Osmolality is de \201 ned as the number of particles per kg. Urine osmolality is determine d by measuring the freez- ing point and physiologically it ranges from 50 to 1200 mosmol/kg . Refractometry is a method based on the measurement of the refractive index which correlates with osmolality. \200 The most common means to determine relative density is dipstick te sting based on dry chemistry with cations in the urine causing the color change of an indic ator substance. This method is not very accurate but is ade- quate for everyday use. As dip stick use is widespread, it is important to pay attention to conditions that may confound t he results (Table 1.2.2). Table 1.2.1 Causes of abnormal color changes in urine Underlying condition Possible urine coloration Pink, red, brown or black Hematuria, hemoglobinuria, myoglobinuria Chemical characteristics of urine pH Urine pH is useful in evaluating acid \200\223base dis orders and for the interpretation of certain dipstick tests and microscopic \201 ndings. The commonly used dipsticks can measure pH values from 5.0 to 9.0. \200 Urine pH normally ranges from 4.8 to 7.6. \200 Acidic urine may be due to high meat intake or acidosis. \200 Alkaline urine may be due to vegetarianism, infections of the urinary tract with ammo nia-producing bacteria (e.g. Proteus spp.), prolonged storage of the specimen or alkalosis. Dark yellow to brown Jaundice White and milky Chyluria Pink Massive uric acid crystalluria Red (possibly darkening upon standing or \202 uorescence in UV light) Porphyrinuria Brown to black Alkaptonuria Drugs Rifampicin Phenytoin Chloroquine Triamterene Methylene blue Metronidazole, imipenem/ cilastatin, methyldopa Orange to red Red Brown Green Blue Darkening upon standing Hemoglobin The detection of hemoglobin by dipsticks utilizes the pseu- doperoxidase activit y of the heme moiety to produce a colored product. In the presence of intact erythrocytes g reen spots become visible, whereas a homogeneous color change may occur following hemolysis due to longer stor- age of the specimen, alkaline urine or low relative urine density. Wit h a high erythrocyte concentration there may also be a diffuse color change. Foods Beetroot Rhubarb, senna Carotene Red Yellowish-brown or red Brown CHAPTER 1.2 Urinalysis and microscopy 9 and therefore the latter a used routinely in everyday clini- cal practice to quantify prote inuria. To analyze proteinuria qualitatively, electrophoretic protein separation can be don e on the basis of molecular weight. Thus information can be obtained about the origin of th e excreted proteins and help to inform possible underlying disorders. Alternatively, speci \201 c marker proteins can indi- cate tubular damage (see below). Prerenal (overload) prot einuria. An increased serum con- centration of a protein results in urinary excretion witho ut a glomerular or tubular defect. Examples are Bence \200\223Jones proteinuria in monoclon al gammopathy, myoglobinuria following rhabdomyolysis or hemoglob inuria due to hemolytic c risis. In the case of Bence \200\223Jones proteinuria, immuno \201 xation of serum should be performed. Glomerular proteinuria. Increased permeability of the glomer- ular filtration membrane causes pathologic proteinuria. Further differentiation is made based on the patte rn of protein excretion. \200 Selective glomerular proteinuria means that predomi- nantly albumin and transferrin are detected, which sug- gests the presence of a moderate glomerul ar disorder (e.g. minimal change nephritis). Table 1.2.2 Main causes for false dipstick results Measurement False-negative False-positive results results Underestimation in glucosuria, pH >6.5 Overestimation in proteinuria >7 g/L, ketoaciduria Speci \201 c gravity Reduced values in presence of formaldehyde pH Myoglobinuria, bacterial peroxidases, intravascular hemolysis, hydrochloric acid Ascorbic acid, standing before examination, high relative density, formaldehyde Hemoglobin Ascorbic acid, bacteria Hydrochloric acid, Glucose oxidizing agents pH >9, quaternary ammonium detergents Light chains, tubular proteins, hydrochloric acid Albumin Oxidizing detergents High relative density, vitamin C, proteinuria >5 g/L, glucosuria >20 g/L, cephalosporins Leukocyte esterase Nonselective glomerular proteinuria implies that higher molecular weight proteins (e.g. IgG ) appear in the urine, which indicates severe glomerular damage. No vegetable intake, vitamin C, bacteria that do not reduce nitrates Nitrites The selectivity can be assessed by determining the ratio of IgG clearance to transferrin cl earance. Values <0.1 re \202 ect selective proteinuria, whereas values >0.2 indi- cate non selective proteinuria. Drugs containing free sulfhydryl groups Ketones This dipstick method has low sensitivity but high speci \201 city. False-positive results may be seen in patients with intravas- cular hemolysis, myoglobinuria or infection with bac teria showing pseudoperoxidase activity (Enterobacteriaceae, staphylococci and streptococci ). Therefore phase-contrast microscopy of a fresh specimen of urine may be useful to con \201 rm positive cases or those where the result is unclear. Microalbuminuria implies an albumin excretion of 30 \200\223300 mg/day or 20 \200\223200 mg /L (ACR of 2.5 \200\22330 mg/mmol in men, or 3.5 \200\22330 mg/mmol in women). It is a typi cal early albeit nonspecific feature of diabetic or hypertensive nephropathy. Tubular prote inuria. This is characterized by the presence of low molecular weight proteins, which are n ormally \201 l- tered in the glomeruli and reabsorbed in the tubules, such as 1-microgl obulin or B2-microglobulin. Their increased concentration in the urine indicates tubular da mage. Glucose Dipstick testing is also commonly used for the detection of glucosuria. By using th is method glucose concentrations of 0.5 \200\22320 g/L can be measured. Usually glucosuria is a conse- quence of hyperglycemia due to diabetes mellitus, but renal glucosuria may occu r as a result of certain tubular disorders. See Table 1.2.2 for causes of false-positive or -negative results. Leukocyte esterase Dipstick testing for leukocyturia as a marker for urinary tract infectio n is based on the activity of leukocyte este- rase released from lysed macrophages and neut rophil granulocytes. High glucose or protein concentrations may cause false-negative result s (see Table 1.2.2). Protein Normally, daily urinary protein excretion does not exceed 150 mg (140 mg/m2 for chi ldren), as most of the \201 ltered protein is reabsorbed in the proximal tubules. A dipst ick can provide a rough quanti \201 cation of pro teinuria. This method has a high sensiti vity to albumin, but only low sensitivity to other proteins, e.g. immunoglobulin light chai ns. For more detailed assessment other laboratory techniques are necessary. The most reliab le results are obtained by 24 h urine collection. The results should be denoted as g/L or g /24 h. Alternatively spot or better timed urine collections can be assessed for albumin or protein concentration and this is then normalized for urinary dilution by dividing it by th e urinary creatinine concentration (albumin:creatinine ratio (ACR) or protein:creatinine ra tio (PCR)). There is a good correlation between carefully performed 24 h urine collec- tion for protein estimation and ACR/PCR on spot urine Nitrites Most Gram-negative uropathogenic bacteria are capable of reducing nitrates to nitr ites. Pseudomonas spp., Staphylococcus albus and Enterococcus spp. lack this ability. Thus dipstick testing for nitrite can help to characterize the causative agent of urinary tract infection. The test is depend- ent on an adequate nitrate intake (e.g. from vegetables). Ketones Acetoacetate and acetone are detected using dipsticks. They may occur in the urine due to diabetic ketoacidosis, fasting, vomiting or arduous exercise. Urine microscopy Urine microscopy can complement the physicochemi- cal results and add crit ical information when properly performed. The interpretation is subjective and requires an experienced investigator. J. FLOEGE AND J. KANNMACHER 10 Uroepithelial cells have their origin in the urinary tract from the renal calyces to the bl adder (or proximal urethra in men). Typically a large, round nucleus is visible in the cent er of the cells. Uroepithelium is a multilayered epithelium, which contains small cells in the deep layers and larger cells in the super \201 cial layers. Whereas super \201 cial c ells are visible in many patients, cells of the deep layers may suggest sig- ni \201 cant urological disease, such as neoplasia or calculi. General procedure \200 After collecting the urine as described above it should be examine d as soon as possible \200\223 any cellular elements within the urine will lyse with prolo nged storage. An aliquot of 10 mL is centrifuged for 5 min at 2000 \200\223 3000 rpm. The supernatant is discarded almost completely and the sediment is resuspended in 0.5 mL o f the residual urine. \200 An aliquot of the suspension is transferred to a slide and covered with a coverslip. The use of a phase-contrast microscope is recom- mended. For the proper differentiation of lipids and crystals, polarized light should be used. Several microscopic \201 elds should be examined with both low and high magni \201 cation. Cells Cells may originate from the blood circulation or from the epithelia of the urinary t ract. A cell count is performed at \227400 magni \201 cation. Note that alkaline pH and a low relative density may cause lysis of erythrocytes and leukocytes. Erythrocytes Erythrocytes may appear as round cells with a regular contour and without a nu cleus (Fig. 1.2.1). In these cases they are referred to as isomorphic and most probably ori ginate from the lower urinary tract. Erythrocytes with irregular shapes are called dysmorph ic. Normally the number of erythrocytes does not exceed two per high power microscopic \201 eld. Acanthocytes show a characteristic contour with vesicle- like protrusions poking out of a ring-shaped body. Although they are very speci \201 c for glomerular damage, the sensitiv- ity is low. Furthermore many different types of dysmorphic erythrocytes can be di fferentiated (e.g. anulocytes, echino- cytes or stomatocytes), but these are rather nonspec i \201 c for glomerular bleeding. Fig. 1.2.1 Erythrocyturia. Some isomorphic erythrocytes with a regular smooth surface. Leukocytes Under normal conditions there should not be more than \201 ve leukocytes per h igh power microscopic \201 eld. \200 Neutrophils constitute the majority of leukocytes and can be recognized by a granulated cytoplasm and lobu- lated nucleus. They are indicator s of urinary tract infec- tion and may also be seen in patients with interstitial nephritis or proliferative forms of glomerulonephritis. Urine contamination also frequently results in their appearance in urine (Fig. 1.2.2). Fig. 1.2.2 Granulocyte in urine sediment. Note the size and shape of the granulated cell (a rrow) compared to the smaller erythrocytes (arrowheads). Squamous cells are the largest cells in urinary sediment and contain a round, central nucle us and smooth or granu- lar cytoplasm. They derive from the urethra or external genitalia. Large numbers may be seen due to contamina- tion from genital secretions. Eosinophils are nonspeci \201 c and may be seen in cases of acute interstitial nephritis, glomerulonephritis, chronic pyelonephritis or prostatitis. The proper identi \201 cation of lymphocytes requires spe- ci \201 c staining, which is n ot widely practiced. Their occur- rence in urine is considered an indicator of acute cellul ar rejection after renal transplantation. Casts Casts are cylindrically shaped elements, which form in the lumen of distal renal tubu les and collecting ducts and sub- sequently are eliminated in the urine. Their matrix consi sts mainly of Tamm \200\223Horsfall glycoprotein, which is secreted into the tubules and pr ecipitates in an acidic environment. An alkaline pH prevents the formation of casts. Differ ent types of cast may form due to embedding of different particles. Useful information for establishing a diagnosis may be obtained by regarding the different visible casts together with other abnormal microscopic \201 ndings. \200 Hyaline casts are homogeneous, transl ucent, colorless elements with a smooth surface. They are common and do not necessarily ind icate disease. Macrophages appear as cells of variable size. They may be granular, phagocytic or vacuolar, and in patients with neph- rotic syndrome they may contain lipid droplets ( \200\230oval f at bodies \200\231). In clinical practice they are of low signi \201 cance. Epithelial cells Renal tubular epithelial cells are of variable size and shape, but mostly they have a large nucleus, which is often positioned eccentrically. Their appearance is non speci \201 c and may be due to acute tubular necrosis, interstitial nephritis, acute renal transplant rejection, or intake of acetylsalicylic acid. CHAPTER 1.2 Urinalysis and microscopy 11 Granular casts contain small granules originating from degenerated cells (Fig. 1.2.3). They may be found in a variety of renal diseases. Thus they are of low diagnostic value. \200 Waxy casts have an opaque wax-like matrix and square ends. These casts suggest chronic ren al disease, such as glomerulonephritis, diabetic nephropathy or amyloidosis. \200 Fatty c asts contain lipid droplets in variable forms and typically are visible in patients with ma rked proteinuria or nephrotic syndrome. Under polarized light choles- terol drops appear as \200\230Maltese crosses \200\231. may indicate acute interstitial nephritis or acute tubular necrosis and may also be seen in glomerular disorders. Lipids Lipids may be visible as spherical, translucent or yellow drops of variable size, wh ich may appear free in the urine, within casts, as cholesterol crystals, and intracellular in epi- thelial cells or macrophages. Urinary lipids suggest glomer- ular disorders with ma rked proteinuria, but they may also be detected in sphingolipidoses, such as Fabry disease. Crystals Several different types of crystal can be differentiated by morphology and appeara nce under polarized light. The majority of crystals have hardly any clinical signi \201 ca nce. Their formation depends on urine pH, urine temperature and food intake. The most frequ ently found crystals consist of uric acid, calcium oxalate, phosphates and urates. \200 U ric acid crystals have an amber color and appear in a large variety of shapes. They are det ected only in acidic urine (pH \211 5.8). Erythrocyte casts contain various amounts of intact or dysmorphic erythrocytes (Fig. 1.2.4) . Brownish hemo- globin casts may develop due to degradation of the erythrocytes. These cas ts re \202 ect glomerular hematuria, e.g. in glomerulonephritis. A so-called nephritic sed i- ment as a marker for active glomerulonephritis is char- acterized by erythrocyte casts, dysmorphic erythrocytes, leukocytes and renal tubular epithelial cells. Calcium oxalate crystals can be found at pH 5.4 \200\2236.7. Monohydrated crystals appear i n various shapes, bihy- drated crystals mostly appear bipyramidal. Only the lat- ter polari ze light. Amorphous urates and phosphates are small granules of irregular appearance that are morphol ogically indistin- guishable. Urates, however, form in acid urine and are birefringent, whe reas phosphates precipitate in alkaline urine (pH \211 7.0) and do not polarize light. Some crystals always indicate underlying disease, such as cystine crystals (hexagonal plate s with irregular sides, pre- cipitation in acidic urine) as indicators of cystinuria or cho - lesterol crystals (transparent thin plates with sharp edges) in patients with marked prot einuria. Bacteria and other organisms Bacteria are identi \201 ed frequently, as urine specimens ar e usually not obtained under sterile conditions. The detection of 105 bacteria per mL in an appropriately collected speci- men strongly indicates urinary tract infection. In patients with dysuria or leukocyturia, lower bacteria concentra- tions are regarded as pathologic. Candida and Trichomonas are frequently found due to contamination from genital secretions. The microscopic detection of eggs of Schistosoma hematobium is an essential diagnostic tool in areas endemic for schistosomiasis. Fig. 1.2.3 Granular cast. The cast contains small granules and no included cells can be see n. Further reading Aspevall O, Hallander H, Gant V, Kouri T. European guidelines for urinalysi s: a collaborative document produced by European clini- cal microbiologists and clinical ch emists under ECLM in collabo- ration with ESCMID. Clin Microbiol Infect 2001; 7: 173 \200 \223178. D \200\231Amico G, Bazzi C. Urinary protein and enzyme excretion as markers of tubular dama ge. Curr Opin Nephrol Hypertens 2003; 12: 639 \200\223643. Fogazzi GB, Ponticelli C, Ritz E. The urinary sediment. An integrated view, 2nd edn. Oxford: Oxford University Press; 1999. Fogazzi GB, Saglimbeni L, Ban \201 G, Cantu M, Moroni G, Garigali G, Cesana BM. Urinary se diment features in proliferative and non- proliferative glomerular diseases. J Nephrol 2005 ; 18: 703 \200\223710. K hler H, Wandel E, Brunck B. Acanthocyturia \200\223 a characteri stic marker for glomerular bleeding. Kidney Int 1991; 40: 115 \200\223120. Fig. 1.2.4 Red cell cast. This cast contains many erythrocytes, which are of similar size a s the single erythrocyte on the right (arrowhead). Leukocyte casts contain leukocytes with polymorphic nuclei, sometimes together with epithel ial cells or eryth- rocytes. Their presence suggests pyelonephritis or acute interstitial n ephritis. See also Clinical assessment of renal function, p. 12 Epithelial casts contain exfoliated tubular epithelial cells recognizable by their prominen t nucleus. These casts J. FLOEGE AND J. KANNMACHER 12 Clinical assessment of renal function Urinary clearance is smaller than GFR for substances, Glomerular \201 ltration rate (GFR) Measurement of the GFR is considered the \200\230gol d standard \200\231 measure of excretory renal function. It is dif \201 cult to meas- ure in routine clinical practice and the most commonly used marker of kidney function is an est imated GFR (see below) which is reported automatically alongside the serum creati- nine con centration (this is universal in UK laboratories). GFR is the multiplication product of the average \201 ltration rate of single nephrons and the total number of nephrons in both k idneys. The normal GFR level ranges from 80 to 120 mL/min/1.73 m2, with signi \201 cant i nterindividual varia- tion depending on many different factors, such as age, body size, phy sical activity and diet. GFR also changes during pregnancy, increasing by 50% in the \201 rst trimester and normalizing soon after delivery. After the fourth decade of life, GF R declines by 0.75 mL/ min/1.73 m2 per year. In an individual patient GFR is a relatively constant parameter. A reduction in GFR may result from a decline in the num- ber of nephron s or from a decline in the GFR of single nephrons (SNGFR). As a reduction in the number of neph- rons may be compensated by an increased SNGFR due to glomerular hypertrophy or elevat ed glomerular capillary pressure, early substantial kidney damage is not always associated with a decline in the GFR. In patients who are at increased risk for chronic kidney disease (CKD), investigations should be made to detect albuminuria or a decrease in GFR. The GFR i s also used as a parameter to stage the severity of CKD. which are \201 ltered and reabsorbed. Exogenous \201 ltration markers For the measurement of urinary clearance these sub- stanc es have to be administered to the patient by infusion or injection. Inulin (5.2 kDa) is a p olymer of fructose, which meets the aforementioned criteria for ideal GFR markers, is nonto xic, and is not metabolized. It is therefore regarded as the gold standard marker, which ca n be used for the evaluation of other markers. Since the protocol for the measurement of in ulin clearance is labor intensive (requiring continuous infusion of the substance to genera te a constant serum concentration and accurate assessment of urine \202 ow with multiple urine col- lections) it cannot be used in clinical practice. There are several other suitab le exogenous marker sub- stances, such as iohexol, [51Cr]EDTA, [99mTc]DTPA and [125I]iothal amate, but due to the complex measurement protocols GFR is commonly estimated using endogen ous \201 ltration markers. Endogenous \201 ltration markers For the calculation of the urinary clearance of a substa nce according to the aforementioned equation, the determina- tion of serum concentration an d a timed urine collection are required. The results depend on a complete timed urine colle ction. In patients in whom the serum levels of the marker are not constant, the precision o f the investiga- tion can be increased by taking multiple blood samples to calculate the av erage serum concentration. GFR measurement Urinary clearance Direct measurement of the GFR is not a routine clinical t est and remains a research tool only. GFR can be deter- mined from the urinary clearance of certain suitable marker substances. In general, clearance of a substance is de \201 ned a s the virtual volume of plasma cleared of this substance by excretion per unit of time. For a substance \200\230x \200\231 it is de \201 ned as: Creatinine Creatinine is the most commonly used marker for the assessment of renal function . \200 It derives from muscle catabolism, and meat or creatine intake, and hence varies a ccording to body muscle mass and food intake. With a molecular weight of 113 Da it is freely \201 ltered by the glomerulus. As it is ad ditionally secreted by the tubules, creatinine clearance systemically overestimates the GFR , particularly in patients with impaired renal function. Cx = Ax/Px where Ax is the amount of x eliminated from the plasma and Px is the plasma conc entration. The value re \202 ects the ef \201 cacy of the elimination of a certain substa nce and is expressed in units of volume per time. It comprises both urinary and extrarenal excretion. For the assessment of renal excretion, urinary clearance is determined. For a su bstance x it can be calculated as follows: The serum creatinine concentration varies inversely with the GFR, but this relationship is not linear. Renal function may be signi \201 cantly impaired before increased creatinine l evels are measured. In patients with early renal dysfunction small changes in serum creatin ine may re \202 ect considerable diminutions of GFR. However, in advanced renal failure gr eat changes in creatinine level re \202 ect only small changes in GFR. Furthermore there i s a great variability of GFR for a given serum creatinine con- centration due to individual variation in muscle mass. \200 To a negligible degree, creatinine is secreted by the int estine and can be degraded by bacteria. This extrare- nal excretion is increased when the G FR is reduced. Antibiotics may inhibit this pathway due to elimination of the intestinal \202 ora, and thereby increase serum creati- nine levels. CU(x) = (Ux \227 V)/Px where Ux is the urinary concentration of x, V is the urinary \202 ow rate and Px is the plasma concentration of x. It depends on glomerular \201 ltration, tubular secretion and tubular reabsorption. \200 The urinary clearance of a substance th at is \201 ltered, but neither secreted nor reabsorbed, equals GFR. Thus sub- stances tha t ful \201 ll these criteria constitute ideal markers for the measurement of GFR. Several factors have been identi \201 ed, which are associ- ated with an increase or decre ase in production and excretion of creatinine, and hence may affect the serum concentration (see Table 1.3.1). Urinary clearance is larger than GFR for substances, which are both \201 ltered and secreted. CHAPTER 1.3 Clinical assessment of renal function 13 Table 1.3.1 Extrarenal factors which may in \202 uence serum creatinine levels Increase of serum creatinine Decrease of serum creatinine Reduced production Increased production Reduced muscle mass due to malnutrition or following amputation Decline in muscle mass with age Lower muscle mass in females Higher muscle mass due to physical exercise Higher average muscle mass in African-Americans Meat-rich diet Vegetarian diet Impaired tubular secretion Drugs, such as trimethoprim, cimetidine or certain \201 brates Impaired intestinal elimination Destruction of the intestinal \202 ora due to antibiotics Table 1.3.2 Interpretation of the \202 uid deprivation test Plasma osmolality Urine osmolality Urine osmolality after DDAVP <296 mOsmol/kg >900 mOsmol/kg no increase Primary polydipsia >296 mOsmol/kg increase of <10 mOsmol/kg/h increase of >10% Central diabetes insipidus >296 mOsmol/kg increase of <10 mOsmol/kg/h no increase Nephrogenic diabetes insipidus Creatinine measurement by the Jaffe reaction may pro- duce falsely elevated values in the p resence of acetoac- etate due to diabetic ketoacidosis. the Cockroft \200\223Gault formula or measurement of creati- nine clearance. As both equations assume a steady state, quick changes in GFR may cause incorrect results, thus overestimating a rapidly declining GFR and underestimating a rapidly rising one. For the calculation of creatinine clearance (CrCl) the above-mentioned equation can be modi \201 ed as follows: CU(crea) = (Ucrea \227 V)/(Pcrea \227 t) where Ucrea is the urinary creatinine concentrat ion [mg/dL], V is the volume of collected urine [mL], Pcrea is the plasma creatinine concen tration [mg/dL] and t is the col- lection time [min]. Assuming a steady state with equal pr oduction and excre- tion of creatinine, GFR can also be estimated from serum levels using e quations that incorporate additional demo- graphic and clinical variables, such as age, sex , race and body size. \200 The Cockcroft \200\223Gault formula (see below) incorporates a ge, sex and body weight. In patients who are overweight and edematous this equation overest imates GFR, whereas it is probably underestimated in patients of African origin. As the for mula estimates creatinine clear- ance there is a systematic overestimation of GFR. (140 \200\223 age) \227 lean body weight [kg] (Creat [ mol/L]/88.4) \227 72 It should be kept in mind that both formulas are not reli- able for GFR values >60 mL/min/1.73 m2. In patients with very high or low creatinine generation (see Table 1.3.1) both equations ar e of limited accuracy. \200 There is considerable variability in measured serum cre- atin ine levels between different laboratories. Therefore GFR estimations based on serum creatin ine are not nec- essarily directly comparable, if they are obtained from dif- ferent labora tories, unless a correction has been applied to allow for this variation (in the UK all lab oratories report the four-variable MDRD eGFR appropriately cor- rected to allow for the dif ferent creatinine assays in use). Urea Urea is a 60 Da product that is derived from hepatic protein catabolism. It is freely \201 ltered in the glomerulus and reabsorbed to a variable extent in the proximal and dis tal tubules. Its generation is increased in states of hyperalimentation, after the absorpti on of blood due to gastrointestinal hemorrhage or in catabolic situations due to infection or chemotherapy. Malnutrition or liver disease may result in decreased urea generation. A r educ- tion in kidney perfusion may cause increased tubular reabsorption. The variability in generation and reabsorption of urea mean that it is not of value for estimating GFR. CrCl [mL/min] = For women multiply the result by 0.85. \200 The MDRD (Modi \201 cation of Diet in Renal Disease) Study formula incorporates a correction for race. The equation underestimates GFR in patients with high levels of GFR, e.g. people evaluated as kidney transplant donors. The re are several population groups, for which the equation has not yet been validated, e.g. c hildren, patients older than 70 years or pregnant women. The four-variable MDRD formula is: Cystatin C Cystatin C is a 1300 Da protein produced by all nucleated human cells. It appear s to be generated at a constant rate. It is freely \201 ltered by the glomerulus and subs equently rea- bsorbed and degraded in the tubular epithelial cells. Therefore urinary excre tion is minimal. Thus it is not pos- sible to measure a urinary clearance for cystatin C. GFR [mL/min/1.73 m2] = 186 \227 (Creat [ mol/L]/88.4) \200\2231.154 \227 (age) \200\2230 .203 \227 (0.742 if female) \227 (1.21 if Black). \200 In some studies it has been show n that GFR estimation by using the MDRD Study formula is more precise than J. FLOEGE AND J. KANNMACHER 14 excretion. The substance is commonly regarded as a marker for early drug nephrotoxicity. In patients with glomerular proteinuria, NAG may partially originate from the plasma. Normal serum levels range from 0.54 to 1.55 mg/L with a constant concentration from the age of 1 \200\22350 years. Furthermore the levels are not in \202 uenced by muscle mass, gen der or body height. After the seventh decade cystatin C levels increase, possibly due to th e decline in GFR in older people. It has been shown that in patients with acute renal failu re increased cystatin C levels can be detected prior to an increase in serum creatinine. Co mpared to serum creatinine, cystatin C seems to be a better marker for GFR, but in comparis on to estimations based on creatinine no signi \201 - cant superiority of cystatin C has b een shown and its meas- urement is considerably more costly. As further research and standa rdization of the laboratory assay is required, the measurement of cystatin C in everyday cl inical practice is not currently recommended. Urine osmolality The measurement of urine osmolality reveals information about the ability of the kidneys to adjust the urine concen- tration in response to water balance. In patient s with an osmolality of \211 800 mOsmol/kg in an early morning urine, a defect of renal co ncentrating ability is very unlikely. For the further investigation of patients with unexpl ained polyuria a \202 uid deprivation test can be administered, after having excluded glu cosuria or hypercalcemia as causes for increased diuresis. After a period of thirst for 12 h, urine osmolality nor- mally rises to >1000 mosmol/kg and plasma osmolality is <295 mOsm ol/kg. To prevent severe dehydration, regular measurements of urine excretion, urine osmola lity and body weight are required during the test. At the end of the test period, plasma os molality and sodium are also deter- mined. Desmopressin (DDAVP) is then administered to the patient. The interpretation of the test is summarized in Table 1.3.2. Markers of tubular damage The urinary excretion of certain proteins is associated with tubu lar disorders. A variety of such substances have been identi \201 ed, but only some of the m have so far made their way into clinical practice. Low molecular weight proteins These proteins are freely \201 ltered by the glomerulus. In healthy individuals they are reabsorbed by the proximal tubule. The urinary excretion of t hese substances increases in cases of impaired proximal tubular reabsorption. \200 A1-Mic roglobulin (33 kDa) is a glycosylated protein originating from the liver, which is stable i n urine and therefore suitable for measurement in clinical practice. In patients with chron ic glomerulonephritis the meas- urement may allow prediction about the outcome of the disea se, as in these cases the level of urinary loss is pro- portional to the severity of tubulo interstitial damage. \200 B2-Microglobulin (11.8 kDa) constitutes the light chain of clas s I major histocompatibility antigens. It is unstable in acidic urine (pH <6.0) and hence b icarbonate needs to be administered to the patient for it to be measured. \200 Retinol-bi nding protein (21 kDa) binds retinol circulating in the plasma and also appears to be a sui table marker for proximal tubular damage, since it is stable in urine. Further reading D \200\231Amico G, Bazzi C. Urinary protein and enzyme excretion as markers of tubular damage. Curr Opin Nephrol Hypertens 2003; 12: 639 \200\223643. Filler G, Bokenkamp A, Hofmann W, Le Bricon T, Martinez-Bru C, Grubb A. Cystatin C as a mar ker of GFR \200\223 history, indications, and future research. Clin Biochem 2005; 38: 1 \200\2238. K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classi \201 ca tion, and strati \201 cation. Am J Kidney Dis 2002; 39: 1 \200\223266. Perrone RD, Madias NE, Levey AS. Serum creatinine as an index of renal function: new insigh ts into old concepts. Clin Chem 1992; 38: 1933 \200\2231953. Rule AD, Larson TS, Bergstralh EJ, Slezak JM, Jacobsen SJ, Cosio FG. Using serum creatinine to estimate glomerular \201 ltration rate: accuracy in good health and in chronic kidney disease. Ann Intern Med 2004; 141: 929 \200\223937. N-Acetyl-b-glucosaminidase (NAG) NAG is a hydrolytic enzyme with activity in the whole neph ron, particularly in the proximal tubule. With a molec- ular weight of 130 kDa it is not \201 ltered by the glomerulus, so NAG measured in the urine mainly derives from the tubule s. Tubular damage, e.g. in tubulointerstitial nephritis or renal transplant rejection, resu lts in an increase in urinary NAG Stevens LA, Levey AS. Measurement of kidney function. Med Clin North Am 2005; 89: 457 \200\223473. See also The aging kidney, p. 18 Urinalysis and microscopy, p. 8 Nephrogenic diabetes insip idus, p. 220 This page intentionally left blank C. JONES 16 Renal function in the newborn infant Sodium homeostasis Neonates have a limited capacity to excrete a Na+ load or to conserve Na + if there is Na+ restriction. In the \201 rst week of life fractional Na+ excretion is h igh and this is greater in the more immature neonates. Early hyponatremia (occurring shortl y after birth) can be caused by: \200 excessive \202 uid given to the mother; \200 re lease of antidiuretic hormone secondary to neonatal The neonatal kidney can adapt to the usual physiological processes occurring after birth. I t is less able to adapt to endogenous and exogenous stress such as hypotension, hypoxia and \202 uid overload and this is more pronounced in the more immature infants. The understa nding of renal function in the neonate will aid the clinician in the appro- priate manageme nt of \202 uid, electrolytes, nutrition and acid \200\223base in either the sick or prete rm neonate. It helps in the interpretation of biochemical investigations and drug handling in the neonate. Normative biochemical data for newborn infants are listed in Table 1.4.1. pathology such as asphyxia; inadequate Na+ content of feed/ \202 uid. Late hyponatremia can be caused by: \200 inade quate Na+ content of feed/ \202 uid; \200 deranged renal tubular function; \200 Na+ lo sses from the gastrointestinal tract. Prolonged hyponatremia is associated with a reduction in brain function and growth. Hypernatremia can be caused by: \200 sodium bicarbonate in fusions for acidosis; \200 insuf \201 cient free water administration when insensible Table 1.4.1 Normative biochemical data for newborn infants Age Reference range Serum Bicarbonate <1 month 17 \200\22326 mmol/L Base excess Newborn \200\22310 to \200\2232 mmol/L Calcium (total) Premature \211 2 weeks 1.5 \200\2232.5 mmol/L 1.9 \200\2232.8 mmol/L water losses are high. Phosphate 1 month 1.4 \200\2232.8 mmol/L Hypernatremia induces a \202 uid shift from the intracellular compartment causing cell sh rinkage and tearing of the cer- ebral capillaries. This mechanism reduces the physiologic c ontraction of the ECF, increasing the risk of patent ductus arteriosus, cardiac failure, br onchopulmonary dysplasia and intracranial hemorrhage. Potassium 1 month 3.5 \200\2236.0 mmol/L Sodium <1 month 130 \200\223145 mmol/L Creatinine 1 week 2 weeks 40 \200\223125 mol/L 35 \200\223105 mol/L 3 weeks 25 \200\22390 mol/L Potassium homeostasis In the neonate the rate of K+ excretion is low. This reduc- tion in K + excretion is partly explained by the low activity of the Na+K+-ATPase in the K+ secreting epithelium, and a reduction in the number of K+ channels. Plasma K+ in term infants falls in the \201 rst week of life. In preterm infants plasma K+ reaches its maximum in the 3rd or 4th week of life as a result of metabolic acidosis, oliguric renal failure and cataboli c activity. 4 weeks 20 \200\22380 mol/L 26 \200\22360 mL/min/1.73 m2 28 \200\22368 mL/min/1.73 m2 GFR 1 week 1 month Urine Maximal osmolality 1 \200\22314 days 2 \200\2234 weeks 210 \200\223650 mosm/kg 780 \200\2231100 mosm/kg Acid \200\223base balance Following birth, neonates have a mixed respiratory/ metabolic aci dosis as a result of changes in the cardiopul- monary circulation and the effects of the bi rthing process. Later they develop a self-limited metabolic acidosis which is in \202 uenc ed by the protein load, weight gain, ability of the renal tubules to excrete an acid load, and the renal thresh- old for bicarbonate reabsorption. This has usually resolved by the en d of the fourth week. Water homeostasis In early postnatal life there is a reduction in total body water, extrace llular water, and extracellular sodium (Na+) and chloride (Cl \200\223). There is an increa se in intracellular water and its main components protein, magnesium and potassium (K+). To tal body water as a percentage of body weight is 75% and the extracellular \202 uid (ECF ) is 35 \200\22340% of total body water. The reduction in total body water is re \202 ecte d in the weight loss observed in early postnatal life. This is greater in low birth weight premature infants where a weight loss of up to 10% can occur. Fluid prescriptions need to b e adjusted for increased insen- sible water loss because of: \200 increased relative body surface area; \200 increased metabolic rate; \200 increased transepidermal water loss, particularly in the premature infant exposed to phototherapy and over- head heaters. Glomerular \201 ltration rate The glomerular filtration rate is low after birth and is cl osely correlated to gestational age. It is 5 mL/m2 in neonates of 28 weeks gestation and 1 2 mL/m2 in term infants. Postnatally there is a rapid increase in GFR with a two-fold incre ase in GFR occurring in the \201 rst 2 weeks of life. Adult values are achieved between 1 and 2 years of age. Factors involved in the maturation of GFR include: \200 increase in renal blood \202 ow as a consequence of an increase in BP and a fall in intravascular resistance; CHAPTER 1.4 Renal function in the newborn infant 17 increase in the number of super \201 cial nephrons involved The location of angiotensin receptors within the kidney changes during development and the observation that the use of angiotensin-II receptor blockers and angiotensin- converting en zyme inhibitors in the mother cause renal dysplasia in the neonate indicates that the renin \200\223angiotensin system has a role in nephrogenesis. in \201 ltration; increase in the surface area of the glomerular basement membrane.