Leucocytes
Neutrophils: leucocytes are a prominent feature of urinary infection but may also be present in infl ammatory renal conditions (GN, TIN). Sterile pyuria refers to the situation wherein leucocytes are seen consistently on microscopy, but subsequent culture is sterile.
Causes of sterile pyuria
• Partially treated UTI or fastidious organism (e.g. Chlamydia ).
• Calculi.
• Prostatitis.
• Bladder tumour.
• Papillary necrosis.
• TIN.
• TB (send 3 x EMUs, b p. 696).
• Appendicitis.
Lymphocytes: may be a feature of chronic tubulointerstitial disease.
Eosinophils: identifi ed with Hansel ’ s or Wright ’ s stain. Associated with TIN ( b p. 580) but also occasionally present in other conditions, including RPGN, prostatitis, and atheroemboli. Renal tubular cells: large, oval cells.
Present in normal urine but i in tubular damage (ATN or TIN). Squamous epithelial cells: large cells with small nuclei, often of urethral origin (or skin/
vaginal contaminant). Transitional epithelial (urothelial) cells: suggest cystitis.
Malignant cells: special stains, immunocytochemistry, and fl ow cytometry all assist detection ( b p. 751).
Microorganisms
• Bacteriuria: normal urine is sterile. Simultaneous presence of leucocytes suggests true infection, rather than contamination. Gram staining enables initial identifi cation and cell count while culture and sensitivity results are awaited.
• Fungi: Candida species are most frequently encountered. Typical appearance is that of a small pale green cell, often with visible budding.
May result from genital contamination. Risk factors for colonization are: indwelling foreign body (ureteric stents, bladder catheter), DM, antibiotic therapy, and immunosuppression.
• Trichomonas: oval and fl agellate (motile if alive). Usually a genital contaminant.
• Schistosoma haematobium: ova detection is an important technique in endemic areas.
URINALYSIS: CELLS, ORGANISMS, AND CASTS 25
Urine culture
M,C+S differentiates contamination from true infection and guides treat-ment. A pure growth of >10 5 colony-forming units (cfu)/mL is the conven-tional diagnostic criterion for urinary tract infection ( b p. 707).
Casts
Casts are plugs of Tamm – Horsfall mucoprotein within the renal tubules, with a characteristic cylindrical shape. They are classifi ed according to appearance and the cellular elements embedded in them. Though pro-duced in normal kidneys, they can be valuable clues to the presence of renal disease (see Fig. 1.4).
Non-cellular casts
• Hyaline casts: mucoprotein alone and virtually transparent.
A non-specifi c fi nding that occurs in concentrated urine.
• Granular casts: granular material (aggregates of protein or cellular remnants) is embedded in the cast. Often pathological but non-specifi c.
• Broad or waxy casts: hyaline material with a waxy appearance under the microscope. Form in dilated, poorly functioning tubules of advanced CKD.
Cellular casts
• Red cell casts: 2 virtually diagnostic of GN.
• White cell casts: characteristic of acute pyelonephritis — may help to distinguish upper from lower tract infection. Also occur in TIN.
• Epithelial cell casts: sloughed epithelial cells embedded in mucoprotein. A non-specifi c feature of ATN. Also found in GN.
• Fatty casts: contain either lipid-fi lled tubular epithelial cells or free lipid globules. Distinguished from other casts by ‘Maltese cross ’ appearance under polarized light. Occur in the lipid-laden urine of the nephrotic syndrome. Lipids may also appear as droplets or crystals. When clumped, these are referred to as oval fat bodies .
• Other casts: under the right conditions, any constituent of the urine (microorganisms, crystals, bilirubin, or myoglobin) may become entrapped in a mucoprotein cast.
(a)
(b)
Fig. 1.4 a) Granular cast. b) Red cell cast. Reproduced from Barrett, J, Harris, K, Topham, P. Oxford Desk Reference of Nephrology (2008), with permission from Oxford University Press.
URINALYSIS: CELLS, ORGANISMS, AND CASTS 27
Urinalysis: crystals
Detected by examining the urine under polarized light (see Fig. 1.5).
Most crystals are clinically irrelevant.
Uric acid
Usually lozenges with a yellow-brown hue. Precipitate at acid pH. A few may be normal (e.g. high meat intake), but i quantities may indicate hyperuricosuria. May be present in acute urate nephropathy (tumour lysis syndrome, b p. 160).
Calcium oxalate
May be monohydrated (ovoid) or bihydrated (pyramidal — like the back of an envelope). Prefer an acidic pH but not always. A few may be normal (spinach and chocolate ingestion) but can denote hypercalciuria or hyper-oxaluria ( b p. 721). A diagnostic clue in ethylene glycol poisoning in both real life and exams ( b p. 910).
Calcium phosphate
Heterogeneous in their appearance ( l needles, prisms, stars). Favoured by alkaline pH. Might be a risk factor for calcium stone formation.
Magnesium ammonium phosphate (triple phosphate) Birefringent prisms ( ‘ coffi n lids ’ ). Prefer alkaline pH. If present, exclude a Proteus UTI.
Amorphous phosphates
Unattractive clumps in (alkaline) urine cooled for storage. No clinical signifi cance.
Cystine
Hexagonal. Cystine is not a constituent of normal urine so always signifi -cant. Prefer acid urine. A marker of cystinuria ( b p. 719).
Cholesterol
Thin plates with sharp edges. Occur with heavy proteinuria.
Drug-induced crystalluria
Many drugs can precipitate in the renal tubule. In severe cases this may cause AKI.
• Antibiotics: sulfadiazine, amoxicillin.
• Antiviral agents: aciclovir, valaciclovir, famciclovir, ganciclovir, valganciclovir, and indinavir.
• Methotrexate.
• Primidone (a barbiturate).
• Triamterene.
• Vitamin C (calcium oxalate deposition).
URINALYSIS: CRYSTALS 29
Uric acid
Bihydrated Monohydrated
Calcium phosphate
Magnesium ammonium phosphate
Cystine Calcium oxalate
Fig. 1.5 Urinary crystals.
Determining renal function
Introduction
Several aspects of renal function can be measured. The most important is glomerular fi ltration rate (GFR). Glomerular fi ltrate refers to the ultrafi l-trate of plasma that crosses the glomerular barrier into the urinary space.
GFR is measured per unit time (usually expressed mL/min) and represents the sum of fi ltration rates in all functioning nephrons ( 6 a surrogate for the amount of functioning renal tissue).
GFR is relatively constant in an individual. d GFR may result from a reduction in nephron number or a reduction in the GFR of single nephrons ( d SNGFR). A reduction in number can be compensated for by an increase in SNGFR through elevated glomerular capillary pressure or glomerular hypertrophy ( this means signifi cant kidney damage may not initially be associated with d GFR).
GFR is useful for:
• Providing a consistent measure of kidney function.
• Monitoring progression of CKD (and response to treatment).
• Forecasting the need for dialysis and transplantation.
• Determining appropriate drug dosing in renal impairment ( b p. 874).
It provides no information regarding the cause of renal insuffi ciency.
Measurement of GFR
• Measured indirectly by evaluating clearance from plasma of a (renally excreted) marker substance.
• Clearance: the volume of plasma from which this substance is removed per unit time.
• Suitable markers require certain characteristics shown in the following list and may be endogenous (e.g. Cr) or exogenous (e.g. inulin).
• Inulin, a fructose polysaccharide for which (along with fl atulence) we have the Jerusalem artichoke to thank, remains the gold standard. Cost and technical considerations (it requires a continuous infusion) prohibit routine use.
Characteristics of an ideal clearance marker
• Safe to administer, economical, and easy to measure.
• Freely fi ltered at the glomerulus.
• Not protein-bound (able to distribute in the extracellular space).
• Present at a stable plasma concentration.
• No extra-renal elimination.
• Not reabsorbed, secreted, or metabolized by the kidney.
DETERMINING RENAL FUNCTION 31
Aspects of renal function
• Glomerular fi ltration rate (GFR).
• Tubular function (including Na + and K + handling and urinary concentrating/diluting capacity).
• Acid – base balance.
• Endocrine function:
• Renin – angiotensin system (RAS).
• Erythropoietin production.
• Vitamin D metabolism.
Not measured in clinical practice
• Autocrine ( l production of endothelins, prostaglandins, natriuretic peptides, nitric oxide).
• Protein and polypeptide metabolism (e.g. insulin catabolism).
In clinical practice, GFR is estimated by one of the following means:
• Serum Cr (and, to a lesser extent, urea).
• Formulae based on the serum creatinine (estimated or eGFR).
• Creatinine clearance (CrCl) from a 24h urine collection.
• Isotopic clearance (e.g. EDTA-GFR or DTPA-GFR).
Creatinine
Serum creatinine
• Convenient and inexpensive — the most commonly used indirect measure of GFR. It is estimated that it is measured over 300 million times annually in the USA.
• Generated from non-enzymatic metabolism of creatine in skeletal muscle. Production is proportional to muscle mass (20g muscle l 7 1mg Cr). Little short-term variation in an individual.
• 7 25% is derived from dietary meat intake (creatine mostly; creatinine if the meat is stewed).
• d muscle mass (elderly, cachectic) l i Cr production l overestimation of GFR.
U cr x V is relatively constant in the formula for CrCl (where V is volume).
So CrCl = (urineCr x V)/plasmaCr Becomes CrCl = constant/plasmaCr
Hence, serum Cr varies inversely with GFR: d GFR l i SCr (until a new steady state is reached).
• Cr meets many, but not all, of the criteria for a clearance marker.
Shortcomings:
• It is secreted by the proximal tubule ( 7 10 – 20% when GFR is normal), so the amount excreted in the urine exceeds the amount fi ltered. As GFR falls, there is a progressive i in tubular secretion until saturation occurs at SCr 7 132 – 176 μ mol/L (1.5–2.0 mg/dL);
beyond this point, SCr rises as expected (see Fig. 1.6).
• It undergoes extra-renal elimination by secretion and degradation in the GI tract. This becomes more important as GFR falls.
• When d GFR is rapid, it takes time for a steady state to be reached and for SCr to i , i.e. SCr may initially be normal after a catastrophic renal insult.
• Traditionally measured using the Jaff é alkaline pictrate colorimetric assay. However, interference by non-creatinine chromogens created a tradition of overestimating Cr. Enzymatic methods on modern auto-analysers are generally more accurate, with less variability.
• Certain substances interfere with Cr, either through competitive inhibition of tubular secretion (cimetidine, trimethoprim, amiloride, spironolactone, triamterene) or assay interference (in the Jaff é reaction: bilirubin, ketoacids, vitamin C, glucose, and cephalosporins).
CREATININE 33
0 40 80 120 160
Glomerular filtration rate, mL/min
Plasma creatinine, mg/dL
8
6
4
2
0
Fig. 1.6 The relationship between Cr concentration and GFR (measured as inulin clearance) in 171 patients with glomerular disease. The hypothetical relationship between GFR and Cr is shown in the continuous line, assuming that only fi ltration of Cr takes place. The broken horizontal line represents the upper limit of normal of serum Cr (1.4mg/dL or 115 μ mol/L). It can be seen that, because of Cr secretion, serum Cr consistently overestimates GFR (reproduced from Shemesh O, Golbertz H, Kriss JP, et al . (1985) Limitations of creatinine as a fi ltration marker in glomerulopathic patients. Kidney Int 28 : 830 – 8, with permission from Nature Publishing Group.
Creatinine clearance CrCl is calculated as:
CrCl x plasma creatinine (P Cr ) = urine creatinine (U Cr ) x volume (V) 6 CrCl = (U Cr x V) / P Cr
Example
• Serum Cr = 106 μ mol/L
• Urine Cr = 8800 μ mol/L
• Urine volume = 1.2L • CrCl = (U Cr x V)/P Cr
• CrCl = (8800 x 1.2) ÷ 106 = 99.6L/day
• Conventionally, CrCl is shown in mL/min 6 x 1000/1440 • (99.6 x 1000)/1440 = 69.2mL/min
(normal range: ♀ : 95 9 20mL/min; ♂ : 120 9 25mL/min)
Limitations
• Tubular secretion of Cr means that GFR is overestimated.
• Requires accurate urine collection.
• Heir to all the pitfalls inherent in Cr measurement.
• Even if collections are accurate, there is marked serial variation (15 – 20%).
CG tends to overestimate and MDRD underestimate GFR. Up to 25% of patients will be misclassifi ed where either is used to categorize patients according to the KDOQI CKD scheme ( b p. 192).
eGFR
Introduction (see also b p. 196)
In the last few years, it has become routine to estimate and classify kid-ney function and disease using equations based on the serum Cr. This is termed estimated or eGFR. Such equations attempt to correct for the confounding effects of body weight, age, sex, race, and muscle mass.
Limitations
Still based on Cr and do not take into account tubular secretion, extra-renal elimination, or differences in production between individuals of the same age and sex, or the same individual over time.
MDRD
Widely used internationally and the current basis of CKD classifi ca-tion. Developed from data in the Modifi cation of Diet in Renal Disease (MDRD) study. However, remains poorly validated in children (age
<18 years), elderly (age >70 years), pregnancy, ethnic groups other than Caucasians and African Americans, and those without CKD.
eGFR , in mL/min per 1.73m 2 =
(170 x (P Cr [mg/dL]) exp[ – 0.999]) x (Age exp[ – 0.176]) x ((S Urea [mg/dL]) exp[ – 0.170]) x ((Albumin [g/dL]) exp[+0.318]) • Multiply x 0.762 if the patient is ♀
• Multiply x 1.180 if the patient is black.
• Simplifi ed version:
eGFR = 186.3 x ((serum creatinine) exp[ – 1.154]) x (Age exp[ – 0.203]) x (0.742 if ♀ ) x (1.21 if African American)
Notes: (i) to convert Cr from mg/dL to μ mol/L, x 88.4. To convert urea from mg/dL to mmol/L, x 0.357; (ii) exp = exponential.
Cockcroft–Gault (CG)
eGFR (mL/min) = 1.2 x {140 – age (yr)} x weight (kg) Cr (μmol/L)
Multiply x 0.85 in ♀ to correct for reduced creatinine production.
eGFR 35
CKD-EPI equation
• The CKD Epidemiology Collaboration has pooled data from multiple studies to produce the CKD-EPI equation.
• Uses the same variables as MDRD.
• The study population included individuals with and without kidney disease over a wide range of GFRs.
• The equation provides a more accurate assessment of GFR in individuals with normal or only slightly d GFR (particularly GFR
>60mL/min), resulting in:
• Lower estimates of population CKD prevalence.
• More accurate prediction of adverse outcomes.
• There are still important limitations: 7 50% will have an eGFR that is >16mL/min different from true measured GFR (although this represents a slightly improvement over MDRD).
• Although most laboratories currently report an MDRD eGFR, a move to adopt CKD-EPI for routine clinical use is likely to be increasingly advocated.
• The eGFR formulae are available as web-based and downloadable calculators.
• M http://www.kidney.org/professionals/tools • M http://www.renal.org/eGFRcalc
• Several useful Apps are also available, e.g. National Kidney Foundation eGFR calculator, MedCalc, QxMD, MedMath, Epocrates.
Reciprocal of plasma creatinine
• There is an inverse relationship between GFR and SCr:
CrCl = constant / SCr
• Plotting the reciprocal of SCr (1/SCr) against time will often, though not always, produce a straight line, the slope of the curve representing change in GFR with time.
• A logarithmic plot of SCr can be used in a similar way.
• This may be useful in two settings:
• Extrapolation of the line can help predict when CKD is likely to reach ESRD and 6 assist timely planning of RRT.
• A change in the slope of the curve can be used to monitor treatment: a d in the slope indicates slowed progression, while an i may indicate a 2nd insult (acute-on-chronic kidney injury).
GFR measurement: other methods
Isotopic GFR
Several radiopharmaceuticals ( 51 Cr-EDTA, 99m Tc-DTPA, 125 I-iothalamate) compare favourably to inulin for the measurement of GFR. Isotopic tech-niques are expensive.
Method
A single IV injection, followed by venous sampling at regular intervals (intervals i , as expected GFR d ). Post-injection, plasma isotope activ-ity d rapidly, as it distributes throughout the ECF. A slower exponential decline (renal elimination) then follows, allowing GFR to be determined.
Protocols based on urine collection are also described.
With DTPA, renal elimination is often measured with a gamma cam-era positioned directly over the kidneys. While not as precise as venous sampling, it enables assessment of each individual kidney ’ s contribution to total GFR (termed ‘split function ’ ) ( b p. 54).
Cystatin C
• The variable nature of Cr production has encouraged the search for alternative endogenous compounds that might help estimate GFR.
• Cystatin C, a 13kDa cysteine protease inhibitor, is the most developed of these.
• Produced by all nucleated cells and freely fi ltered at the glomerulus before tubular reabsorption and metabolism. Unlike Cr, it does not undergo tubular secretion, but a degree of extra-renal elimination is likely.
• Unfortunately, despite early optimism, several important ‘non-GFR ’ determinants of cystatin C concentration have been identifi ed.
• Advancing age, male sex, diabetes, i BMI, i albumin, and i CRP are all associated with increased levels.
• Relative high or low cell turnover, e.g. i / d thyroidism or steroid treatment ( transplant patients!), also cause variability.
• Several cystatin C-based eGFR equations have been formulated (as have equations that incorporate both cystatin C and SCr).
• At present, there remains considerable uncertainty as to any advantages for GFR estimation, both in the general population and existing CKD. Available data are frustratingly inconsistent.
• A shorter half-life and smaller volume of distribution led to an expectation that it might be a more useful marker in the non-steady state, particularly AKI. However, results have proved variable.
• There also appears to be no clear-cut advantage in the context of low muscle mass.
• Nonetheless, cystatin C appears to be a better predictor of adverse outcomes than either SCr or eGFR based on the SCr.
• There is also signifi cant current inter-assay and inter-laboratory variability, although progress is being made toward standardization.
• Overall, it remains to be seen whether it will yet prove itself in selected circumstances and 6 move into the clinical mainstream.
GFR MEASUREMENT: OTHER METHODS 37
Urea
• Synthesized in the liver as a means of ammonia excretion.
• Rate of production not constant (unlike SCr).
• Inverse relationship with GFR.
• Infl uenced by a number of factors independent of GFR (see Table 1.4).
• Freely fi ltered at the glomerulus but reabsorbed in the tubules:
• Urea movement is linked to water (under vasopressin infl uence) in the distal nephron.
• d renal perfusion l i urea reabsorption l disproportionate i urea compared to SCr.
• May help to differentiate ‘pre-renal ’ renal dysfunction ( b p. 96).
Table 1.4 Infl uences on urea
i Ur d Ur
• High dietary protein intake
• GI bleeding
• Catabolic states
• Haemorrhage
• Trauma
• Corticosteroids
• Tetracyclines
• Low protein diet
• Liver disease
• Pregnancy
Renal function in the elderly
The kidneys suffer signifi cantly at the hands of the ageing process, with senescence associated with progressive glomerular and tubulointerstitial scarring, nephron loss, declining renal function, and downstream conse-quences for systemic haemodynamics. These changes begin (depressingly) in the 4th decade and hasten during the 5th and 6th, with increasing clinical relevance.
Pathology
Macroscopic features include thinning of the renal cortex and an overall decrease in renal size.
Glomerular
Basement membrane thickening, mesangial matrix expansion, capillary loop changes, periglomerular fi brosis, glomerular hypertrophy, glomeru-losclerosis ( 7 30% by age 80).
Tubular
Dilatation and atrophy (particularly in outer medulla), macrophage infi ltra-tion, and collagen deposition l interstitial fi brosis.
Vascular
Intimal hyperplasia ( l stiffness) of interlobular arteries and afferent arterioles.
Pathophysiology
• Glomerular hyperfi ltration and hypertrophy l glomerular sclerosis and scarring l further glomerular hyperfi ltration and hypertrophy.
• Intrarenal RAS activation l i A2 l intrarenal (including podocyte) injury.
• Endothelial dysfunction l d NO production l hypoxic and ischaemic damage.
• TGF- B overexpression l fi brosis.
• i oxidative stress l tissue damage.
• AGE accumulation.
• Arteriolar hyalinosis l failure of autoregulation l glomerular injury.
• General biological senescence: telomere shortening, mitochondrial loss, and enhanced apoptosis.
Effect on renal function GFR
The Baltimore longitudinal study (1958 – 1981) used serial measurements of CrCl to show a d GFR of 0.75mL/min/year. Recent studies have used inulin clearances to show that, although GFR progressively falls in older age groups ( ♂ > ♀ ), it is not necessarily an inevitability. There may be no demonstrable decline in normotensive, otherwise healthy, individuals, so it remains important to consider the effect associated comorbidities ( i BP, vascular disease, CCF, etc.) may be having over and above age itself.
RENAL FUNCTION IN THE ELDERLY 39
Assessment of GFR
SCr is less useful in the elderly because of (i) d muscle mass and (ii) d urinary Cr excretion. This means that formulae based on SCr (including MDRD, Cockcroft – Gault, and CKD-EPI) tend to underestimate GFR in those age >65 (and are not actually robustly validated in this population).
Clinical relevance
• d muscle mass in the elderly l d SCr.
• 2 i SCr in an elderly patient usually represents a signifi cant d GFR.
• Although age-related d GFR is common anyway, it will be exaggerated in the presence of comorbidity, such as i BP and vascular disease.
• X CKD is common in the elderly:
• In NHANES (1999 – 2004), nearly 7 45% of elderly subjects had CKD ( b p. 194) by current criteria, with those aged >70 accounting for 50% of CKD overall.
• However, is early CKD relevant in this age group? For example, GFR 50 – 59mL/min does not increase CV mortality in comparison to >60mL/min?
• In view of this, many believe CKD is an unhelpful term in the majority of individuals in this age group.
• The incidence and prevalence of ESRD is higher in the elderly (mean age of RRT is mid-60s).
• AKI and acute-on-chronic kidney disease are both more common.
• The elderly are more vulnerable to the effects of nephrotoxic drugs.
• The elderly are also more prone to electrolyte imbalances occurring through disease or inappropriate medical management (e.g.
indisciplined perioperative fl uid therapy).
Additional effects of renal ageing
• d renal plasma fl ow.
• Microalbuminuria and overt albuminuria more common.
• Regulation of Na + balance is impaired ( d excretion l contributes to i BP in the context of a high-salt western diet).
• i K + more common ( d excretion and d aldosterone). The elderly are 6 vulnerable to the effect of any medication interfering with K + excretion (such as ACE-I).
• Water handling (both diluting and concentrating) ability is impaired.
Potential consequences:
• Nocturia is very common.
• Inappropriately dilute urine (and reduced thirst response) despite i plasma osmolality l dehydration.
• Inadequate excretion following water load l hyponatraemia.
• Impaired distal tubular acidifi cation ( l mild acidosis).
• i EPO production (although the increase in response to a low Hb is actually blunted).
• Effects on mineral metabolism: i PTH, d calcitriol, and d PO 4 .