI.

In conclusion, the described results indicate that NGAL represents an earlier and more sensitive marker of renal injury, compared to creatinine and that the concept and the definition of AKI (currently still based on the measurement of serum creatinine and urinary flow) might need reassessment.

Chronic, as well as acute, heart diseases can affect the function of the liver, and vice versa liver dysfunction affects the heart [74]. Liver injury encountered in patients suffering from CVDs is arbitrarily divided into acute and chronic, based on the duration or persistence of the process.

Acute insults may arise from a transient deprivation of blood flow and O₂, and the return of blood flow during reperfusion (e.g. in AMI and cardiogenic shock (CS)). Chronic heart disease, namely haemodynamic changes due to right or left cardiac failure, can lead to reduced liver perfusion, and secondarily to the impairment of liver function. In particular, liver damage in congestive heart failure may further progress to liver fibrosis and cirrhosis.

Ischaemia and reperfusion injury (IRI) represents a complex series of events that result in cellular and tissue damage of the liver. In particular, cardiogenic ischaemic hepatitis (CIH) is often seen in the face of acute CS. (See also Chapter 17.)

CIH is defined as a rapid and marked increase of serum transaminases and is diagnosed when there are extremely high levels of ALT and/or AST (>1000 U/L), acute arterial hypertension is present, and primary liver failure is excluded [75]. The liver enzymes fall rapidly to normal levels, once the circulation is restored. Oxygen free radicals (OFRs), NO, and several chemokines and cytokines may mediate and modulate this process [76].

OFRs are considered one of the most significant components of cell and tissue damage during ischaemia and reperfusion. Among OFRs, hydrogen peroxide, superoxide anion, and hydroxyl represent the main aggressive components, while, among the OFR scavengers and inhibitors, superoxide dismutase (SOD) represents an important, and even measurable, biomarker [77].

Leucocytes are highly involved in liver IRI and function both to amplify the molecular pathways and to directly cause cellular damage [78, 79]. A number of mechanisms may account for leucocyte infiltration during liver IRI: P-selectin, phosphatidyl-serine, various cytokines/chemokines (CXCL10, RANTES, MCP-1, MIP-1, MIP-MMP, namely MMP-9) [80].

Moreover, nitric oxide (NO) plays a significant role in the microcirculation and organ ischaemia reperfusion injury (IRI), exerting both beneficial and harmful effects [81].

The new insights and experimental evidence previously described have not been translated into clinical practice, and currently none of the described mediators, other than transaminases, is used as a valuable biomarker for detecting acute liver damage in CVD.

The distinction between acute and chronic liver injury is a mechanistic oversimplification. Chronic liver injury reflects, in part, continuous acute liver injury extended over time; however, it leads to progressive fibrosis that can eventually result in cirrhosis, liver failure, or hepatocellular carcinoma [82]. Liver fibrosis represents a hallmark of chronic liver diseases and cirrhosis, and staging of hepatic fibrosis is of paramount clinical importance for the prognostic assessment in the individual patient. Liver biopsy still represents the gold standard for evaluating the presence, type and stage of liver fibrosis, but this procedure is invasive, costly, and difficult to standardize [83]. In recent years, there has been increasing interest in identifying and describing liver fibrosis by using non-invasive biochemical markers measurable in the peripheral blood.

Biochemical markers of liver fibrosis may be classified into two broad groups that are: (1) direct markers of fibrogenesis which are the direct expression of either the deposition or the removal of extracellular matrix in the liver; and (2) indirect markers that are single or combined haematological or biochemical parameters that reflect the stage of liver disease.

These include several glycoproteins (hyaluronan, laminin, YKL-40), the collagen family (procollagen III, type IV collagen), collagenases and their inhibitors, and a number of cytokines connected with the fibrogenetic process (TGF-β1, TNF-β). While some of these biochemical markers, namely hyaluronan and type III procollagen, have demonstrated a good accuracy in excluding cirrhosis, their performance in defining the stage of liver fibrosis greatly varies from one study to another, with a wide range of sensitivity and specificity [83].

The first indirect markers of liver fibrosis are the transaminases, particularly when reported as aspartate aminotranferase (AST) to alanine aminotransferase (ALT) ratio (AAR). A further evolution of this index is the so-called APRI (AST to platelet ratio index). In the last few years, many other test panels have been proposed, but the most widely investigated combination set of non-invasive markers of liver fibrosis is the FibroTest^®. This is a combination of five blood tests, including gamma glutamyltransferase (GGT), bilirubin, haptoglobin, apolipoprotein A1, and α2-macroglobulin, adjusted for gender and age by using a patent algorithm [84]. FibroTest^® has been extensively tested in chronic hepatitis, namely virus C hepatitis, where the area under the curve (AUC) resulted to be around 0.85 for significant fibrosis. Recently, a new combination algorithm of non-invasive markers of liver fibrosis in chronic hepatitis C has been proposed, with a high diagnostic accuracy (>94%), allowing a reduction by 50% of the number of liver biopsies [85].

The Enhanced Liver Fibrosis Test (ELF^®) is an algorithm consisting of direct markers of fibrogenesis, namely hyaluronic acid, the amino terminal propeptide of type III collagen, and tissue inhibitor of MMP-1. A systematic review has shown comparable results for ELF^® and FibroTest^®, with an AUC of 0.90 for cirrhosis and 0.82 for >stage 2 fibrosis [86, 87].

Cirrhotic cardiomyopathy is defined as ‘a cardiac dysfunction in patients with cirrhosis that is characterized by impaired contractile responsiveness to stress and/or altered diastolic relaxation with electrophysiological abnormalities in the absence of other known cardiac disease’ [88].

Brain natriuretic peptide (BNP) and NT-proBNP elevations correlate with the severity of cirrhosis and with the degree of cardiac dysfunction, as well as with the intraventricular septal and LV wall thickness [89]. A possible role for adrenomedullin, a hormone involved in the regulation of vascular tone and natriuresis, was recently reported [90]; adrenomedullin may contribute to myocardial dysfunction in cirrhosis, via the negative inotropic effect of NO. Serum levels of this hormone are higher in cirrhotic, particularly in ascitic, than non-ascitic patients.

Recent cross-sectional and prospective studies on the association between non-alcoholic fatty liver disease (NAFLD) and intermediate markers of CVD or clinical outcomes indicate not only a link, but also a causal role of NAFLD in the pathogenesis of CVD [91]. Current understanding of the pathogenesis of NAFLD implies that lipids accumulate in hepatocytes in the presence of insulin resistance and that multiple factors, including non-esterified fatty acids (NEFAs), hormones, pro-inflammatory cytokines, and adipocytokines, are involved in the atherogenetic process, as shown in Figure 39.2.

The liver is central to the production of classical biomarkers of inflammation and endothelial dysfunction, the secretion of which depends, at least in part, on factors that are upregulated in the presence of insulin resistance and the metabolic syndrome. Fibrinogen and CRP are increased in NAFLD patients, particularly in those with steatohepatitis (non-alcoholic steatohepatitis, NASH). In the last few years, the importance of adiponectin has been increasingly recognized, and hypoadiponectinaemia is independently associated with NASH and with more severe hepatic steatosis and necroinflammation [92]. The measurement of these biochemical markers should be suggested to allow an early diagnosis and effective treatment of these diseases.

The discovery of molecular and pathophysiological mechanisms underlying the complex interactions between the heart, kidney, and liver paved the way to the development and implementation of new biomarkers. In particular, a body of evidence has been collected to recommend the reporting of eGFR, as well as the inclusion of cystatin C assay, for cardiovascular risk prediction. The ACR, a very simple and inexpensive test, is associated with an increased risk of CKD complications, as well as with a subsequent risk of cardiovascular mortality, and NGAL represents a very promising marker in AKI. Concerning the heart and liver interaction, the measurement of simple tests, such as serum transaminases, is still of value in acute liver diseases, while a promising field of translational research is in the development of non-invasive markers of fibrosis.