Clinical Investigation of Renal Disease

Clinical investigation of kidney disease - technical

Topics covered:

  • Summary
  • Introduction
  • Examination of the urine
  • Estimation of glomerular filtration rate
  • Estimation of renal blood flow
  • Investigation of tubular function
  • Imaging of patients with renal disease
  • Renal biopsy

Summary

An accurate history and careful examination will determine the sequence and spectrum of clinical investigations required to make a diagnosis or decide on prognosis or treatment.

Examination of the urine

Midstream urine (MSU) sample—this standard investigation requires consideration of: (1) macroscopic appearance—this may be suggestive of a diagnosis, e.g. frothy urine suggests heavy proteinuria; (2) stick testing—including for pH (<5.3 in an early-morning specimen makes a renal acidification defect unlikely), glycosuria, specific gravity (should be >1.024 in an early-morning or concentrated sample), nitrite (>90% of common urinary pathogens produce nitrite) and leucocyte esterase; and (3) microscopy—for cellular elements (in particular red cells, with the presence of dysmorphic red cells detected by experienced observers indicative of glomerular bleeding), casts (cellular casts indicate renal inflammation), and crystals.

Quantification of proteinuria—this is important because the risk for progression of underlying kidney disease to endstage renal failure is related to the amount of protein in the urine. Quantification by 24-h urinary collection is cumbersome and unreliable in many patients, and has been replaced by estimation of the urinary albumin:creatinine ratio (ACR; normal is <2.5 mg/mmol for men and less than 3.5 mg/mmol for women) or protein:creatinine ratio (PCR; normal is <13 mg/mmol) on a spot sample. An ACR of 100 mg/mmol approximately corresponds to proteinuria of 1.5 g/day, and 350 mg/mmol to nephrotic-range proteinuria.

Low-molecular-weight proteinuria—is caused by proximal tubular injury and can be detected with markers including α-glutathione-S-transferase, α1-macroglobulin, and retinol-binding protein.

Estimation of glomerular filtration rate

Knowledge of the glomerular filtration rate (GFR) is of crucial importance in the management of patients, not only for detecting the presence of renal impairment, but also in the monitoring of all patients with or at risk of renal impairment, and in determining appropriate dosing of those drugs cleared by the kidney. Measurement of plasma creatinine remains the standard biochemical test used to assess renal function.

Estimating the glomerular filtration rate (eGFR)—from a measurement of plasma creatinine concentration, the standard method uses the simplified Modification of Diet in Renal Disease (sMDRD) formula, which requires knowledge of the patient’s sex, age, and ethnicity (but not their weight). On the basis of the eGFR, stages of chronic kidney disease (CKD) are classified as follows:

Limitations of the eGFR—this has not been validated in people below 18 years of age, hospitalized patients, or those with acute kidney injury, pregnancy, oedematous states, muscle-wasting disorders, amputations, or malnourishment. Similarly, it has not been validated for extremes of age or body weight, or for ethnic groups other than whites of northern European origin and African-Americans. Because of the inaccuracy of the MDRD equation, particularly for those with eGFRs greater than 60 ml/min, a revised version (CKD-EPI) has been introduced.

Other methods of measuring GFR—isotopic methods can provide the most accurate determination of GFR, but are not often required in routine clinical practice. Estimation of creatinine clearance with a 24-h urinary collection remains a useful test, particularly when there is reason to doubt the validity of the eGFR.

CKD stagea eGFR (ml/min per 1.73 m2 body surface area)
1 >90, with other evidence of renal disease
2 60–89, with other evidence of renal disease
3A 45–59
3B 30–44
4 15–29
5 <15, or receiving renal replacement therapy

a The suffix (p) can be used to denote the presence of proteinuria as defined by a spot urinary albumin:creatinine ratio (ACR) of ≥30 mg/mmol, which is approximately equivalent to a protein:creatinine ratio (PCR) of ≥50 mg/mmol (≥0.5 g/24 h).

Investigation of tubular function

Proximal tubule—analysis of excretion of the following substances can assist in the diagnosis of proximal tubular disorders: (1) glucose—the maximum reabsorption rate for glucose (TmG) in the proximal tubule can be determined following infusion of 20% dextrose and is normally about 15 mmol/litre (TmG/GFR); (2) phosphate—the theoretical maximum tubular threshold of phosphate (TMP/GFR) can be estimated by formula from the plasma and urinary phosphate and creatinine concentrations, or can be measured directly following infusion of phosphate; and (3) amino acids—five types of renal aminoaciduria are distinguished: dibasic amino acids, neutral amino acids (monoaminomonocarboxylic acids), glycine and imino acids, dicarboxylic amino acids, and generalized amino aciduria (Fanconi’s syndrome).

Distal tubule—a water-deprivation test can help to distinguish patients with primary or secondary nephrogenic or cranial diabetes insipidus from those with primary polydipsia, who may all present with polyuria.

Renal-induced electrolyte and acid–base imbalances— (1) estimation of urinary free-water clearance is useful in the analysis of patients with hyponatraemia (see Chapter 21.2.1); (2) estimation of transtubular potassium gradient (TTKG) is advocated by some as useful in analysis of disorders of potassium homeostasis (see Chapter 21.2.2); (3) tests of urinary acidification are discussed in Chapters 21.14 and 21.15.

Renal imaging

Ultrasonography—this noninvasive, safe, versatile and (relatively) inexpensive technique is the first-line method for imaging the kidney and urinary tract in many clinical circumstances.

Ultrafast multislice CT scanning—this allows resolution of 2 to 3 mm or less and has become the mainstay of renal imaging. CT urography can be performed with a combination of unenhanced, nephrogenic-phase, and excretory-phase imaging: the unenhanced images are ideal for detecting urinary calculi; renal masses can be detected and characterized with the combination of unenhanced, nephrogenic- and excretory-phase imaging; the excretory phase provides imaging of the urothelium. CT angiography is the first-line investigation in the evaluation of acute renal trauma, assessment of tumour blood supply in cases of nephron-sparing surgery, and for the diagnosis of renal artery stenosis and/or aneurysms.

MRI—this is an alternative to CT scanning in patients who are allergic to conventional iodine-based radiocontrast media and has particular value in the staging of renal carcinoma and assessment of complex renal cysts. Magnetic resonance angiography (MRA) tends to overemphasize the significance of stenoses. Gadolinium contrast scanning should be carefully considered in patients with eGFR below 30 ml/min because of the risk of nephrogenic systemic fibrosis, which limits the utility of magnetic resonance techniques for many renal patients.

Renal nuclear medicine scanning—(1) dimercaptosuccinic acid (DMSA), used in estimation of differential renal function and detection of scarring (usually associated with reflux); (2) mercaptoacetyltriglycine (MAG3), used in detection of functionally significant obstruction, estimation of differential renal function, screening for renal artery stenosis, and monitoring of renal transplants.

Invasive techniques—these can allow therapeutic intervention as well as diagnosis, including antegrade or retrograde ureteropyelography (insertion of stents to relieve urinary obstruction) and angiography (angioplasty or stenting of the renal artery).

Renal biopsy

A renal biopsy should be considered in any patient with disease affecting the kidney when the clinical information and other laboratory investigations have failed to establish a definitive diagnosis or prognosis, or when there is doubt as to the optimal therapy. However, renal biopsy has the potential to cause morbidity and (on rare occasions) mortality, hence its risk must be outweighed by the potential advantages of the result to the individual patient. Biopsies which would be ‘of interest’ but ‘not in the patient’s interest’ should not be performed.

Introduction

The key to making any correct diagnosis depends on a careful history and thorough examination. In patients with kidney disease, the history and examination should attempt to differentiate acute from chronic kidney disease, single-organ system involvement from multisystem disease, and obstruction from intrinsic or prerenal disease. Kidney disease may be associated with preceding infections and the ingestion of drugs or herbal remedies. An accurate history and careful examination will determine the sequence and spectrum of clinical investigations required to make a diagnosis or decide on prognosis or treatment.

Examination of the urine

Urine collection

To minimize contamination, standard investigation is of a midstream urine (MSU) sample. Voiding from a full bladder containing at least 200 ml of urine should remove urethral organisms before the MSU is collected. Even so, in women, vaginal leucocytes and bacteria may contaminate the urine, and men should retract the foreskin to minimize contamination. Suprapubic aspiration is the technique of choice in babies and infants, and occasionally in adults who cannot cooperate to provide an MSU. The second urine of the morning is the best for microscopy as it is still acidic and concentrated, but without the overnight stay in the bladder that results in the degeneration of casts and cells. Cell lysis can occur in both hypotonic and alkaline urine. Only the first 10 ml of the stream should be collected in cases of suspected urethritis.

Macroscopic appearance

Fresh urine usually has a yellow colour due to the presence of urochromes, but occasionally it will have a milky appearance due to pus, spermatozoa, insoluble phosphates in alkaline urine (sometimes seen following heavy meals), or occasionally in cases of chyluria, or urate crystals in acid urine. Foamy or frothy urine is typical of heavy proteinuria. Certain agents and conditions can discolour urine.

Pink to red coloration

Haematuria may result in a range of colours from smoky pink through to port-wine red in cases of frank macroscopic haematuria. Other causes of a pink or red urine include eating sweets containing aniline dyes, beetroot or other foodstuffs containing anthocyanins; haemoglobin; myoglobin; some drugs such as phenindione and phenolphthalein; and (if the urine is left to stand) porphyrins in cases of acute intermittent porphyria.

Blue or green coloration

Blue or green coloration can be caused by pseudomonas urinary sepsis, methylene blue, biliverdin, triamterene, amitriptyline, chlorophyll-containing breath mints (Clorets), excessive use of mouthwash and deodorants, magnesium salicylate (Doan’s pills), phenyl salicylate, guaiacol (in cough remedies), thymol (in volatile oils and horesemint), iodochlorhydroxyquin, tolonium, Evans blue, methocarbamol, Diagnex blue, indigo blue, resorcinol, azuresin, bromoform, and occasionally propofol. Phenol and lysol can result in a green or black discolouration.

Orange coloration

Orange coloration can be caused by anthraquinone-containing laxatives, rifampicin, and excess urobilinogen.

Yellow urine

Yellow urine may be found in patients prescribed mepacrine or phenacetin, those taking excessive amounts of riboflavin, and icteric patients with conjugated hyperbilirubinaemia.

Black or brown urine

Alkaptonuria results in black or brown urine, whereas myoglobin and melanin only lead to black urine on standing. Other causes of brown urine include bilirubin, L-dopa, niridazole, furazolidone, and phenazopyridine, and—after standing—haemoglobin and myoglobin. As mentioned above, phenol and lysol can result in a black or green discolouration.

Stick testing

The upper limit of normal for protein excretion in the urine is 128 mg/24 h. Although albumin is the largest single component, more than half of the protein content comprises low-molecular-weight proteins and protein fragments. Commercial sticks such as Albustix are very sensitive, detecting protein in urine starting at concentrations around 100 mg/litre. Since these sticks detect protein on a concentration basis using bromocresol green as an indicator dye, the results they give are affected by urine flow rate and urine dilution or concentration. The sticks are treated with a buffer to keep their pH constant: an elevated urinary protein concentration can erroneously be recorded if the buffer is washed off by leaving the stick in the urine for too long, and with very alkaline urine. Some antiseptics used to clean the skin, including cetrimide and chlorhexidine, may also react and cause a false-positive result. More recently, antibody-based dipsticks have been developed for detecting microalbuminuria.

pH

Normal urine is slightly acidic, but can vary between pH 4.5 and 8.0. If an early-morning urine specimen is under pH 5.3, then there is unlikely to be a significant defect in urinary acidification. Alkaline pH is often found in urine infected with urea-splitting bacteria. In some cases of urinary stone disease, particularly in cystinuria and urate nephropathy, crystal solubility is greater in alkaline urine, and patients should regularly check their urine pH. Haemoglobin and myoglobin are also more soluble in alkaline urine, hence maintaining a forced alkaline diuresis is important in the management of patients following tumour lysis and those with rhabdomyolysis or haemoglobinuria.

Glycosuria

The stick reaction is based on glucose oxidase, which releases hydrogen peroxide from glucose, so producing a graded colour change by oxidizing an indicator. This reaction is specific for glucose and does not detect other sugars. The reaction can be blocked by large doses of ascorbic acid. A positive stick test for glucose must be interpreted in light of the plasma glucose level as glycosuria may reflect a defect in renal tubular glucose absorption.

Specific gravity

Specific gravity is a measure of the number of particles dissolved in a litre, whereas osmolality is the number of particles per kilogram. Protein and glucose increase the specific gravity more than the osmolality as they are dense particles. In normal patients, the early-morning, or concentrated, urine sample should have a specific gravity of 1.024 or more.

Nitrite stick test

Nitrite sticks contain an aromatic amine that reacts with nitrites, which are produced by bacterial reduction of nitrate, to form a pink-coloured diazonium complex. More than 90% of the common urinary pathogens are nitrite-forming bacteria. However, pseudomonas, Staphylococcus albus, Staphylococcus saprophyticus, and Enterococcus faecalis may have minimal or no nitrite-producing capacity. Other false-negative results may be obtained in alkaline urine, in patients taking large doses of vitamin C, and with frequent voiding of dilute urine when the urinary nitrite concentration is too low.

Leucocyte esterase stick test

This stick test is based on the presence of a leucocyte esterase and is very specific for the presence of urinary leucocytes, both intact and lysed. This test may be more accurate than microscopy when the urine is alkaline or hypotonic. However, the test can be inhibited by high concentrations of glucose (20 g/litre or more), ketones, and antibiotics including cefalexin, cefalotin, nitrofurantoin, tetracycline, and tobramycin. The sensitivity of this test is also reduced when the specific gravity of the urine is high, for instance in the presence of a heavy proteinuria.

Urine microscopy

To obtain reproducible results, urine should be processed in a standard manner and examined under the microscope as soon as possible. In our own institution, a few drops of acetic acid (10% v/v) are added to ensure a pH of 6.0 or less; then 10 ml of urine is centrifuged for 5 min at 1500 rev/min (750 g), following which 9.5 ml of supernatant is removed and the deposit resuspended. One drop (50 µl) is placed on a microscope slide and covered with a standard coverslip (24 × 32 mm). Although phase-contrast microscopy is an advantage in identifying red cells and casts, a standard microscope will suffice. A semiquantitative assessment of casts is made at low power (160×) and other elements at high power (400×), expressing the counts as numbers per field. Normal urine contains 1 or 2 leucocytes per high-power field (HPF), 1 erythrocyte per 2 or 3 HPF, 1 tubular cell per 10 HPF, and both hyaline casts (1 per low-power field, LPF) and granular casts (1 per LPF). Physical exercise can result in haematuria and cylindruria for several hours. Stains such as modified Sternheimer’s stain (Sedi-stain) can be used to help differentiate renal tubular cells from leucocytes. To improve the detection of casts, urine can be filtered through a 5-µm Millipore filter, and the retained casts stained with Papanicolaou’s stain.

Cellular elements

The morphology of the erythrocytes in the urine can give valuable information as to the source of bleeding. Those which have passed through the glomerulus and then along the renal tubule can become distorted or dysmorphic, whereas those originating from other sources within the urinary tract, such as the bladder, typically show much less evidence of damage so that they more closely resemble erythrocytes in the peripheral blood and are termed isomorphic. To establish a diagnosis of glomerular haematuria there should be a minimum of three different forms of dysmorphic erythrocytes present. One particular type of dysmorphic erythrocyte, the acanthocyte, is reported to have 52% specificity and 98% sensitivity for glomerular haematuria when the acanthocyte count is 5% or more. However, not all workers have found erythrocyte morphology to be useful in discriminating glomerular from nonglomerular bleeding, and the physician who only occasionally examines urine under the microscope is unlikely to obtain clear, reproducible, and useful discrimination between dysmorphic and isomorphic cells.

Some centres use automated haematological cell counters (Coulter counter) to assess red cell morphology in both urine and peripheral blood. The red cell size–distribution pattern for lower urinary tract haematuria is similar to that of the peripheral blood, with a relatively narrow size range and a high frequency distribution curve, whereas the typical pattern for dysmorphic haematuria is one of a broader range of red cell sizes, with a lower frequency distribution. To have any reliability, urine samples must be processed rapidly by those who do it regularly.

Microscopy may also reveal renal tubular epithelial cells. These cells are shed into the urine in acute tubular necrosis, in response to certain drugs (both nephrotoxic and ischaemic), and also in acute renal allograft rejection. In patients with nephrotic syndrome, these cells are seen as oval fat bodies, laden with lipid droplets. Squamous epithelial cells from the urethra and vagina and transitional cells from the ureter and bladder may also be present in normal urine. Urine cytology may reveal malignant transitional epithelial and/or metaplastic sqamous cells from the bladder.

During infection, the urine may contain large numbers of leucocytes and bacteria. When large numbers of leucocytes are present in the absence of bacteria (so-called sterile pyuria), then a variety of conditions should be considered: urinary stone disease, analgesic nephropathy, interstitial nephropathy, proliferative glomerulonephritis (rarely), renal tuberculosis, schistosomiasis, and partially treated bacterial urinary tract infection. Phase-contrast microscopy can distinguish lymphocytes from neutrophils, but eosinophils can only be identified with specific stains (Hansel’s stain). Urinary eosinophilia classically occurs in cases of acute interstitial nephritis, typically due to drugs, and also in cholesterol atheroembolic disease.

Urinary casts

Casts form from the transformation of Tamm–Horsfall glycoprotein, secreted by the distal tubular cells, into a gel matrix. They typically assume a tubular structure. Hyaline casts only contain Tamm–Horsfall glycoprotein and are found in a variable amount in the urine of normal subjects (Fig. 21.4.1). Fever, cardiac failure, strenuous exercise, and some drugs, such as furosemide and ethacrynic acid, increase hyaline cast excretion. During passage through the distal tubule and collecting duct, a variety of proteins, pigments, and cells can adhere to the Tamm–Horsfall protein, producing a wide variety of casts. Granular casts have deposits of either fine or coarse protein granules (Fig. 21.4.2). Although they may occur in normal subjects, or after exercise, they are typically found in cases of parenchymal renal disease. In patients with proteinuria, the protein deposited comes from the glomerulus, whereas in acute tubular necrosis, the protein comes from degenerate tubular cells. Broad waxy casts are much larger than normal casts and have clear-cut edges: they are formed in dilated hypertrophied tubules, as found in patients with chronic renal failure. Casts containing erythrocytes (red cell casts) indicate renal bleeding and are typically found when there is acute glomerular inflammation caused by glomerulonephritis or vasculitis (Fig. 21.4.3). White cell casts (containing leucocytes) can be found in proliferative glomerulonephritis, acute interstitial nephritis, and acute pyelonephritis.

Crystals

Urine may contain several types of crystals, depending on the pH. The presence of a few crystals of uric acid, calcium oxalate, or calcium phosphate is usually not clinically relevant, although thin hexagonal crystals of cystine are a marker of cystinuria. In a few cases, crystalluria may be associated with intratubular obstruction and acute kidney injury. Such cases would include acute uric acid nephropathy, ethylene glycol poisoning, and drugs including aciclovir, amoxicillin, indinavir, naftidrofuryl oxalate, sulfadiazine, and vitamin C.

Measurement of proteinuria

Quantification of proteinuria is important as the risk for progression of underlying kidney disease to endstage renal failure is related to the amount of protein in the urine. Traditionally, proteinuria has been measured using 24-h urine collections and expressed as grams per day. This has the advantage that it averages out protein excretion and is not therefore affected by its normal diurnal variation (less overnight and first thing in the morning) or urine concentration. Several different methods are used to measure the protein content of 24-h urine collections, ranging from the biuret method, which uses a copper-based method to precipitate proteins, to dye-binding methods using Coomassie Brilliant Blue as the indicator. These are more accurate than the turbidimetric methods, which use trichloroacetic or sulphosalicylic acid and measure turbidity with a densitometer. Radiocontrast media and some drugs (including penicillin, sulphonamides, and tolbutamide) may give false-positive results for proteinuria with the sulphosalicylic acid method. The biuret method measures total proteins, whereas the turbidimetric method provides different readings for albumin and globulins, as may do the dye-binding methods.

However, because of the inherent problems of accuracy and reliability with 24-h urine collections, the assessment of protein in spot urine samples has become the standard method of assessing proteinuria in routine clinical practice. Urinary albumin concentration is measured by a variety of methods based on an antibody technique for detecting serum albumin, including radioimmunoassay, nephelometry, immunoturbidity, and enzyme-linked immunosorbent assay (ELISA). Under resting conditions, urinary creatinine excretion is relatively constant throughout the day, hence to overcome the problems of timing of urinary collections, proteinuria in spot urine samples is expressed as an albumin:creatinine ratio (ACR, normal is less than 2.5 mg/mmol for men and less than 3.5 mg/mmol for women in a daytime urine or 24-h collection, and less than 1.5 mg/mmol for an overnight or early-morning sample). An ACR of 100 mg/mmol approximately corresponds to 1.5 g/day, and 350 mg/mmol to nephrotic-range proteinuria. However, albumin is not the only protein in urine and the relationship between albumin and total urinary protein is not linear, with a ratio of 50% albumin with a urinary protein of 300 mg/litre increasing to 70% at 1000 mg/litre.

As the measurement of protein in spot urine samples is cheaper than albumin, it has been suggested that, for patients with more than 1+ proteinuria on dipstick testing, the protein/creatinine ratio (PCR) should be used in routine clinical practice. The normal PCR is less than 13 mg/mmol, with a dipstick value of 1+ roughly equivalent to a PCR of 45 to 149 mg/mmol and ACR above 30 mg/mmol, a dipstick of 2+ to a PCR of 150 to 449 mg/mmol, and 3+ to 450 mg/mmol or more.

Aside from their use to replace 24-h urine collections, spot urine collections are particularly useful in the diagnosis of orthostatic proteinuria, in other words where the patient has a normal urinary protein excretion when recumbent, or overnight, but has marginally increased proteinuria in the ambulant or daytime sample.

The ACR should not be measured during acute illness, menstruation, or intercurrent illness as these will temporarily increase the degree of proteinuria.

Microalbuminuria

Various antibody based assays for albumin can detect an increased urinary albumin excretion in patients with normal levels of proteinuria. High-performance liquid chromatography (HPLC) detects more urinary albumin than the radioimmunoassay and other serum antibody-based tests. Normoalbuminuria is defined as an excretion rate of 20 µg/min or less. Proteinuria is usually detectable on dipstick testing at rates of over 200 µg/min, hence microalbuminuria is defined as an excretion rate between 20 and 200 µg/min. The albumin excretion rate (AER) is some 25% higher during the day than the night. The classification of abnormal urinary albumin excretion is shown in Table 1.

Table 1 Classification of abnormal urinary albumin excretion
  24-h urine albumin (mg/24 h) Overnight albumin (µg/min) Spot albumin (mg/litre) Spot urine
ACR (mg/mmol) PCR (mg/mmol)
Normal <15 <10 <10 M <1.25, F <1.75 <13
Microalbuminuria 30 to <300 20 to <200 20 to <200 M 2.5 -<25, F 3.5 to <35 16 to <160
Macroalbuminuria >300 >200 >200 M >25, F >35 >160

ACR, albumin:creatinine ratio; F, female; M, male; PCR, protein:creatinine ratio.

The normal ACR ratio is lower in men than women owing to higher urinary creatinine excretion.

There is a good correlation between the morning AER and the ACR in the first urine sample of the morning. A further advantage of spot urines is that patients can provide a sample when they attend the clinic: provided these are taken at the same time and the patient’s dietary intake is relatively constant, they are very useful in assessing patients over time. A further advantage of measuring the ACR instead of AER is that the former, but not the latter, eliminates the need for timing of urinary samples.

Microalbuminuria is not only an adverse factor for the progression of diabetic renal disease, but is also predictive of cardiovascular events in both the diabetic and nondiabetic population. In addition to those with diabetes, microalbuminuria may be found in patients with hypertension, cardiac failure, and following a pyrexial or viral illness. Similarly, microalbuminuria may be present in healthy subjects after exercise and during normal pregnancy.

Selectivity of proteinuria

Patients with glomerular disease typically have a nonselective proteinuria, with a similar clearance of both high- and low-molecular-weight plasma proteins. However, those with minimal change disease may have selective proteinuria, with clearance of predominantly low-molecular-weight proteins, the demonstration of which is useful in paediatric practice where patients are often treated with steroids without a renal biopsy.

Most laboratories compare the clearance of IgG as the large-molecular-weight protein (150 kDa) to that of albumin (or transferrin, 88 kDa) as the low-molecular-weight protein. Both plasma and spot urine samples are required. Protein concentrations are measured either by laser nephelometry or radial immunodiffusion. Nonselective proteinuria is taken as an ([IgG]urine/[IgG]plasma) × ([transferrin]plasma/[transferrin]urine) ratio of 0.2 or more, whereas selective proteinuria is taken as a ratio of 0.1 or less.

Spill-over proteinuria

Patients with myeloma, some types of amyloidosis, and those with reticuloendothelial disorders may have a spill-over proteinuria due to glomerular filtration of complete and incomplete κ and λ chains and immunoglobulin light chains. These low-molecular-weight proteins are not detected by simple urine stick testing, or by standard biochemical methods to determine urine protein concentration. Thus, when clinically appropriate, urine should specifically be sent for immunoelectrophoresis to exclude myeloma. However, light chains in particular may still not be detected, hence further investigation with specific antisera may be required if their presence is suspected.

Renal tubular proteinuria

Interstitial renal disease can result in proteinuria, usually of less than 2 g/day. Proximal tubular injury leads to increased low- molecular-weight proteinuria, characterized by an excess of intestinal alkaline phosphatase, N-acetylglucosaminidase, retinol-binding protein, tissue-specific alkaline phosphatase, α-glutathione-S-transferase (α-GST), α1-macroglobulin, and β2-microglobulin. By contrast, Tamm–Horsfall glycoprotein and α-GST are increased in distal tubular injury.

β2-Microglobulin is freely filtered at the glomerulus and then reabsorbed in the proximal tubule, such that less than 1% of the filtered load is excreted in the urine of normal subjects (normal is <370 µg/24 h). Thus urinary β2-microglobulin excretion has been used as a marker of proximal tubular damage. However, β2-microglobulin is unstable in urine, and its excretion can be affected both by an increased production rate (found in cases of myeloproliferative disease, chronic inflammatory states, and acute liver disease) and by saturation of β2-microglobulin tubular uptake due to an excess of dibasic amino acids.

More-reliable markers of tubular proteinuria are now available. These include α-GST, α1-macroglobulin, and retinol-binding protein. Turbidimetric or enzyme assays are now available. Results are expressed as either excretion rates (e.g. normal α-GST is <12.5 ng/min or <11.5 µg/litre) or as a ratio to urinary creatinine (e.g. normal reference range for retinol-binding protein:creatinine is <0.019 mg/mmol). Typically in cases of renal tubular proteinuria the ratio of retinol-binding protein:creatinine is greater than that of albumin:creatinine.

These tests of renal tubular proteinuria are helpful in investigating patients with suspected Chinese herbal nephropathy, Asian subcontinent nephropathy, and Balkan nephropathy, and they may also be useful in monitoring progression of these diseases and other tubulointerstital diseases such as adult polycystic kidney disease. Industrial workers exposed to heavy metals and organic chemicals, such as those used in the dry-cleaning industry, may develop interstitial renal disease characterized by increased urinary low-molecular-weight proteinuria.

Urinary biomarkers in acute renal injury

Advances in urinary proteomics have led to the search for biomarkers of acute kidney injury and acute renal transplant rejection. Urinary biomarkers fall into three main categories: (1) markers of kidney function, similar to creatinine, typified by cystatin C; (2) markers of the severity of the inflammatory response made by the individual to the insult, including neutrophil gelatinase-associated lipocalin (NGAL) and liver type fatty acid binding protein; and (3) markers of kidney damage, such as kidney injury molecule (KIM-1), urinary cytokines (IL-18), urinary enzymes (α1-microglobulin, α1-acid glycoprotein, N-acetyl-β-D-glucosaminidase, gamma-glutamyltranspeptidase and alkaline phosphatase) and albumin.

Preliminary single-centre studies in children with pre-existing normal renal function have shown that urinary biomarkers are more effective in predicting both renal injury and severity than changes in serum creatinine. However, these encouraging findings have not been replicated in multicentre studies that included patients with established chronic kidney disease, hence urinary biomarkers currently remain a research tool in acute kidney injury. Similarly, further studies are required to determine the role of urinary biomarkers such as granzyme in distinguishing renal transplant rejection from ischaemic renal injury. Studies evaluating these urinary biomarkers in assessing progression of chronic kidney disease are under way.

Estimation of glomerular filtration rate

Biochemical tests

Knowledge of the GFR is of crucial importance in the management of patients, not only for detecting the presence of renal impairment but also in the monitoring of all patients with or at risk of renal impairment, and in determining appropriate dosing of those drugs cleared by the kidney. Measurement of plasma creatinine remains the standard biochemical test used to assess renal function. Unfortunately, the plasma creatinine concentration is not linearly related to the GFR, hence some 30% of patients with significantly impaired renal function still have a plasma creatinine value within the normal range (<120 µmol/litre).

Creatinine

Creatine, which is endogenously synthesized in the liver or exogenously supplied by meat in the diet, is transported to muscle and converted to creatinine by nonenzymatic dehydration. Muscle mass represents some 98% of the total body creatine pool. Thus gender, racial and age-related differences in body composition, physical training and exercise, muscle-wasting diseases, paralysis, and intercurrent illnesses will all affect the production rate of creatinine and therefore both the plasma creatinine concentration and urinary creatinine excretion (Table 2). Hence in young children there is a steady increase in the plasma creatinine level as their muscle mass increases. Dietary influences will affect plasma creatinine levels, with a reduction in strict vegans and increased values in those with a high meat intake (particularly stewed meat: cooking leads to the conversion of creatine to creatinine) or those taking creatine supplements. For any individual, the plasma creatinine level is relatively constant throughout the day, although there is a tendency for it to increase slightly in the afternoon.

Table 2 Factors affecting creatinine generation
Factor Effect on serum creatinine
Ageing Decreased
Female sex Decreased
Race or ethnic group (compared with white)
Black Increased
Hispanic Decreased
Oriental Decreased
Body habitus
Muscular Increased
Amputation Decreased
Obesity Decreased
Chronic illness
Cirrhosis, malnutrition, chronic inflammation, cancer, severe cardiovascular or respiratory disease, hospitalized patients Decreased
Neuromuscular diseases Decreased
Hypothyroidism Increased
Diet
Vegetarian diet Decreased
Ingestion of cooked meat Increased

Creatinine is not only freely filtered by the glomerulus but is also secreted into the renal tubule. Creatinine reabsorption may occur at low urinary flow rates, such as in congestive cardiac failure. The relative proportion of renal tubular creatinine secretion to that filtered increases as renal function declines. In addition, in oedematous states such as nephrotic syndrome, calculated creatinine clearance exceeds inulin clearance, suggesting increased tubular creatinine secretion. Several drugs are known to block the tubular secretion of creatinine and thus cause an increase in the serum creatinine level: these include the diuretics amiloride, spironolactone, and triamterene; also cimetidine, aspirin, probenecid, and trimethoprim.

The most accurate method of measuring plasma creatinine is by isotope dilution mass spectrometry (IDMS), followed by enzymatic methods, but these are costly compared with the standard Jaffé assay. Most laboratories therefore measure plasma creatinine using standard automated analysers that assess the chromogenic product of creatinine and alkaline picrate (Jaffé reaction). Table 3 lists some substances which in high concentration can act directly or indirectly as chromogens, or affect the background control blanks, and so result in a spurious increase in the plasma creatinine level. In clinical practice these may lead to an overestimation of creatinine in people with poorly controlled diabetes, and an underestimation in deeply jaundiced patients, such as those with primary biliary cirrhosis. Compensated Jaffe rate reactions have been introduced in an attempt to overcome some of these technical problems, but other enzymatic methods may provide greater accuracy, although at potentially greater cost.

Table 3 Compounds that can affect the measurement of plasma or urinary creatinine concentration
Endogenous compounds Exogenous compounds
Protein Acetohexamide
Ketones Cephalosporins
Ketoacids 5-Fluorocytosine
Glucose Methanol metabolites
Fatty acids Phenylacetylurea
Urate Dopamine
Urea  
Bilirubin
Reciprocal creatinine or logarithm of creatinine values

As the plasma creatinine level roughly doubles for every 50% reduction in GFR, expressing (transforming) the results as the reciprocal or logarithm is useful in assessing serial plasma values, which changes the graph from an exponential to a straight-line plot. The advantage of using a straight-line plot of plasma creatinine is that it allows the rate of renal decline to be calculated, which can then be used to predict the onset of endstage renal failure and the requirement for dialysis treatment in many patients. The reciprocal creatinine plot assumes a constant rate of loss, whereas the logarithm a constant fractional loss of renal function.

Patients with diabetic nephropathy tend to have a faster rate of decline in renal function than those with glomerular disease, who tend to have a faster rate than those with tubulointerstitial renal disease. In addition, it is easier to assess the effect of treatment interventions on the progression of renal disease by analysing transformed data, and also to recognize when there has been a sudden and unexpected deterioration in function that requires urgent investigation.

Prediction of creatinine clearance from the plasma creatinine level and estimation of GFR (eGFR)

Despite the potential inaccuracies in the determination of plasma creatinine, variations in endogenous creatinine production rates, and the relative increase in renal tubular and intestinal creatinine secretion with deteriorating renal function, formulas based on the plasma creatinine level are used in clinical practice to estimate creatinine clearance. The first commonly used equation, validated in adults, was the formula of Cockcroft and Gault, later modified by Gault:

creatine clearance formula

or

creatine clearance formula

In the original formula there was a different equation for women, with a factor of 0.85 (instead of 1.2) to allow for the lower rate of creatinine production in women due to differences in their body composition. Similar equations were developed for children. Although these formulas may be helpful in clinical practice to provide an estimation of renal function (eGFR), they are not always accurate, particularly in people with diabetes and African-Americans (owing to differences in body composition).

Another equation was developed in 1999 following the Modification of Diet in Renal Disease (MDRD) trial, based on 1628 adult patients in the United States of America, and this was further revised in 2005 as the simplified MDRD equation (sMDRD):

eGFR(ml/min per1.73 m2) = 175×[serum creatinine(mg/dl)]-1.154 × (age in years)-0.203 × 0.742 (if female) × 1.212(if black)

eGFR(ml/min per 1.73 m2) = 175 × ([serum creatinine (μmol/litre)/1.004] × 0.011312)-1.154 × (age in years)-0.203 × 0.742 (if female) × × 1.212 (if black)

The sMDRD equation has the advantage over the Cockcroft-Gault and many other formulas in that it does not require knowledge of the patient's weight, and their sex and age are routine demographics collected for sample identification. Use of the sMDRD equation has now been introduced into standard clinical practice in the United States of America, the United Kingdom, and Australia to define stages of chronic kidney disease, the intention being to encourage recognition of renal impairment at an early stage in the population at large, and therefore allow management of risk factors to reduce both renal progression and cardiovascular risk (Table 4).

Table 4 The stages of chronic kidney disease (CKD)
CKD stagea eGFR (ml/min per 1.73 m2 body surface area)
1 >90, with other evidence of renal disease
2 60–89, with other evidence of renal disease
3A 45–59
3B 30–44
4 15–29
5 <15, or receiving renal replacement therapy

a The suffix (p) can be used to denote the presence of proteinuria as defined by a spot urinary albumin:creatinine ratio (ACR) of ≥30 mg/mmol, which is approximately equivalent to a protein:creatinine ratio (PCR) of ≥50 mg/mmol (≥0.5 g/24 h).

Patients with CKD stages 3A, 3B, 4, and 5 may or may not have any other evidence of renal disease.

Because of the inaccuracy of the MDRD equation for patients with an eGFR of ≥60 ml/min per 1.73m2 or more, a further modification has been published, termed CKD-EPI:

For men with serum creatinine <0.9 mg/dl: GFR ml/min per 1.73m2 = 141 × (serum creatinine / 0.9) −0.411 = 0.993age = 1.159 (if black)

For men with serum creatinine >>0.9 mg/dl: GFR ml/min per 1.73m2=141 × (serum creatinine / 0.9) −1.209 = 0.993age = 1.159 (if black)

For women with serum creatinine <0.7 mg/dl: GFR ml/min per 1.73m2 = 144 × (serum creatinine / 0.7) −0.329 = 0.993age = 1.159 (if black)

For women with serum creatinine >0.7 mg/dl: GFR ml/min per 1.73m2 = 144 × (serum creatinine / 0.7) −1.209 = 0.993age = 1.159 (if black)

Divide by 88.4 to convert serum creatinine µmol/litre to mg/dl.

However, these equations have not been validated for elderly patients or those from the ethnic minorities. Furthermore, they were derived based on iothalamate urinary clearances, which themselves have inherent inaccuracies, both because of the requirement for urinary collections, and also the relative importance of non-renal excretion at low levels of GFR. Further refinements to the predictive equations will probably be developed.

The first problem in rolling out such a program of population screening was to standardize the measurement of plasma creatinine. For example, in the United Kingdom alone there were 31 different modifications of the standard Jaffé reaction used in routine clinical practice. Rather than each laboratory changing its method/analyser, each individual laboratory had to develop correction factors from the IDMS-traceable version of the MDRD equation. Thus in the United Kingdom the following equation is employed using an IDMS-based national external quality assessment service. eGFR (ml/min per 1.73m2) = 175 × (([creatinine (μmol/l) - intercept]/slope) × 0.011312)-1.154 × (age in years)-0.203 × 0.742 (if female) × 1.212 (if black) where intercept and slope are the individual laboratory correction factors for the IDMS method.

The eGFR has not been validated in people younger than 18 years, hospitalized patients, or those with acute kidney injury, pregnancy, oedematous states, muscle-wasting disorders, amputations, or malnourishment. Similarly, it has not been validated for extremes of age or body weight, or for ethnic groups other than whites of northern European origin and African-Americans.

In the United Kingdom, the value of the eGFR falls within 30% of the true GFR in 90% of patients. Typically, the eGFR underestimates true renal function in patients with hyperfiltration, with its accuracy improving as renal function deteriorates. In the United States of America, Australia, and Scotland, laboratories were initially instructed to report all eGFR values higher than 60 ml/min per 1.73 m2 simply as ‘>60’ because of increased inaccuracy at higher eGFR, whereas in the United Kingdom the advice to laboratories is to report values up to 90 ml/min per 1.73 m2, and then ‘>90’.

Although the eGFR, however estimated, has inherent inaccuracies, it is now universally employed, may prove useful in assessing stability or progression of renal function over time in the general population, and can allow a rational basis for referral to specialist renal physicians.

Due to the inaccuracy of the MDRD equation for patients with an eGFR of ≥60 ml/min/1.73m2, a further modification has been published, termed CKD-EPI:

For men with serum creatinine <0.9 mg/dl: GFR ml/min/1.73m2 = 141 x (Serum creatinine / 0.9) -0.411 x 0.993age x 1.159 (if black).

For men with serum creatinine >0.9 mg/dl: GFR ml/min/1.73m2 = 141 x (Serum creatinine / 0.9) -1.209 x 0.993age x 1.159 (if black).

For women with serum creatinine <0.7 mg/dl: GFR ml/min/1.73m2 = 144 x (Serum creatinine / 0.7) -0.329 x 0.993age x 1.159 (if black).

For women with serum creatinine >0.7 mg/dl: GFR ml/min/1.73m2 = 144 x (Serum creatinine / 0.7) -1.209 x 0.993age x 1.159 (if black).

(For serum creatinine umol/l, divide by 88.4 to convert to mg/dl).

However, these equations have not been validated for elderly patients or those from the ethnic minorities. Furthermore, they were derived based on iothalamate urinary clearances, which themselves have inherent inaccuracies, both because of the requirement for urinary collections, and also the relative importance of non-renal excretion at low levels of GFR. Further refinements to the predictive equations will probably be developed.

Creatinine clearance

In clinical practice, creatinine clearance is now being replaced by the eGFR, as the accuracy of the creatinine clearance method depends on patient compliance to provide an accurate 24-h urine collection. Even when patients are in a steady state, urinary creatinine excretion varies from day to day, and reliability can be increased by performing consecutive daily clearances.

Creatinine clearance is calculated as follows:

creatinine clearance formula

With regard to the use of the creatinine clearance measurement as an estimate of GFR, two errors tend to balance each other out. The chromogenic assay tends to overestimate the plasma, but not urinary, creatinine concentration, leading to an underestimation of GFR. By contrast, creatinine is not only excreted by glomerular filtration: some is secreted by the renal tubules, leading to an overestimation of the GFR. However, in patients with impaired renal function these contrasting effects are not balanced, and the relative increase in tubular creatinine secretion results in creatinine clearance exceeding GFR. This problem can be overcome by the administration of 400 mg of cimetidine to block renal tubular creatinine secretion, but this manoeuvre is rarely (if ever) performed in clinical practice solely for this purpose. By convention, creatinine clearance values are commonly corrected for body surface area to adjust for differences in muscle mass, assuming a fixed mathematical relationship between body surface area and the relative proportions of fat to muscle. However, body composition is not only age- and gender-dependent, but also varies from race to race, and other inaccuracies occur in oedematous and obese states.

Cystatin C

Cystatin C is a low-molecular-weight basic protein (13.26 kDa) from the cystatin superfamily of cysteine proteinase inhibitors that is produced by all nucleated cells. It is freely filtered by the glomerulus and not reabsorbed, secreted or catabolised by the renal tubules during its passage into the urine. The generation of cystatin C appears to be less variable from person to person than creatinine and is not affected by dietary protein intake, hence it has been advocated as a better marker for GFR than creatinine. Rapid and fully automated immunonephelometric assays are now available, but these are more costly than assays of creatinine.

As with creatinine, several equations have been proposed to allow estimation of GFR (ml/min per 1.73m2) based on serum cystatin C measurements (mg/litre):

  • Larsson equation: GFR = 77.329 × cystatin C−1.2623
  • Hoek equation: GFR = −4.32 + 80.34 × 1/cystatin C
  • Le Bricon equation: GFR = 78 × (1/cystatin C) + 4
  • Rule equation: GFR = 76.6 × cystatin C−1.16 
  • Filler–Lepage equation GFR = 1.962 + [1.123 × log (1/cystatin C)]

In most studies, the accuracy of cystatin C assessment of GFR is superior to that of creatinine-based eGFR (using the sMDRD formula) in those patients in the crucial CKD stage 3 and 4 groups, both in children and adults. However, evidence is accumulating that the serum concentration of cystatin C is influenced by corticosteroid use, sex, age, weight, height, smoking status, proteinuric states, chronic liver disease, malignancy, and the level of C-reactive protein, even after adjustment for creatinine clearance. Cystatin C has also been reported to be reduced in renal transplant recipients, with some drugs (including valsartan), in bone marrow transplant patients, following myeloablative chemotherapy, and in hypothyroid states, and to be increased in thyrotoxicosis. For these reasons, amongst others, cystatin C has failed to replace creatinine as a biomarker of renal function in routine clinical practice.

Carbamylation

Urea accumulates with deteriorating renal function and in plasma can spontaneously dissociate to form a reactive cyanate species that can react with the terminal valine of haemoglobin α and β chains (and also similar valine molecules in other proteins). This reaction is termed ‘carbamylation’ and the product ‘carbamylated haemoglobin’ (or other protein). Whereas glycosylated haemoglobin has proved useful in clinical practice for assessing time-averaged diabetic control, carbamylated haemoglobin or carbamyl-lysine adducts have not been shown to be superior to simple serum creatinine measurements in determining stable renal function. However, they are useful in helping to differentiate acute from chronic renal failure, because of the time course of the carbamylation reaction, and also in the assessment of time-averaged urea levels in the dialysis patient with endstage renal failure. However, until the relevant assays are commercially available, their use will remain experimental.

Other methods

Isotopic methods

The GFR can be determined by the clearance of a compound which is freely filtered by the glomerulus and then passes through the nephron without tubular reabsorption or secretion. Traditionally, inulin—a naturally occurring polyfructose—was given as a constant infusion to achieve a constant plasma concentration, and then clearance determined from timed urinary collections. This was a laborious technique. Furthermore, the biochemical estimation of inulin was initially tedious and difficult, with significant interassay variation, and accurate timed urine collections are unreliable in patients with urinary tract anomalies. To overcome these and other difficulties, compounds other than inulin are generally used to estimate GFR, and methods other than constant infusion.

Following a single bolus injection, depending on the compound used, the fall in plasma concentration follows either a single- or two-compartment model related to renal clearance. Chromium-labelled ethylenediaminetetraacetic acid ([51Cr]EDTA) is the most commonly used isotope. After the single injection, three timed plasma samples are taken to calculate the plasma decay rate and thereby the GFR. More recently it has been showed that only a single blood sample at 4 h is required for a GFR over 30 ml/min. At GFRs above 30 ml/min there is a very good correlation between inulin and [51Cr]EDTA clearance, but below 30 ml/min the accuracy of the isotope technique is reduced, there being some renal tubular reabsorption. Accuracy can be improved in this situation by taking a delayed (24-h) plasma sample.

Other isotopes that have been used to estimate GFR include [125I]iothalamate, which when given as a subcutaneous injection results in a constant plasma concentration equivalent to the infusion technique, and 99Tcm-diethylenetriaminepentaacetic acid (DTPA), which is less accurate because of its short half-life (6 h) and dissociation of DTPA from the radionuclide.

With all the isotopic methods, it is conventional for the GFR to be corrected for the size of the patient. This correction assumes a fixed relationship between the weight and height of an individual: hence serial estimations to detect a change in renal function are more likely to be accurate than single estimations. Single bolus isotopic determinations are determined by the area under the curve, and as such will over estimate the GFR in patients who are fluid overloaded, and also those with ascites, such as patients with cirrhosis, due to redistribution of the tracer.

Radiological methods

Iohexol is a nonionic, low-osmolality radiocontrast dye. It can be used to estimate GFR following a single bolus injection of between 2 and 5 ml. In patients with a clearance of over 30 ml/min, a single plasma sample taken 3 h after injection provides an accurate estimation, whereas additional later samples are required to improve the accuracy in those with severely impaired renal function.

Summary

Because of the difficulty in interpreting plasma creatinine concentrations below 150 µmol/litre as an assessment of renal function, the eGFR has been introduced in clinical practice in the United States of America, Australia, and the United Kingdom to detect patients with early stages of chronic kidney disease. It is an appropriate and adequate technique for most clinical purposes. When more precise estimation of GFR is required, an isotopic assessment is the most accurate method of determination, otherwise two 24-h urine collections with corresponding plasma samples should be used to calculate the GFR by creatinine clearance. Cystatin C has failed to replace serum creatinine in clinical practice and remains a research tool. To examine changes in renal function, where eGFR measurements are not available, plasma creatinine concentrations should be transformed to either the reciprocal or the logarithm to assess trends in serial results.

Estimation of renal blood flow

Renal blood flow can be estimated noninvasively using Doppler flow probes, provided there is a single renal artery and adequate imaging is possible. This is technically easier for a transplanted kidney than a native kidney. The recent development of contrast agents for ultrasonography may increase the reliability of these estimations. Alternatively, renal blood flow can be estimated from the measurement of the renal plasma flow and the haematocrit. However, the haematocrit of peripheral venous blood may not be the same as that entering the renal artery. More recently, the development of positron emission tomography (PET) coupled with CT has allowed assessment of renal blood flow using 15O-labelled water, 82Rb, and other tracers.

Renal plasma flow

Ideally, any compound used to assess renal plasma flow should have 100% uptake by the kidney, with any fraction not filtered by the glomerulus being extracted by the tubules and secreted. p-Aminohippurate is the most commonly used compound, but is only 85% extracted during a single passage through the kidney, and thus at best only provides an estimate of renal plasma flow. Continuous infusion of p-aminohippurate provides a more accurate estimation of renal plasma flow than single-injection techniques.

Renal blood flow varies in normal subjects with pain, stress, physical exercise, and normal pregnancy, and following a high-protein meal. In patients with impaired renal function, the decline in renal plasma flow generally corresponds to the decrease in GFR. However, in some conditions where there may be renal tubular hypoxia or toxicity, such as in patients with severe heart disease or those with ciclosporin nephrotoxicity, the reduction in estimated renal plasma flow is greater than that expected for the change in GFR, due to a reduction in the renal tubular uptake of p-aminohippurate. Similarly, p-aminohippurate uptake is reduced in small children. [125I]o-Iodohippurate has also been used to estimate renal plasma flow, but this has a lower extraction than p-aminohippurate (75%), and is less reliable.

Investigation of tubular function

In a normal subject, some 180 litres of glomerular filtrate is produced each day and less than 3% of this is excreted, owing to reabsorption by the tubules. The proximal and distal tubules have different functions, and traditionally each is considered separately.

Proximal tubular function

Defects in proximal tubular function may be isolated or generalized, as in the Fanconi syndrome. Glucose, phosphate, amino acids, and organic ions are reabsorbed by the apical border of proximal renal tubular cells by sodium-dependent cotransporters, and are then transported across the basolateral membrane by different, sodium-independent, cotransporters.

Glucose

There is a maximum reabsorption rate for glucose (TmG) in the proximal tubule of 15.1 ± 2.5 mmol/litre (TmG/GFR), above which glycosuria will be present. To determine TmG/GFR, a 20% dextrose infusion is administered at increasing rates to produce a slow rise in the plasma glucose up to a maximum of 30 mmol/litre, which is maintained for a minimum of 1 h. Plasma and urine samples are collected every 30 min. Renal function is determined by [51Cr]EDTA-GFR. The glucose absorption rate is calculated as the difference between the filtered load in urine (urine volume × [glucose]urine) and the filtered load in plasma (GFR × [glucose]plasma). Patients with type A renal glycosuria typically have a reduced threshold of around 5 mmol/litre.

Phosphate

Phosphate is normally filtered at the glomerulus and reabsorbed in the proximal tubule, with only 10 to 20% of the filtered load being excreted. The normal tubular reabsorption of phosphate (TRP) is above 85% and can be calculated from:

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