Page 239 – Clinical Laboratory int.

Chronic kidney disease: an ‘invisible epidemic’ ?

, 26 August 2020/in Featured Articles /by 3wmedia

A growing challenge for nephrologists is to quickly identify patients with chronic kidney disease (CKD). Experts believe there is now “coherent, undisputable evidence” that timely treatment of CKD can delay the onset of end-stage renal failure (ESRF).

The challenge of timely diagnosis
Several studies suggest that early identification and management of CKD can reduce the risk of kidney failure progression and halve co-morbidities such as cardiovascular disease. The latter is a significant achievement on its own, since cardiovascular complications are estimated to increase mortality in CKD patients by 2-4 times over that of the general population.
Nevertheless, early diagnosis of CKD has not proven to be easy. Chronic kidney disease tends to develop slowly, with few evident symptoms. As a result, most patients seek clinical expertise only after the disease has advanced to a stage where dialysis becomes unavoidable.

Epidemiological data limited
Confounding the problem is a lack of epidemiological data. In the US, the Centers for Disease Control (CDC) can only estimate that “more than 20 million people may have CKD.”
The picture is even less encouraging in Europe. In a first-of-its-kind effort in 2010 to explore what they labelled a “silent epidemic,” Dutch and Italian researchers were “struck by the fact that epidemiological data for CKD existed only in a tiny minority of European countries.” More puzzling was official data from the European Commission or the bulk of EU Member States, which “did not even mention CKD as an issue of public health concern.”
Attempts to establish aggregated CKD data in Europe have led to mixed results. The ambitious EUGLOREH (Status of Health in the European Union) survey established prevalence rates of (higher risk) Stages 3-5 CKD ranging from 3.6% in Norway to 7.2% in Germany, but little about earlier Stages (1 and 2) where treatment best delays progression.
Prevalence of Stages 3-5 CKD was similar for males and females in Germany and Italy, but higher for females in other countries – Belgium, England, Iceland and Norway. In countries for which data was available, prevalence also rose with age.
However, EUGLOREH underlined the total lack of time-trend epidemiological data in Europe, unlike the US where the so-called NHANES III and IV surveys showed prevalence of Stages 1-5 CKD rising from 14.5% in 1988-94 to 16.8% in 1999-2004, while that of Stages 3-5 remained near unchanged, at about 6%.

Delaying dialysis
Recent clinical practices, which advise delaying the onset of dialysis as far as possible, are driving the need for early identification of CKD. In 2003, ‘Kidney International’, the Official Journal of the International Society of Nephrology, stated that “dialysis delayed is death prevented.”
The authors noted that hundreds of thousands of renal failure patients lived in developing countries with inadequate resources or infrastructure; however, many Western patients too were unsuitable for dialysis, “because of age, frailty, or a co-morbid illness with poor prognostic outcome.”

Costs of CKD
The financial impact of CKD is high. In the US, the cost of CKD and its co-morbidities in the pre-ESRF phase are estimated to be as much as $26,000 per patient each year.
Delaying the onset of ESRF provides significant, additional benefits. For example, Italy spends 1.8% of its total health care budget on ESRF patients, who represent only 0.083% of the general population. In the UK, ESRF accounts for 2% of health spending.
The situation in the US is dramatic, with kidney treatment accounting for 24% of Medicare spending, higher than congestive heart failure.
Testing for CKD
Typically, detection and monitoring of CKD requires both urine and blood tests in the laboratory. The former typically focus on kidney function and the latter on damage, according to the US National Institutes of Health (NIH). Key markers for CKD are abnormal urinary levels of albumin (albuminuria) and other proteins (proteinuria), along with a persistent reduction in the glomerular filtration rate (GFR). Variations in urine concentration are corrected by urine albumin-to-creatinine ratio (ACR) or protein-to-creatinine ratio (PCR) on a spot specimen. However, there is considerable debate about the clinical and cost-effectiveness evidence of ACR versus PCR.
So far, CKD testing has largely been driven by diabetes patients, with best practices based on care standards from professional bodies such as the US National Kidney Foundation, the American Diabetes Association and the National Institute for Health and Care Excellence (NICE) in the UK. At-risk groups considered to be typical targets for screening consist of patients with hypertension, cardiovascular disease or a family history of kidney failure. In the US, the CDC estimates that about “1 of 3 adults with diabetes and 1 of 5 adults with high blood pressure has CKD.”

Limitations to screening
With some exceptions, testing urine is not recommended in the general population. In the US, the NIH explains that the “benefit of CKD screening in the general population is unclear.”
The reasons for this restraint are also practical. Albumin levels fluctuate over the day due to metabolism and diet. As a result, urine samples are collected on a random, 4-hourly, or overnight basis. If abnormalities are indicated, re-confirmation is required via a timed, 24-hour collection cycle, accompanied by the labelling and refrigerated storage of the samples. All this clearly poses logistical challenges for large scale CKD screening.
Secondly, any mass screening would clearly use colour-changing dipsticks, the first point of call for urine tests. Though the National Kidney Foundation considers dipsticks satisfactory for a first screening, it warns clinicians to be especially cognizant of false negative results, and specifies the need for lab analyses to quantify urine PCR and ACR ratios. Indeed, while ACR of over 30 mg/g is considered to indicate CKD, the sensitivity level of dipsticks begins at 300 mg/g.
The only alternative to urine is a blood sample, but this too has evident limitations as a mass screening tool for CKD.

Expanding screening: recent developments
Nevertheless, there is growing evidence that larger-scale screening for CKD is likely in the future.
In 2005, Dutch researchers sought to address the challenge of a 24-hour urine collection cycle. They reported that a level of about 11 mg/L urinary albumin concentration (UAC) in a single spot morning urine sample could identify microalbuminuria, and do this as effectively as ACR. They suggested the above as a cut-off point for requiring subsequent urine sample collections, to avoid “huge numbers of individuals” having to undergo “the cumbersome procedure of a 24-hour urine collection” in any mass screening program.
In 2008, the UK’s NICE formally enlarged the scope of testing. It recommended that apart from diabetes, screening for CKD be undertaken in patients with hypertension, cardiovascular diseases, structural renal tract disease, renal calculi or prostatic hypertrophy and multisystem diseases such as systemic lupus erythematosus. The list extended to people with a family history of Stage 5 CKD or hereditary kidney disease.
In its guidelines, NICE also attempted to come to grips with vexing questions on CKD markers such as albuminuria and proteinuria, and the selection and sequencing of tests. The Institute however left open room for further interpretation. Its consensus recommendation was that “ACR should be the test of choice” , but also said there would “often be good clinical reasons for subsequently using PCR to quantify and monitor” proteinuria.
In 2012, the Australasian Proteinuria Consensus Working Group took what may be one of the biggest steps so far to scale up screening. Based on a retrospective longitudinal cohort study of 5,586 CKD patients, it extended the UK NICE screening envelope to include obesity and smoking, and “strongly” advocated targeted opportunistic testing for CKD risk in “all adults attending an appointment with their health care practitioner”. The Working Group also concluded that ACR from a morning (or random) spot urine sample was as good as PCR and 24-hour urinary albumin and protein measurements, and recommended testing for albuminuria rather than proteinuria in individuals at risk of CKD.
The group advised “all pathology laboratories in Australia” to implement its recommendations as part of an “integrated national approach to CKD detection.”

The UK: A ‘World Leader’ in early detection of CKD
Although the Australian recommendations appear to be much more explicit than the NICE guidelines, the UK has ensured that CKD is incorporated into its wider Quality and Outcomes Framework (QOF). As a result, GPs are now rewarded based on how well they identify and manage patients with CKD. CKD is given 27 points in QOF, alongside 9 points for diabetes related to kidney disease. This is considered to provide significant support for the Renal National Service Framework (NSF), which aims to minimize the impact of kidney disease in its early stages.
Indeed, the EUGLOREH report mentioned previously considers the CKD-directed steps in the Quality and Outcomes Framework to have made the UK “a world leader in this field.”

Telediagnosis may offer breakthrough in mass CKD screening
Meanwhile, a breakthrough in the US could remove the final barriers to a true mass screening program for CKD.
In August 2013, researchers from the University of California at Los Angeles (UCLA) announced they had developed a compact, field-portable device to conduct albumin tests and transmit data via smartphone, thus reducing the need for “frequent office visits by people with diabetes and others with chronic kidney ailments” or the use of “bulky and costly benchtop urine analysers” which limit testing and diagnosis to laboratory settings. The new system projects beams of visible light through two attached fluorescent tubes, one of which contains a control liquid and the other a urine sample mixed with fluorescent dyes. The smartphone camera captures the fluorescent light after it passes through an additional lens.
The device measures albumin concentration to less than 10 micrograms per millilitre, which its inventor states is “more than 3 times lower than the clinically accepted normal range.” It weighs less than 1 kg and is estimated to cost $50 to $100, with tests taking about 5 minutes.

Measuring renal function: traditional methods and iohexol clearance

, 26 August 2020/in Featured Articles /by 3wmedia

An accurate measure of glomerular filtration rate (GFR) is required in a variety of clinical situations. Plasma creatinine and creatinine-based equations have served as convenient estimates of GFR but they are subject to limitations. Formal assessment of GFR using clearance methods traditionally necessitates the use of radioactive traces such as ⁵¹Cr-EDTA. More recently, iohexol has become increasingly well recognized as an alternative exogenous marker which can now be used routinely, providing a safe, efficient and cost-effective measure.

by Zoe Maunsell and Prof. Tim James

Background
Glomerular filtration rate (GFR) describes the amount of fluid filtered by the glomerulus per unit time, and is therefore a useful indicator of renal function. Expressed in mL/min, it can be accurately measured from the rate of disappearance of an injected substance from the plasma or by collecting urine over a defined time period. The clearance formula, UV/P (where U represents urine concentration, V is urine volume per unit time and P is plasma concentration) can be used to calculate GFR when an ideal marker is used. Such a marker should be freely filtered at the glomerulus, and not secreted, absorbed or metabolized by the renal tubules. Since renal function is proportional to kidney size (which is proportional to body surface area), when estimating GFR, values are usually standardized to an average body surface area of 1.73 m². Having an accurate yet convenient means to monitor renal function is extremely important in clinical practice. A compromise exists between highly accurate but time consuming, technically difficult reference methods and more accessible, readily available markers of renal function.

Gold standard methods
The ‘gold standard’ for the measurement of GFR is inulin clearance. Inulin is a polysaccharide derived from plants, which can be introduced into the body either by intravenous infusion or bolus dose and its rate of clearance measured. Since inulin is freely filtered by the glomerulus, is not absorbed, secreted or metabolized by the tubules, its elimination is proportional to GFR. Although it is the ‘gold standard’ for assessment of GFR, assay of inulin is technically demanding, and so this technique is not suitable for routine clinical practice.
Similarly, the radioactive tracer ⁵¹Cr-EDTA can be administered intravenously and its elimination rate monitored. This marker is widely used to measure GFR, but presents a risk to patients and healthcare workers since it involves exposure to ionizing radiation.

Iohexol clearance is an alternative to inulin clearance [1]. Iohexol is an iodine-containing, non-isotopic contrast agent, and studies have shown close agreement between GFR values obtained by iohexol and inulin clearance.

Creatinine and endogenous markers
Creatinine is a widely used clearance marker but suffers from well-described problems including the difficulty and inconvenience of 24-hour urine collection.

GFR has therefore been estimated without urine collection using endogenous markers, the most widely used of which is plasma/serum creatinine. Improvements have been made to the methodology used for creatinine measurement, including standardization of kinetic Jaffe creatinine assays, defining their traceability to isotope-dilution reference assays. However, the Jaffe method remains subject to numerous interferences and therefore laboratories may select the more specific enzymatic creatinine methods. Using creatinine as a marker of GFR has several limitations, including the relationship of creatinine concentration to muscle mass, meaning that estimation of GFR using creatinine alone is particularly a problem in children. It is for this reason that corrections for body surface area have been made. The major limitation of creatinine alone as a marker of GFR is that it is a relatively insensitive marker, and a large decline in renal function can occur before a change in plasma creatinine concentration is observed.

Cystatin C is another endogenous marker that can be used for the estimation of GFR. It is a cysteine protease inhibitor, is produced at a constant rate (independent of muscle mass, sex, age when older than 12 months and inflammatory conditions) and is freely filtered by the kidneys. It is almost completely reabsorbed by the proximal renal tubular cells so that little is normally excreted in the urine. The reciprocal of plasma cystatin C concentration has been shown to be correlated with GFR. Cystatin C has significant advantages over creatinine as a marker of renal function, since increases in serum concentration become apparent with mild renal impairment, such as GFR of 60–90 mL/min, and may be more useful than creatinine in detecting acute kidney injury. Like creatinine, the measurement of cystatin C suffers to some extent from problems with standardization, in particular with reported differences [2] in measured concentrations using turbidimetric versus nephelometric assays by different manufacturers, presumably due to the use of different antibody and detection systems. The availability of an international reference preparation (ERM–DA471/IFCC) is likely to lead to greater agreement between cystatin C methods.

Calculated estimates of GFR
In order to overcome the limitations of measuring creatinine and cystatin C concentrations alone, calculations have been devised. These involve corrections for body surface area, sex, age and ethnicity. Among the most well known are the 4- or 6-parameter MDRD (Modification of Diet in Renal Disease study), Cockroft–Gault (which estimates creatinine clearance) and CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equations for adults and the Schwartz and the Counahan–Barratt prediction equations for children.

The Cockroft–Gault equation [3] provides an estimate of creatinine clearance. Devised in the 1970s, the equation was calculated by examining the mean of two 24-hour creatinine clearances from 249 adult men.

The 4-variable MDRD equation [4] (incorporating creatinine, age, sex and race) is the most widely used in the UK, being reported by laboratories in the form of eGFR, and forms the basis of the staging system for CKD. The formula was developed from the Modification of Diet in Renal Disease study, using data from 1628 patients. The patients had already been diagnosed with CKD, and the study aims were to slow the progression of CKD with a low protein diet and blood pressure control. However, significant limitations to the use of the equation exist, mainly connected with the use of creatinine as a marker of GFR. These include inaccuracies of the equation at extremes of body type such as malnourished or amputee patients and the unsuitability of the equation for use in pregnancy. The formula includes a correction factor for use in African American populations but its validity in other ethnic groups has not been established. The MDRD formula tends to underestimate function at normal GFR, therefore slight reductions in eGFR should not be over-interpreted and reporting eGFR >90mLs/min/1.73m² is not recommended.

The CKD-EPI equation was developed in 2008 [5] and updated in 2012 [6]. The 2008 equation was developed using data from over 8000 patients from 10 studies. The 2012 versions used over 5000 patients from 13 studies. Importantly, both studies included patients with normal GFR, as well as those with CRF. It was found that the CKD-EPI equations performed better than the MDRD equation, particularly at higher levels of estimated GFR. The limitations of the studies include a limited number of elderly patients and those from ethnic groups other than black race.

In terms of estimation of GFR in children, the Schwartz [7] and Counahan–Barratt [8] equations were developed in the 1970s and have been widely used to estimate GFR from serum creatinine and height (length in infants). The Schwartz equation was modified in 2009 [ 9, 10]. Based on new studies of iohexol clearance, the original formula was found to overestimate GFR. It was postulated that this was in part due to the use of new, standardized creatinine assays. In 2009 the equation was updated, based on studies of 349 patients aged 1–16 years with mild to moderate chronic renal disease. The formula uses creatinine determined using an enzymatic method, urea, height and cystatin C.
Various modifications of these equations, using creatinine, cystatin C [11] or both [12] have been published and optimized in various patient cohorts. When using creatinine-based assays, it is important to know which creatinine assay is being used, since equations have been devised which include different coefficients depending on the methodology used.

Iohexol clearance: a gold standard method in routine use
Oxford University Hospitals NHS Trust has been routinely offering an iohexol clearance service for the measurement of GFR since June 2011. The service is widely used by the hospital’s pediatric service, and is used in a variety of clinical situations, including determining renal function in surgical patients and for chemotherapy dosing. Before the introduction of this service, GFR was assessed using ⁵¹Cr EDTA clearance. Although this provided an accurate measurement of GFR, a long waiting list and difficult patient preparation made this technique suboptimal for use in children. In addition, where GFR results are required before administration of chemotherapy agents, timely result availability is critical to prevent delayed treatment. With iohexol clearance we have been able to improve GFR turnaround time compared to ⁵¹Cr-EDTA clearance and audit of our first 2 years of service demonstrated that 89% of results were reported within 2 working days and 99% within 3 working days. Iohexol clearance also avoids the use of radioactive isotopes, reducing exposure for patients, carers and hospital staff. Therefore the service has several distinct advantages over conventional isotopic clearance methods (Table 1).

Iohexol clearance is measured according to a standardized protocol, as described previously [13]. In summary, the patient is cannulated, a baseline blood specimen is collected and a standard dose of Omnipaque containing 300 mg iodine/mL is administered through the cannula, (2 mL in patients weighing <40 kg, 5 mL in patients weighing >40 kg). Blood (1 mL) is collected into lithium heparin tubes at 120, 180 and 240 min after iohexol administration.

The iohexol concentration in each specimen is measured by a UPLC (ultra-high performance liquid chromatography) method involving precipitation of plasma samples with equal volumes of perchloric acid and injection of the supernatant onto a Waters 50 x 2.1 mm 1.8 μm reversed phase column with isocratic acetonitrile-based solvent elution. The assay uses a one-point (604 mg/L) calibration through zero. The assay is straighfoward, reliable and rapid; chromatography time is 6 min per specimen. The assay demonstrates excellent inter-assay CVs: 2.2%, 1.9% and 1.9% at 39, 163 and 322 mg/L respectively. The laboratory participates in external quality assessment through the Scandinavian EQUALIS scheme.

The two-point model is used to calculate iohexol clearance, according to the Brochner–Mortensen method [14]. The model assumes a one-compartment model where iohexol is cleared by first-order kinetics. The estimated clearance (Cl ) of the GFR marker is expressed as:

Cl= Q• b/c1 (ml/min)

Where Q = amount of injected marker, b = disappearance rate of marker (min⁻¹), c₁= intercept on the y-axis.

The clearance correction for non-immediate mixing of the tracer substance is expressed as:
Cl = 0.990778 x estimated Cl−0.001218• estimated Cl²

Alternatively, single point estimates of GFR at appropriate time points can be calculated using the Jacobsson model. This method can be used in cases where the two-point method cannot be used, for example when contamination has occurred due to poor flushing of lines between sample collection. Here:

GFR = ln(D/VCt) / (t/V + 0.0016)

Where V = volume of distribution in mL (V = 187 x weight in kg + 732), t = time of sampling in min, D = dose of iohexol administered, Ct = concentration of iohexol at time t.

Iohexol clearance is corrected for actual body surface area, using the Dubois and Dubois method, calculated as 0.007184 x (weight in kg)^0.425 x (height in cm)^0.72, to yield a measure of GFR in mL/min/1.73 m².

Since initiating the iohexol GFR service in 2011, we have analysed over 400 sets of patient samples. The use of UPLC over HPLC allows for smaller sample volumes to be collected, making the technique particularly suited to pediatrics. Ongoing close liaison with clinicians and nursing staff has revealed that patients and their families have responded favourably to the new procedure and in particular prefer being able to remain on the Daycare Ward throughout the investigation, which is a child-friendly environment.

Iohexol clearance has the added benefit of being significantly less costly than ⁵¹Cr-EDTA clearance. The approximate cost is £80 compared to £250 for ⁵¹Cr-EDTA.

Conclusions
The measurement of glomerular filtration rate, GFR, is important in a number of clinical scenarios. Much work has been undertaken to develop methods of estimating GFR in order to avoid the time-consuming and relatively invasive formal measurement of renal function using clearance methods. These include the development of eGFR equations based on factors such as plasma creatinine, cystatin C, age, race and body surface area. Equations such as the MDRD and CKD-EPI formulae for adults, and Schwartz and Counahan–Barratt equations for children have been widely used in clinical practice.

However, despite their widespread use, limitations of these equations have been described, including the problems of creatinine-based equations tending to underestimate GFR at normal GFR levels and the issue of creatinine-based formulae being unsuitable for use in patients with non-normal muscle mass.
For the formal measurement of GFR, clearance studies must be performed which have traditionally used radioactive tracers such as 51Chromium-EDTA. However, in recent years the use of iohexol has increased. Thanks to its stability in vitro and relative ease of measurement, the assay of iohexol is rapid, reliable and can be performed on small specimens. This fits in particularly well with the clinical needs of a pediatric service for GFR measurement and is cheaper, safer and more convenient than traditional methods.

References
1. Ng DK, Schwartz GJ, Jacobson LP, Palella FJ, Margolick JB, Warady BA, Furth SL, Muñoz A. Universal GFR determination based on two time points during plasma iohexol disappearance. Kidney Int. 2011; 80: 423–430.
2. Herget-Rosenthal S, Bökenkamp A, Hofmann W. How to estimate GFR-serum creatinine, serum cystatin C or equations? Clin Biochem. 2007; 40: 153–161.
3. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976; 16: 31–41.
4. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: A new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med. 1999; 130: 461–470.
5. Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF III, Feldman HI, Kusek JW, Eggers P, Van Lente F, Greene T, Coresh J, for the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI). A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009; 150: 604–612.
6. Inker LA, Schmid CH, Tighiouart H, Eckfeldt JH, Feldman HI, Greene T, Kusek JW, Manzi J, Van Lente F, Zhang YL, Coresh J, Levey AS, for the CKD-EPI Investigators. Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med 2012; 367: 20–29.
7. Schwartz GJ, Haycock GB, Edelmann CM Jr, Spitzer A. A simple estimate of glomerular filtration rate in children derived from body length and plasma creatinine. Pediatrics 1976; 58: 259–263.
8. Counahan R, Chantler C, Ghazali S, Kirkwood B, Rose F, Baratt TM. Estimation of glomerular filtration rate from plasma creatinine concentration in children. Arch Dis Child 1976; 51: 875–878.
9. Schwartz GJ, Muñoz A, Schneider MF, Mak RH, Kaskel F, Warady BA, Furth SL. New equations to estimate GFR in children with CKD. J Am Soc Nephrol. 2009; 20: 629–637.
10. Schwartz GJ, Work DF. Measurement and estimation of GFR in children and adolescents. Clin J Am Soc Nephrol 2009; 4: 1832–1843.
11. Grubb A, Nyman Un, Björk J, Lindström V, Rippe BG, Christensson A. Simple cystatin C–based prediction equations for glomerular filtration rate compared with the modification of diet in renal disease prediction equation for adults and the Schwartz and the Counahan–Barratt prediction equations for children. Clinical Chemistry 2005; 51: 1420–1431.
12. The Swedish Council on Health Technology Assessment (SBU). Methods to estimate and measure renal function (glomerular filtration rate). Stockholm: The Swedish Council on Health Technology Assessment (SBU). 2012; Yellow 214.
13. James TJ, Lewis AV, Tan GD, Altmann P, Taylor RP, Levy JC. Validity of simplified protocols to estimate glomerular filtration rate using iohexol clearance Ann Clin Biochem. 2007: 44; 369–376.
14. Bröchner-Mortensen J. A simple method for the determination of glomerular filtration rate. Scand J Clin Lab Invest. 1972; 30(3): 271–274.

The authors
Zoe Maunsell* MBiochem MSc DipRCPath and Tim James PhD
Department of Clinical Biochemistry, Oxford University Hospitals NHS Trust, Oxford, UK
*Corresponding author
E-mail: zoe.maunsell@ouh.nhs.uk

Monitoring kidney function in aminoglycoside therapy

, 26 August 2020/in Featured Articles /by 3wmedia

Aminoglycosides are antibiotics largely used at the hospital. Their nephrotoxicity imposes therapeutic drug monitoring as well as kidney function monitoring. Creatinine is the most widely used biochemical marker; however, new biomarkers such as neutrophil gelatinase associated lipocalin (NGAL), cystatin C (Cys C) or kidney injury molecule-1 (KIM-1) can allow the detection of acute kidney injury more quickly.

by F. Fraissinet, E. Sacchetto and Dr E. Bigot-Corbel

Background
Aminoglycosides are bactericidal antibiotics used for the treatment of Gram-negative or endocarditis infections. The most important adverse effects of aminoglycosides are nephrotoxicity and ototoxicity. The prevalence of aminoglycoside-associated nephrotoxicity is estimated at 10 to 20 %, although it also depends on the patient’s clinical condition and exposure to nephrotoxic drugs such as cyclosporine, anti-inflammatory or iodinated drugs. In intensive care units, nephrotoxicity is most frequent (60% of patients) and associated with a high rate of mortality [1, 2]. Nephrotoxicity mainly results in nonoliguric acute kidney injury which occurs following 7 to 10 days. This toxicity may manifest as a decrease in the glomerular filtration rate with glycosuria, hypokalemia and hypocalcemia. Aminoglycosides are often administered by intravenous drip and are freely eliminated by glomerular filtration and reabsorbed by the proximal tubule. Of the injected dose, 5% is retained by epithelial cells of the proximal tubule. After endocytosis in tubular cells, these molecules accumulate in lysosomes and induce phospholipidosis, alteration of key cellular components and apoptosis. Aminoglycosides also have glomerular effects: gentamicin stimulates mesangial proliferation, produces mesangial contraction and induces neutralization of negative charges of the glomerulus [3]. Gentamicin also induces a reduction in renal blood flow with an increased renal vascular resistance. These factors contribute to decrease the glomerular filtration rate (GFR). Additional risk factors for nephrotoxicity induced by aminoglycoside have been identified as sepsis, prolonged therapy, renal or liver dysfunction, hypokalemia or hypomagnesemia. Nephrotoxicity is less frequent when aminoglycosides are administered once daily compared with 12 h [4].

Methods of detection of acute kidney injury induced by aminoglycoside therapy

Classic markers: creatinine and creatinine clearance
Creatinine is the most widely used marker in the diagnosis of the acute renal insufficiency. For defining AKI, the Risk Injury Failure Loss End stage kidney disease (RIFLE) classification is based on increase of serum creatinine concentration and decrease of glomerular filtration rate. The introduction of the RIFLE classification has increased the conceptual understanding of AKI syndrome, and this classification has been successfully tested in a number of clinical studies [5].
In spite of an easily accessible dosage, creatinine as a marker of AKI has some drawbacks. Creatinine is filtered by the glomerulus and is not bound to plasma proteins. In standard physiological conditions, the daily rate of creatinine production is constant; however, the rate of creatinine production is affected by conditions of muscular pathology or muscular loss (as occurs in intensive care and cirrhosis). In these patients, AKI does not result in an increase of serum creatinine levels. Other factors, such as age, sex, ethnic group and diet, also influence serum concentrations of creatinine. Creatinine is not the ideal marker to estimate GFR, because it is secreted by renal tubule, which artefacutally increases glomerular filtration rate. At low serum creating concentrations, creatinine is lacks sensitivity to estimate GFR. Large changes in GFR may be associated with relatively small changes in serum creatinine (See Figure 2 in Delanaye et al. [6]). The rise of the creatinine is late (occurring after 3–5 days) and is not specific for nephrotoxicity induced by aminoglycosides, and an increase of creatinine in AKI is a function of the initial concentration of creatinine [7].

To estimate GFR, formulas that use creatinine plasma concentration, such as the Modified Diet in Renal Disease formula (MDRD), Chronic Kidney Disease Epidemiology collaboration formula (CKD-EPI) or estimation of clearance creatinine by Cockroft–Gault (CG) equation, were derived in subjects with chronic, not acute, kidney disease. A limitation of the MDRD equation was an underestimation of GFR in the high range. The CKD-EPI equation performs better at high GFR levels (GFR >60 mL/min/1.73 m²). Use of serum creatinine concentration to estimate GFR supposes a steady-state between creatinine production and excretion [8]. In spite of the use of correction factors, it is more difficult to estimate GFR in Asian or African populations as well as in elderly or obese patients [9].

New biomarkers

Cystatin C
Cystatin C (CysC), a 13-kDa endogenous cysteine proteinase inhibitor, plays an important role in intracellular catabolism of various peptides and proteins. CysC is considered to be a good biomarker of decreased kidney function because it is produced at a relatively constant rate and released into plasma, and is filtered by glomeruli without tubular secretion. The influence of muscular mass is less than for creatinine, and CysC allows diagnosis of AKI 48 h before serum creatinine [10]. Equations with serum CysC concentration can also estimate GFR. If GFR is great than 60 mL/min/m², CysC measurement is more powerful than the MDRD equation. CysC is a useful biomarker for early detection of AKI in the pediatric population and for patients in the intensive care unit, as CysC determination can be performed in serum and/or in urine. In spite of efforts to standardize the procedure, there is no reference method. Production of CysC also depends on hormonal factors, so CysC cannot be used in cases of thyroid dysfunction.

Neutrophil gelatinase associated lipocalin
Neutrophil gelatinase associated lipocalin (NGAL) is a protein of 25 kDa protein of the lipocalin family and is covalently bound to matrix metalloproteinase-9. NGAL is expressed early in ischemic kidney impairment in animal models. During AKI, NGAL expression is induced in distal nephron epithelia resulting in elevated plasma and urinary levels of NGAL (Fig. 1) [2]. NGAL determination can be performed on serum and/or urine by immunoturbimetric or immunofluorimetric assays. There is a general agreement on a cut-off value of >150 ng/mL, but a clear cut-off NGAL concentration for AKI has not been reported. Several studies show the importance of NGAL in cardiac surgery or critically ill patients for predicting AKI. NGAL is also useful for the detection of nephrotoxicity induced by contrast agents and has prognostic value for mortality or initiation of renal replacement therapy. Plasma NGAL measurements may be influenced by a number of coexisting variables as chronic hypertension, systemic infections, inflammatory conditions or hypoxia. Changes in NGAL values are potentially associated with septic state or aminoglycoside therapy [11].

Kidney injury molecule-1
Kidney injury molecule-1 (KIM-1) is a glycoprotein localized in the apical membrane of the proximal tubule of kidney, and KIM-1 expression can be induced by nephrotoxic drugs. Urine KIM-1 is a promising biomarker of proximal tubular injury. As with NGAL, urinary KIM-1 levels predicted adverse clinical outcomes such as dialysis requirement and mortality. In a previous study, urinary KIM-1 is correlated with AKI severity in non-critically ill children treated by aminoglycosides [12].

Conclusion
Patients treated with aminoglycosides must be carefully monitored for nephrotoxicity. Creatinine has been the most used biochemical marker of AKI, but new biomarkers, such as NGAL and KIM-1, have been developed in recent years.

References
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The authors
François Fraissinet¹ BSc, Emilie Sacchetto² and Edith Bigot-Corbel²* PhD
¹Laboratoire de Biochimie, 86021 Poitiers, France
²Laboratoire de Biochimie, CHU de Nantes, Hôpital G et R Laënnec, 44800 Saint-Herblain, France

*Corresponding author
E-mail: edith.bigot@chu-nantes.fr

Anti-PLA2R: the serological biomarker for primary MN

, 26 August 2020/in Featured Articles /by 3wmedia

Autoantibodies against phospholipase A2 receptors (PLA₂R) are a new, highly specific diagnostic marker for primary membranous nephropathy (MN). Detection of anti-PLA₂R using easy-to-perform and inexpensive serological assays can indicate primary MN in patients suffering from nephrotic syndrome and secure a differential diagnosis from secondary MN. Anti-PLA₂R analysis is also useful for determining the disease activity, assessing the extent of treatment required and monitoring responses to therapy. Anti-PLA₂R antibodies can be determined using innovative indirect immunofluorescence and ELISA test systems.

by Jacqueline Gosink, PhD

Primary membranous nephropathy
Primary MN, also known as primary membranous glomerulonephritis or primary MGN, is a chronic inflammatory autoimmune disease of the blood-filtering structures of the kidneys (glomeruli). It is accompanied by a progressive reduction in renal function. The disease manifests the complex nephrotic syndrome, which is characterized by heavy proteinuria, hypoalbuminemia, hyperlipidemia, edema and lipiduria. Primary MN is one of the leading causes of nephrotic syndrome in adults. As proteinuria increases, so does the long-term risk of kidney failure with major morbidity and mortality, especially from thromboembolic and cardiovascular complications. Around a third of patients progress to end-stage renal disease, a third exhibit persistent proteinuria without progression to renal failure, and the remainder experience spontaneous remission. Primary MN is prevalent in all ethnic groups and in both genders, with men over 40 years old being the most frequently affected.

Diagnostic challenge
The diagnosis of primary MN is demanding, as the disease must be differentiated from other nephropathies, especially from secondary MN, which is triggered by an underlying cause such as a malignant tumour, an infection, drug intoxication or another autoimmune disease such as systemic lupus erythematosus or diabetes mellitus type 1. Of all MN cases, 20-30% are of secondary genesis, while the remaining 70–80% are classified as primary. Primary cases with no detectable anti-PLA₂R antibodies are subclassified as idiopathic; it has been postulated that these patients may exhibit antibodies against other, as yet unidentified, target antigens. Reliable differentiation of primary and secondary forms of MN is critical because of different treatment regimes: primary MN is treated with immunosuppressants, while therapy for secondary MN is targeted at the underlying disease.

MN is diagnosed by kidney puncture followed by histological examination or electron microscopy of the tissue to identify the characteristic glomerular immune deposits. To obtain a definite diagnosis of primary MN, secondary causes must be excluded, which involves additional time-consuming and often invasive procedures, for example tumour screening. Moreover, in some patients, MN appears before the secondary cause is even detectable, adding an extra layer of complexity to diagnosis and therapeutic decision-making. Primary MN must also be differentiated from other autoimmune diseases with kidney involvement, for example lupus nephritis, vasculitides associated with antibodies against neutrophil cytoplasm (ANCA) and Goodpasture’s syndrome. The availability of reliable serological tests to support the diagnosis of primary MN has been elusive until recently due to lack of knowledge about the target antigen.

New pathognomonic marker
Autoantibodies against PLA₂R were first discovered and described in patients with primary MN in 2009 (1). PLA₂R is a transmembrane glycoprotein (Figure 1) which is expressed in human glomeruli on the surface of podocytes and is involved in regulatory processes in the cell following phospholipase binding (Figure 2). Type M PLA₂R has been identified as the major target antigen of autoantibodies. In patients with primary MN, antigen-antibody complexes form deposits in the glomerular basement membrane, where they trigger local complement activation with overproduction of collagen IV and laminin. This causes damage to the podocytes, via destruction of the cytoskeleton and broadening of the basement membrane. As a result protein enters the primary urine, giving rise to proteinuria and other symptoms.

Differential diagnosis
Autoantibodies of class IgG against PLA₂R are present in the serum of up to 70-80% of patients with primary MN (1, 2), whereas they are not found in healthy blood donors or patients with secondary MN or other kidney diseases such as lupus nephritis (3) or IgA nephritis. The high predictive value of anti-PLA₂R makes this parameter ideally suited as a diagnostic marker (4).

Disease evaluation
Anti-PLA₂R antibodies are, moreover, very sensitive markers of clinical disease activity. They reflect the pathogenic immunological activity of the disease, which is responsible for the clinical expression in the form of proteinuria. High antibody titres indicate a severe disease course (2, 5), while low titres are associated with a decreased risk of renal failure and a greater rate of spontaneous remission (6). Thus, the anti-PLA₂R titre also serves as a prognostic indicator.

Therapy monitoring
Treatment with immunosuppressants results in a drop in the anti-PLA₂R titre, while in relapse the antibody titre increases again. Significantly, changes in the antibody titre typically precede changes in the proteinuria (6, 7). Thus, a titre increase is detectable before proteinuria appears, while a titre decrease is observed before a reduction in the proteinuria. Patients in remission exhibit residual proteinuria months after the anti-PLA2R titre becomes undetectable. Anti-PLA₂R measurements are therefore extremely useful for early therapeutic decision-making and for long-term monitoring of responses to immunotherapy.

A recently published prospective study reinforced the value of anti-PLA₂R antibodies as a marker of clinical outcome (8). In the study 133 patients with primary MN were tracked over a time period of 24 months. In all cases there was a clear correlation between proteinuria and anti-PLA₂R levels. In patients who were given immunotherapy, a significant time lag was observed between the rapid fall in antibody levels and the protracted reduction in proteinuria. Moreover, remission of proteinuria occurred later in individuals with high antibody levels than in those with low levels. In patients who did not receive immunosuppressive therapy, spontaneous remission was also associated with a reduction in anti-PLA₂R, while individuals who did not achieve remission showed continued elevated antibody levels. Thus, anti-PLA₂R proved a reliable biomarker for immunological and clinical activity in primary MN.

Risk assessment
Up to 40% of patients with primary MN experience a relapse after kidney transplantation. The risk of recurrent primary MN is particularly high if anti-PLA₂R antibodies are found prior to transplantation. In a study on a patient with primary MN, who exhibited high anti-PLA₂R levels before and three months after transplantation (7), it could be shown that immunotherapy resulted in a drop in the antibody concentration and also the level of proteinuria. Other studies have shown that anti-PLA₂R or PLA₂R deposits are detected more often in transplant patients with recurrent MN than in those with de novo MN. A retrospective analysis of fifteen transplant patients revealed that a persistently positive anti-PLA₂R activity at six months or later after transplantation was a significant risk factor for relapse, especially in patients on a weak immunosuppressive regimen (9). Thus, the anti-PLA₂R antibody titre is useful for assessing the risk of relapse after transplantation and the extent of immunotherapy needed to prevent a recurrence.

Anti-PLA₂R test systems
Anti-PLA₂R autoantibodies can be determined easily and reliably using standardized indirect immunofluorescence test (IIFT) and ELISA systems. In the IIFT a BIOCHIP of transfected human cells expressing recombinant PLA₂R is used as the antigenic substrate to provide monospecific antibody detection (Figure 3). A second BIOCHIP containing cells transfected with an empty vector serves as a control. The IIFT represents an established test for serodiagnostic screening, providing qualitative and semi-quantitative antibody analysis. The corresponding ELISA is based on purified recombinant PLA₂R and shows the same high-quality characteristics as the IIFT. The ELISA is particularly useful for disease and therapy monitoring as it offers precise quantification of antibody levels in patient sera. The IIFT and ELISA are fast and simple to perform and are suitable for use in any diagnostic laboratory. Both procedures can be automated.

Clinical data
The performance characteristics of the Anti-PLA₂R IIFT and ELISA have been assessed in a multitude of studies. In a retrospective clinical study (10) the Anti-PLA₂R IIFT yielded a prevalence of 52% in a cohort of 100 patients with biopsy-proven primary MN, and a specificity of 100% with respect to healthy controls and patients with secondary MN or non-membranous glomerular injury. In a prospective clinical study (11) the sensitivity amounted to 82% in patients with biopsy-proven MN where no secondary cause could be found. The difference in sensitivities obtained in different study panels is most likely due to factors such as disease remission and the therapy status of the individuals, which can influence the antibody results, especially when studies are performed retrospectively.

Results obtained with the ELISA show a very good correlation with results from the IIFT (Figure 4). In a retrospective study with sera from 198 patients with primary MN and 836 healthy and disease controls, the ELISA showed a sensitivity of 96% with respect to the IIFT, and a specificity of 99.9% with borderline sera included (12). The few discrepant sera that were negative in the ELISA gave only low titres of 1:10 to 1:100 in the IIFT. All sera with titres of over 1:100 in IIFT were also positive in ELISA.

Summary
Antibodies against PLA₂R represent a landmark development in nephrological diagnostics. Their detection can secure a diagnosis of primary MN in patients with nephrotic syndrome, offering a convenient, non-invasive alternative to biopsy. Anti- PLA₂R determination, moreover, yields information about the disease status and evolvement. Serial measurements are especially useful for monitoring therapy responses over the long term and guiding decision-making on the extent of treatment required for individual patients. Serological tests based on state-of-the-art IIFT and ELISA technology provide simple, quick and highly specific anti-PLA₂R antibody detection.

References
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