Scand J Urol N e p h r o l 12: 133-137. 1978

DETERMINATION OF G L O M E R U L A R FILTRATION R A T E IN ADVANCED R E N A L INSUFFICIENCY R . J a g e n b u r g . P - 0 A t t m a n . M . Aurell a n d H . B u c h t From thr Dc~pnrtmc,ntso f Cliniccil Chemistry a n d Mcdicinc. V , Univc~raityc?fGothc,nh/rrg. Sohlgren's Ho.spitc11. Gothc~nhirrg.S w d e n

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(Submitted for publication October 28, 1977)

Ahstruc~t.The glomerular filtration rate (GFR) has been determined in 17 patients with advanced renal insufficiency (GFR< 15 ml/min) by different clearance techniques using creatinine. inulin and "Cr-EDTA as filtration markers. With renal inulin clearance a s reference method for GFR. endogenous renal creatinine clearance overestimated G F R by an average of 3 0 F . Renal clearance of "Cr-EDTA and inulin were closely correlated and thus "Cr-EDTA is a suitable G F R marker even at low filtration rates. However, it was found that the plasma clearance of "Cr-EDTA overestimated the G F R often by more than 100% in the range 2.6-11.2 mlimin. Renal clearance measured during 24 h was lower than 4 h renal clearance with the patient well hydrated and resting in bed. It is concluded that the precise measurement of low glomerular filtration rates requires the use of renal clearance techniques. Four-hour 5'Cr-EDTA renal clearance is a suitable method for measuring and following the development of renal function in advanced renal insufficiency.

Knowledge of residual k i d n e y f u n c t i o n in advanced renal disease is of importance b o t h for d i e t a r y management and for individual a d j u s t m e n t of dialysis t r e a t m e n t . I t is also i m p o r t a n t t o d e t e r m i n e t h e limits of f u n c t i o n a l renal c a p a c i t y for successful c o n s e r v a t i v e t r e a t m e n t . T h i s has led t o a g r o w i n g i n t e r e s t in t h e d e t e r m i n a t i o n of t h e residual glomerular filtration r a t e . This s t u d y has been u n d e r t a k e n t o e v a l u t a t e c u r r e n t m e t h o d s for d e t e r m i n i n g t h e glomerular filtrat i o n r a t e in a d v a n c e d renal d i s e a s e .

tions for dietary treatment were presence of uraemic symptoms mainly nausea, anorexia and pruritus. The patients were otherwise in good general condition without signs of overhydration o r malnutrition. Electrolyte and acid-base balance disturbances were present only to a mild degree with serum bicarbonate levels above 20 mmol/l. The progressive course of the functional impairment of the kidneys measured a s inulin clearance in the 9 patients repeatedly investigated is demonstrated in Fig. 1. In addition 2 anephric women on haemodialysis were investigated with plasma "Cr-EDTA-clearance between two dialysis sessions.

METHODS Glomerular filtration rate (GFR) was determined by conventional clearance technique with collection of urine and measurement of plasma and urine concentrations (here called rend clearance). Endogenous creatinine, inulin and "Cr-EDTA were used a s filtration markers. Clearance of "Cr-EDTA was also calculated by determination of the plasma elimination curve after single injection of the marker (here calledplasmu clearance). By this method the clearance value is obtained from the general formula CI=D/A. where D = d o s e of the marker injected and A =area under the plasma elimination curve. R e n d crcwtininc~.inulin und "Cr-EDTA clcurancc~(160

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MATERIAL Seventeen patients ( I I men, 6 women) aged 1 9 4 4 years were investigated. They were studied immediately before, and in some cases repeatedly during treatment with protein-reduced diet and essential amino acids. IndicaReprint requests to Dr Rudolf Jagenburg, Department of Clinical Chemistry, University of Gothenburg, Sahlgrenska sjukhuset, S-413 45 Goteborg, Sweden.

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Fig. I . Changes in G F R in 9 patients with repeated investigations.

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RENAL INULIN CLEARANCE (160 m i n t m l l m l n

Fig. 2 . Relation between serum creatinine and G F R nieastired a s renal inulin clearance (I60 min).

Fig. 4 . Correlation between renal "Cr-EDTA clearance (I60 min) and renal inulin clearance (160 min).

niin). The renal clearances of creatinine. inulin and 51CrEDTA were determined in the morning during a period of I60 min (4 periods of 40 min duration) with the patients resting in bed. Thc patients were hydrated with 15-20 ml/kg body weight of water before start of the clearance procedure and water was then given to compensate for diuresis which averaged 3-5 ml/min. N o food was given for I2 h preceding the investigation but the patients were allowed to drink freely. Bladder catheters were not used. The collection of urine was started 40 min after the intravenous administration of inulin (Intitesta. 0.2 ml/kg body weight of a 25% solution) and " 0 - E D T A (50-200 pCi, Behringwerke, A . (3.1. Blood samples were drawn 60. 100. 140 and 180 min after the intravenous injection, i.e. in the middle of each clearance period. Renul creurinine und "Cr-EDTA clearance (24 h ) . The renal (24-hour) clearances of creatinine and "Cr-EDTA were determined in the time interval 4-28 hours after the

injection of "Cr-EDTA. The clearance was determined from the plasma creatinine concentration at the beginning of the clearance period and from the mean T r - E I X " concentration obtained from the area under the plasma concentration curve of "Cr-EDTA. The patients were in hospital but not in bed during daytime. Extreme care was taken to ascertain that no urine was lost during the investigation. Plustnn "'C'r-EDTA cleuruncc ( 3 4 11) was determined from the dose of the marker and the plasma elimination curve with blood sampling 180, 200. 220 and 240 min after injection of the marker. The clearance value was then obtained as doselarea under the elimination curve as described by Brochner-Mortensen e t al. (1972). Plusmu "Cr-EDTA cleurunce (4-28 11) was also determined by the equation given by Brochner-Mortensen (1972) but with blood sampling o n only two occasions. 4 h and 28 h after injection of the marker.

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RENAL CLEARANCE (160 rnin) rnllrnin RENAL INULIN CLEARANCE (160 m i n ) m l l m i n

Fig. 3 . Correlation between renal creatinine clearance (I60 min) and renal inulin clearance (I60 min).

Fig. 5 . Correlation between renal clearance (24 h) and

and "Cr-EDTA renal clearance (I60 min) of creatinine (0) (0).

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Fig. 6 . Correlation between plasma clearance (3-4 h) and

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Assay

CreafinincJ was determined by a picrate method (Helger, Rindfrey & Hilgenfeldt, 1974) without precipitation of proteins using an automated system (Vickers M300). The urine was diluted before analysis to give a concentration close to that of the corresponding plasma ( + 2 0 % ) . Inirlin was determined by the method of Hubbard & Loomis (1942). At low renal function undiluted urine or urine diluted 1 : 2 was analysed and this made a blank correction necessary for urinary compunds reacting with the resorcin reagent. As a blank, the urine collected immediately before the injection of inulin was used. However, a s the blank value varied with the concentration of the urine, a correction was made on the basis of the different creatinine concentrations of the samples. "Cr-EDTA was determined in a well scintillation counter (Selektronic. Denmark) in a sample volume of 3 ml and 30 min counting time.

Renal creritininr c~1eurunc.r(160 min) overestimated G F R by on average 30% within the whole range studied (Fig. 3). R e n d "'Cr-EDTA cleurcincc~ (160 min) was closely correlated to the inulin clearance and no systematic deviation was observed (Fig. 4). R e n d crrutinine cleurcinc~c (24 h ) und rcwil J'Cr-EDTA clearunce (24 h ) were poorly correlated to the corresponding clearance determinations performed in the morning when the patients were well hydrated and resting in bed (renal clearance, 160 min), and gave u p to 5 5 % lower values (mean 37%) (Fig. 5). Plusmu "Cr-EDTA clearrrnce w r s u s rcwul "CrEDTA cleurunce. The plasma "Cr-EDTA clearance ( 3 4 h) overestimated the G F R by on average 4.2 ml/min in the range 2.6-1 1.2 ml/min (mean 6.5 ml/min) (Fig. 6). The precision of the plasma clearance method, was not much improved when the precision of the "Cr-EDTA determinations was increased by extending the counting time. However, the plasma and the renal clearances were more closely correlated when they were studied in the time interval 4-28 h after the injection of "CrEDTA (Fig. 7). Plasmu chwrance of " 0 - E D T A in unephric putirnts. Plasma clearance was calculated from the "Cr-activity in plasma at different times after injection of the marker in 2 anephric patients (Table I). The calculated clearance was dependent on the time interval after injection of the marker. Calculations of the elimination in the time interval 1 6 2 4 h after injection of the marker indicated an extrarenal clearance of approximately 2 ml/min.

RESULTS The renal inulin clearance (160 min) was chosen as reference method for the glomerular filtration rate (GFR) measurement. Serum creatinine. As expected, the correlation between serum creatinine and GFR was poor (Fig. 2 ) . A serum creatinine concentration of around 1 000 pmol/l was found at G F R values between 3 and 12 ml/min, and at a G F R of 5-6 ml/min a variation in the serum creatinine concentration between 5W1500 pmol/l was observed. The correlation between serum creatinine and G F R in a single individual was better than in the total material but the changes in the serum creatinine concentration were still of limited value for predicting the changes in GFR.

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Fig. 7. Correlation between plasma clearance (4-28 h) and

renal clearance (24 h) of SICr-EDTA. Sciind J Urol N e p h r d

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DISCUSSION Recent advances in both conservative treatment and dialysis management of patients with advanced renal insufficiency have led to a demand for reliable and convenient methods for measuring residual renal function. Changes in G F R constitute the most important prognostic parameter for the patient with non-oliguric renal failure. Hence, it is important to determine the G F R as accurately as possible particularly for scientific evaluation of different therapeutic measures and to characterize different stages of uraemia. Repeated investigations of G F R in advanced renal insufficiency will also aid in the differential diagnosis between progression of original renal disease and potentially reversible decrements of renal function and permit more objective assessment of the efficacy of the therapeutic measurements employed. Mere measurement of serum creatinine is not a reliable guide to GFR, as is amply demonstrated in the present study. Because of its supposed simplicity, endogenous 24 h creatinine clearance is still widely used for routine GFR determinations, even in advanced renal failure. However, there are several objections to the reliability of this test, apart from the well-known difficulties in urinary collection, e.g. the considerable daily variation in creatinine excretion (Hierholtzer, Butz & Baetke, 1972). Endogenous 24 h creatinine clearance has often been reported to overestimate G F R as measured by inulin clearance, but the ratio of creatinine clearance to inulin clearance is so variable within and between individuals that endogenous creatinine clearance does not allow accurate determination of GFR in advanced renal insufficiency even under supervised hospital conditions (Enger & Blegen, 1964; Hierholtzer et

al.. 1972: Hood, Attman. Ahlrnen & Jagenburg, 1971; Mertz, 1972; Skov, 1970). This view is strongly supported by the data presented here. It has been suggested (Lubowitz. Slatopolsky. Shankel, Rieselbach & Bricker, 1967) that the mean of urea and endogenous creatinine renal clearances performed over one hour would give results similar to that of renal inulin clearance in advanced renal insufficiency. There are several objections to this method from theoretical and methodological points of view and we see no advantage in using the method described by Lubowitz et al. (1967). Thus, precise determination of G F R necessitates the adminstration of filtration marker also in advanced renal insufficiency. Inulin remains the standard marker and the previous objections to its use in renal insufficiency have not been validated, as reviewed by Hierholtzer et al. (1972). The determination of inulin is. however, cumbersome and the use of radioisotope-labelled markers is much more convenient for clinical work and also adds to the accuracy of determination when measuring low clearance values. “Cr-EDTA has been found to fulfil the criteria for a glomerular filtration marker (Aurell & Ditzel, 1970: Brochner-Mortensen & Rodbro. 1976: Chantler. Garnett, Parsons & Veall, 1969: Garnett, Parsons & Veall. 1967, Stacy & Thorburn, 1966). Our study confirms that “CrEDTA is an adequate filtration marker behaving as inulin even in the very low filtration range. Thus. it can serve as a substitute for inulin in conventional clearance technique throughout the whole range of glomerular filtration rate. Radioisotope-labelled substances are also particularly suited for plasma clearance measurements using the plasma disappearance curve, thereby obviating the need for urinary collection. This clearance value is an expression of the total plasma clearance of the marker. However. the plasma clearance is not necessarily identical with the renal clearance as an extrarenal elimination of the marker may exist. We have found that the extrarenal clearance of ”Cr-EDTA amounted to 2 4 ml/min as demonstrated by determination in anephric patients and by the difference between renal and plasma clearances of ”Cr-EDTA. Similar results for patients with normal o r moderately reduced GFR have been reported previously (Aurell & Ditzel. 1970; Brochner-Mortensen, Giese & Rossing, 1969; Brochner-Mortensen & Rodbro, 1976). This differ-

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ence is of little importance with normal o r moderately impaired renal function, but is of course important in advanced renal insufficiency. The use of the disappearance curve for estimating clearance in advanced renal failure may raise a question of methodology. i.e. the adequate measurement of the final slope of the plasma disappearance curve. The slope of the curve is so small, that determination of the disappearance rate is difficult. Thus, it is ciear that the plasma clearance of ”Cr-EDTA, whether it is determined over a few hours o r over 24 hours, is not suitable for a precise determination of very low GFR. It is also worth pointing out that little is known about the interindividual o r day-to-day variations in the extrarenal elimination of “Cr-EDTA in advanced renal failure. Our results suggest that the variations may be greater than previously assumed (Brochner-Mortensen & Rodbro. 1976; Chantler et al.. 1969; Fiegel & Hoffler, 1972). Our data show that 24 h renal clearance gives lower values for GFR than 4 h renal clearance in the morning with the patient well hydrated and resting in bed, the difference averaging 20%. This may be due to several factors, such as variation in renal perfusion with posture and activity and the degree of hydration. This variation in G F R during the day is also important when discussing methods for determining G F R in advanced renal insufficiency. CONCLUSIONS We conclude that only renal clearances measured with external G F R markers are reliable for the accurate determination of G F R in advanced renal failure. 51Cr-EDTA can replace inulin as filtration marker. Four-hour renal clearance in the morning in a well hydrated patient gives the most reliable results while 24 h renal clearance gives lower values for GFR. Plasma clearance is an unreliable method for determination of low GFR. Four-hour renal clearance with 5’Cr-EDTA is well suited for measuring and following the development of renal function in advanced renal insufficiency.

infusion and single injection techniques. Proc. VII Int. Congr. Clin. Chem., GenevalEvian, vol. 111. p. 405. 1970 (Karger. Basel/Munchen/Paris/New York). Brochner-Mortensen. J . 1972. A simple method for the determination of glomerular filtration rate. Srrrncl J Clin Lab Invest 30, 271. Brochner-Mortensen, J . , Giese. J . & Rossing, N . 1969. Renal inulin clearance versus total plasma clearance of “Cr-EDTA. Scund J Clin Lab Inr3Pst 2 2 . 301. Brochner-Mortensen, J . & Rddbro, P. 1976. Comparison between total and renal plasma clearance of “CrEDTA. Scand J Clin Lub Invest 36, 247. Chantler, C . , Garnett. E. S., Parsons, V. & Veal], N. 1969. GFR-measurement in man by the single injection method using W r - E D T A . Clin Sci37. 169. Enger, E. & Blegen, E . M. 1964. The relationship between endogenous creatinine clearance and serum creatinine in renal failure. S a n d J Clin Lob Invest 16, 273. Fiegel. P. & Hoffler. D. 1972. The measurement of reduced glomerular filtration rate with the “Cr-EDTA single shot clearance. In Uremia (ed. R. Kluthe. R. Berlyne and B. Burton), p 126. Georg Thieme. Stuttgart . Garnett, E. S.. Parsons. V. & Veal]. N. 1967. Measurement of G F R in man using a T r - E d e t i c acid conplex. Lancef i , 818. Helger, R., Rindfrey. M. & Hilgenfeldt, J. 1974. Eine Methode zur direkten Bestimmung des Creatinins in Serum und Harn ohne Enteiweissung nach einer modifizierten Jaffe Methode. Z Klin Chem Klin Biochc,m 12, 344. Hierholtzer. K., Butz, M. & Baetke, R. 1972. Evaluation of measurement of reduced glomerular filtration rate in the severely diseased kidney. In Uremia (ed. R. Kluthe, R. Berlyne and B. Burton). p. 98, Georg Thieme. Stuttgart. Hood, B., Attman, P. O., Ahlmen, J. & Jagenburg, R. 1971. Renal hemodynamics and limitations of creatinine clearance in determining filtration rate in glomerular disease. S c a n d J U r o l Nephrol.5. 154. Hubbard. R. S. & Loomis. T. A. 1942. The determination of inulin. J Biol Chem 145. 641. Lubowitz, H., Slatopolsky, E . , Shankel, S., Rieselbach, R. E. & Bricker, N . S . 1967. Glomerular filtration rate. J A M A 199, 100. Mertz. D. P. 1972. Value of clearance tests in advanced renal failure. In U r e m i a (ed. R. Kluthe, R. Berlyne and B. Burton). p. 108, Georg Thieme, Stuttgart. Skov, P. E. 1970. Glomerular filtration rate in patients with severe and very severe renal insufficiency. Actu Med Scund 187. 419. Stacy, B. D. & Thorburn, G . D. 1966. Cromium ‘lethylenediamine-tetraacetate for estimation of glomerular filtration rate. Science 152. 1076.

REFERENCES Aurell, M. & Ditzel, J. 1969. Renal clearance of J’CrEDTA complex. A comparison between continuous

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