Effect of Mannitol on Glomerular Ultrafiltrationin the Hydropenic Rat

Roland C. Blantz

J Clin Invest. 1974;54(5):1135-1143. https://doi.org/10.1172/JCI107857.

The effect of mannitol upon glomerular ultrafiltration was examined in hydropenic Munich-Wistar rats. Superficial nephron filtration rate (sngfr) rose from 32.0±0.9 nl/min/g kidney wt to42.0±1.6 (P < 0.001) in eight rats. Hydrostatic pressure gradients acting across theglomerular capillary (ΔP) were measured in glomerular capillaries and Bowman's spacewith a servo-nulling device, systemic (pA) and efferent arteriolar oncotic pressures (pE)were determined by microprotein analysis. These data were applied to a computer-basedmathematical model of glomerular ultrafiltration to determine the profile of effective filtrationpressure (EFP = ΔP — p) and total glomerular permeability (LpA) in both states. Filtrationequilibrium obtained in hydropenia (LpA ≥ 0.099±0.006 nl/s/g kidney wt/mm Hg) and sngfrrose because EFP increased from a maximum value of 4.2±1.1 to 12.8±0.5 mm Hg aftermannitol (P <0.01). This increase was due to both increased nephron plasma flow anddecreased pA. Computer analysis of these data revealed that more than half (>58%) of thisincrease was due to decreased pA, consequent to dilution of protein. Since EFP wasdisequilibrated after mannitol, LpA could be calculated accurately (0.065 ± 0.003 nl/s/gkidney wt/mm Hg) and was significantly lower than the minimum estimate in hydropenia.

Therefore, sngfr does increase with mannitol and this increase is not wholly dependentupon an increase in nephron plasma flow since the major factor […]

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Effect of Mannitol on Glomerular Ultrafiltration

in the Hydropenic Rat

ROLANDC. BLANIZ

From the Departments of Medicine, University of California and VeteransAdministration Hospital, San Diego, California 92161

A B S T R A C T The effect of mannitol upon glomerularultrafiltration was examined in hydropenic Munich-Wistar rats. Superficial nephron filtration rate (sngfr)rose from 32.0±:0.9 nl/min/g kidney wt to 42.0±1.6(P < 0.001) in eight rats. Hydrostatic pressuregradients acting across the glomerular capillary (AP)were measured in glomerular capillaries and Bowman'sspace with a servo-nulling device, systemic (IrA) andefferent arteriolar oncotic pressures (7rE) were deter-mined by microprotein analysis. These data were ap-plied to a computer-based mathematical model ofglomerular ultrafiltration to determine the profile ofeffective filtration pressure (EFP = AP - 7r) and totalglomerular permeability (LpA) in both states. Filtra-tion equilibrium obtained in hydropenia (LpA )0.099± 0.006 nl/s/g kidney wt/mm Hg) and sngfrrose because EFP increased from a maximum value of4.2±t 1.1 to 12.8±0.5mm Hgaftermannitol (P <0.01).This increase was due to both increased nephron plasmaflow and decreased 7rA. Computer analysis of thesedata revealed that more than half (> 58%) of thisincrease was due to decreased WA, consequent to dilu-tion of protein. Since EFP was disequilibrated aftermannitol, LpA could be calculated accurately (0.065± 0.003 nl/s/g kidney wt/mm Hg) and was signif-icantly lower than the minimum estimate in hydropenia.

Therefore, sngfr does increase with mannitol and thisincrease is not wholly dependent upon an increasein nephron plasma flow since the major factor increas-ing EFP was decreased 7rA

INTRODUCTION

There is evidence from both the reijal physiologylaboratory (1-4) and the clinical experience (5, 6)

This work was presented in part at The 6th Annual Meet-ing of The American Society of Nephrology, Washington,D. C., November 1973 and published as an abstract in the1973 Abstracts of the American Society of Nephrology; 6:12.

Received for publication 12 February 1974 and in revisedform 6 June 1974.

that osmotically active agents such as mannitol canaugment or maintain the rate of glomerular filtrationin both normal and hypotensive animals. Therefore,these agents may promote or restore urinary excretionof water and electrolytes through activities both as anosmotic diuretic within the tubules (7, 8) and byincreasing or maintaining the load of filtrate to eachnephron.

Recent studies which directly evaluate the factorsgoverning filtration (9, 10) suggest that glomerularfiltration rate may be increased by four potentialmechanisms: (a) an increase in plasma flow, (b) anincrease in the hydrostatic pressure gradient (AP)'acting across the glomerular capillary membrane, (c)and increase in total glomerular permeability (LpA)(effective only if filtration equilibrium is not attained),and (d) a decrease in the systemic oncotic pressure(WA). Previous studies examining the mechanism ofaction of osmotically active agents favor the first twoexplanations, increased plasma flow and increased AP,both of which could be mediated by a reduction inafferent arteriolar resistance (AR) (3, 11-14). However,mannitol appears more effective in restoring glomerularfiltration than other agents which possess the capacityto vasodilate the renal vasculature (13), and this

1 Abbreviations used in this paper: AR, afferent arteriolarresistance (mm Hg/nl/min/g kidney wt); C, protein con-centration; CA, systemic protein concentration; CE, efferentpertibular capillary protein concentration; AIP, hydrostaticpressure gradient across glomerular capillary (AP = PG - Pt)(mm Hg); EFP, effective filtration pressure (EFP = AP - r)(mm Hg); EFP, mean EFP; EFPA, afferent EFP; EFPE,

efferent EFP; LpA, total glomerular permeability (60 s/min-LpA = sngfr/EFP) (nl/s/g kidney wt/mm Hg); PG, glomeru-lar capillary hydrostatic pressure (mm Hg); Pt, Bowman'sspace or proximal tubular hydrostatic pressure (mm Hg); ir,oncotic pressure (mm Hg); TrE, efferent arteriolar pressure;7rA, systemic oncotic pressure; QO, rpf, nephron plasma flow(nl/min/g kidney wt); snbf, single nephron blood flow; snff,

superficial nephron filtration fraction; sngfr, nephron filtra-tion rate (nl/min/g kidney wt); x*, normalized glomerularcapillary length (dimensionless parameter).

The Journal of Clinical Investigation Volume 54 November 1974* 1135-1143 1135

restoration can occur in clinical situations in whichincreases in renal blood flow are severely limited.

Utilizing recently developed microtechniques fordirect analysis of both the hydrostatic and oncoticforces which determine the rate of glomerular ultra-filtration (15, 16), we have examined the specificmechanisms whereby mannitol exerts its influence uponthe kidney (17). When data was obtained and analyzedby a computer-based mathematical model of glomerularultrafiltration (16), a reduction in IrA attendant to theinfusion of mannitol, was the major and most consistentfactor causing a rise in filtration rate in superficialnephrons of the rat.

METHODS

Micropuncture studies were performed upon male rats ofthe Munich-Wistar strain which were grown in a colony atthe San Diego Veterans Administration Hospital in La Jolla,Calif. Original stock for this strain were obtained from Dr.Klaus Thurau of the Physiologic Institute of Munich. Theweight of the rats ranged between 150 and 225 g at the time ofmicropuncture and were maintained upon standard PurinaRat Chow diets (Ralston Purina Co., St. Louis, Mo.) andad lib water intake until the time of micropuncture. Theunique value of this strain of rats is that they possess severalglomeruli directly on the surface of the kidney, immediatelyunder the capsule, which are accessible to micropuncturetechniques and measurement of pressure within the glomerularcapillaries.

The animals were anesthetized with Inactin (100 mg/kgi.p., Promonta, Hamburg, West Germany). A tracheostomytube (PE 240) was inserted. Polyethylene catheters were thenplaced in the left jugular vein and left femoral artery for boththe infusion of solutions and the monitoring of blood pressure.Blood pressure was monitored with a Statham P 23dB pres-sure transducer and recorded on a Statham four-channel re-corder (Statham Instruments, Inc., Oxnard, Calif.). A bladdercatheter was placed (PE 50) to collect urine from the rightkidney. The animal was then turned onto the right side anda left subcostal flank incision was made. The animal's tem-perature was constantly controlled on a heated micropunctureboard by a servo-controlled thermoregulator at approximately37.50-380C utilizing a rectal thermister probe. After the inci-sion the perirenal fat and connective tissue were carefullydissected from the left kidney and the adrenal gland dissectedfree. The kidney was then placed in a Lucite cup with caresuch that the pedicle was not stretched. A PE-50 catheterwas inserted into the left ureter near the pelvis of the kidneyto collect urine from the micropuncture kidney. The Lucitecup was then lined and the kidney surrounded loosely withcotton. Clear agar at 370 to 39°C was then placed around thekidney leaving the dorsal surface exposed. The kidney wasthen covered with heated 37°C isotonic NaCl-NaHCO3 solu-tion. At the completion of surgery, [14C]inulin (New EnglandNuclear, Boston, Mass.) was infused at a rate of 40-50 uCi/hfor the measurement of glomerular filtration rate. There wasno attempt to replace surgical losses, but the infusion ofisotonic NaCl-NaHCO3 was maintained at approximately0.5% body wt/h.

In the control situation after equilibration of the inulinmarker, glomerular capillary pressures, pressures in Bowman'sspace and proximal tubules were measured with the servo-nulling pressure sensor device (Instrumentation for Physiologyand Medicine, San Diego, Calif.). The specific operation of the

servo-nulling device has been described in detail in a previouspublication (9). The basic principle is that of an electricalWheatstone bridge, one arm of which is a micropipette witha 1-pum (OD) tip. It is filled with 1 MNaCl. Both during thecontrol hydropenic periods and after the institution of themannitol infusion, pressures were measured in all glomerularcapillaries accessible on the surface of the kidney. Bowman'sspace pressures (Pt) were measured in the same nephron asthe glomerular capillary pressure (PG), and these valuesutilized to calculate the AP in each state. The validity of thismethod has been discussed in detail in a previous publication(9), but it should be restated that if evidence of disruption ofthe glomerular capillary was noted as evidenced by bleedingeither into Bowman's space or onto the surface, the glomeruluswas rejected and the pressure measurement not utilized.

After measurement of PG and Pt at least three efferentarterioles were localized and punctured with 14-16-pum (OD)coated pipettes and samples of blood were obtained. At leastsix sngfr were obtained in six separate nephrons utilizingtechniques previously described (9, 16). Total glomerularfiltration rate was estimated from the count rate of [14C]-inulin from both left and right kidneys in at least two periodsin both the control and experimental states. After the measure-ment of sngfr and conclusion of clearance periods, a sample ofleft renal venous blood was obtained with a heparinizedmicropipette (35-pum OD tip). This blood sample was utilizedto determine the extraction of inulin across the kidney andthereby kidney filtration fraction.

During hydropenia, it has been demonstrated by severalauthors that superficial nephron filtration fraction (snff) isequal to total kidney filtration fraction (18-20). Also in arecent study from this laboratory, snff was nearly identicalto the total kidney filtration fraction in hydropenic rats (16).Wehave therefore elected to calculate efferent protein con-centration (CE) from the total kidney filtration fractionduring hydropenia. To determine whether filtration equili-brium occurs, it is imperative to define the filtration fractionand the CE by the method with the smallest possible error.

After completion of the control hydropenic periods, a solu-tion of 10 g/100 ml mannitol in 0.45% NaCl was infused at arate sufficient to deliver a volume equal to 1.5% body wt overa period of 20 min. A stable maximum urine flow was obtainedusually within 10 min such that the net addition of fluid tothe animal was usually less than 1 ml total. At the end of the20-min period, the infusion rate was readjusted (Sage Instru-ments Div., Orion Research, Inc., Cambridge, Mass.) to equalthe rate of urine output throughout the remainder of theexperimental period. All measurements that were obtainedin the control periods were then repeated during the continuousinfusion of mannitol. Total glomerular filtration rates andtotal renal extraction of inulin were also evaluated duringthe infusion of mannitol.

Analytical methods. Urine was collected in preweighedcontainers under oil. Arterial plasma samples were collectedin heparinized capillary tubes. Arterial blood samples forprotein determinations were collected in presiliconized glasscapillary tubes. 14C counts in plasma, urine, venous, andtubular fluid samples were monitored on a Packard liquidscintillation counter (model 2425, Packard Instrument Co.,Inc., Downers Grove, Ill.) and total glomerular filtration rate,renal plasma flow, and total filtration fraction calculated aspreviously described (16, 21). Nephron filtration rate (sngfr= UV/P) was calculated as the total count rate collectedper minute (UV) divided by the plasma count rate (P)(corrected for plasma water).

The microprotein determinations were performed bymodification of the Lowry protein method (22) similar to that

1136 R. C. Blantz

described by Brenner, Falchuk, Keimowitz, and Berliner (23)and as previously described in modified form from thislaboratory (16). Microcapillary pipettes were specially coatedto prevent clotting of blood (Dow Corning 1107 [DowCorning Corp., Midland, Mich.], 2.5% in trichloroethyleneand then baked at 200'C for 3 h). The peritubular capillaryblood sample was sealed at the tip with Eastman 910 (East-man Chemical Products, Inc., New York) and red cellsseparated from the plasma by centrifugation. At least three7-nl plasma samples were obtained from each of three collec-tions and each sample run in triplicate. Three 7-nl sampleswere taken from the aortic blood collection obtained con-currently with the peritubular capillary samples and alsoeach sample read in triplicate. Concentrations from thesesamples were estimated by comparison with a standard curveof known protein concentrations handled in identical fashionwith 7-nl plasma protein samples. The standard curve wasdetermined by linear regression analysis to best fit for acurve utilizing all standard readings. Correlation coefficientfor the standard curves was usually greater than r = 0.99 andalways greater than 0.98. As a further check on proteinvalues, another modification of Lowry protein method wasperformed on all aortic samples which utilized 2-.ul plasmasamples. Samples were rejected if obvious hemolysis occurred.Concentrations of mannitol up to 12.5 g/100 ml had no effectupon the protein determination by this method.

Urine and plasma sodium concentrations were determinedon an Instrumentation Laboratory flame photometer (In-strumentation Laboratory, Inc., Lexington, Mass.). Urineosmolalities were measured on an Advanced Instrumentosmometer (Advanced Instruments, Inc., Needham Heights,Mass.)

Calculations. snff was calculated as follows:

snff = 1 - CA/CE,

where CA = systemic protein concentration and CE equalsefferent peritubular capillary protein concentration. Singlenephron plasma flow (rpf) is calculated as follows:

rpf = sngfr/snff.

Single nephron blood flow (snbf) is calculated as follows:

snbf = rpf/(1 - hct),

where hct equals hematocrit as a fraction of 1. AR is cal-culated as follows:

AR = MAP- PG/snbf,

where MAP= mean arterial blood pressure in millimeters ofHg and PG equals glomerular capillary hydrostatic pressure.

The relationship between protein concentration (C) andoncotic pressure (7r) was described by Landis and Pappen-heimer (24), where:

r = 2.1C + 0.16C2 + 0.009C3,

(r) is expressed in millimeters (Hg). Wehave simplified thisexpression for use in the mathematical calculations by a bestfit analysis to the Landis-Pappenheimer equation by leastsquares using well-established computer methods. (Programson file at the University of California at San Diego ComputerFacility.) where:

7r = 1.736C + 0.281C2 (reference 16).

The above coefficients for Cand C2 are utilized in all calcula-tions in this study during hydropenia and mannitol. Wehave

60 -

50 -

40 -._

_ 30 -

20 -

10o

SNGFR 300 -1

250 -

200 -

50 -

100 -

50 -

HYDROPENIA MANNITOL

RPF

1584±9.8

HYDROPENIA MANNITOL

FIGURE 1 On the left is demonstrated as the mean sngfr ineach of the 11 studies in hydropenia and during mannitolinfusion. The values are expressed as the mean of each animalmean value ±SE. On the right is depicted the rpf and theresponse to mannitol in each animal. The variation in responseto mannitol is greater with rpf than sngfr.

clearly demonstrated that total protein is approximately 50%albumin during hydropenia and it is assumed that the infusionof mannitol does not alter this distribution of total proteinbetween globulin and albumin (16).

Determination of the profile of EFP profile along the lengthof the glomerular capillary (x*), where EFP = AP -7r,requires a method of generating this curve accurately andintegrating the curve to define a EFP. A value for total LpAcan also be calculated by this technique (sngfr = LpA-EFP).Deen, Robertson, and Brenner have described such a set ofequations (10). We have also developed a method utilizingiterative techniques to determine the EFP profile and calculateLpA. Wehave calculated EFP and LpA by both methods inthis study and utilized the iterative method to separate andquantitate the individual factors which act to increase thesngfr. Data required for such calculations are the CA and CE,rpf, and the AP. The iterative method is described in detailin the accompanying Appendix.

Statistical analysis. Calculation of the EFP profile andLpA were conducted for each animal. Values for AP, rpf,CA/CE, and sngfr were analyzed by analysis of variance (25).Animal mean values for these computer input data were carriedthrough the mathematical calculations were their estimatesof variance to define the exact variance of both EFP and LpAin each animal and overall. Statistical analyses of this typeare also required to determine with confidence if filtrationequilibrium occurs of if EFPE was significantly positive.

RESULTS

With the infusion of mannitol, urine volume increasedfrom 1.5+40.1 yl/min in hydropenia to 70.2+6.4. Serumsodium concentration was 133+2 meq/liter, potassium5.0+40.3 meq/liter, and osmolality 2734±5 mosmolafter mannitol.

Effect of mannitol on the sngfr. Mannitol had aconsistent and profound effect upon the sngfr in hydro-penic animals, increasing the filtration rate from 32.0+0.9 (n = 47) to 42.0+41.6 nl/min/g kidney wt(n = 46) (P < .001). The results of filtration rates aredisplayed in Fig. 1 and Table I as a mean value for each

Effect of Mannitol on Glomerular Ultrafiltration 1137

TABLE I

sngfr and rpf before and after Mannitol Infusion

Hydropenia MannitolRatno. sngfr rpf sngfr rpf

(nl/min/g (nl/min/g (nl/min/g (nl/min/gkidney wt kidney wt kidney wt kidney wt

9 27.84-1.7* 76.8 44.6 50.5 ±2.8 265.0±4 15.1

10 29.5 42.6 78.1±-6.8 39.7 41.7 158.7 46.6

13 25.4±1.3 87.5 ±4.4 29.7 ±1.7 57.1±-3.2

17 34.3 ±3.0 103.3 ±8.9 38.9 ±2.5 204.3 ±13.3

2 1 36.8 ±-1.9 116.7 i6.0 51.3±43.9 116.6 ±-8.9

22 30.7 ±2.1 85.9 ±5.9 39.1 ±3.1 166.7 ±13.4

26 36.4 ±1.5 89.7 ±3.7 47.7 ±1.8 207.5 ±7.6

27 34.6±2.2 86.6i5.6 36.0±1.9 97.3±5.1

Overall 32.0 ±0.9 90.7 ±2.7 42.01 ± 1.6 158.4$4±9.8mean

(n = 47) (n = 47) (n = 46) (n = 46)

* SEM.:P <0.01.

animal. Sngfr is expressed per gram kidney weight tonormalize the variation between animals of varyingsize.

Hydrostatic factors affecting glomerular filtration. PGduring hydropenia was 43+ 1.0 (n = 23) and rose to47.2+1.6 mm Hg (n = 17) after the infusion ofmannitol (P < 0.01).2 The AP between the glomerularcapillary and Bowman's space was 30.5± 1.1 (n = 23)during hydropenia and 28.7±41.4 mmHg (n = 17)after mannitol infusion (P > 0.05). The individualvalues for PG and AP in each animal are depicted inTable II. Therefore the AP across the glomerular capil-lary does not contribute to the increase in sngfr.Tubular pressure rose from 12.8+0.9 to 19.1+0.9 mmHg after the infusion of mannitol (P < 0.001).

Hemodynamic effects of mannitol infusion. Singlerpf increased from 90.7+2.7 (n = 47) to 158.4+9.8nl/min/g kidney wt (n = 46) (P < 0.01). Although astatistically significant rise, the variation in the increaseof plasma flow was rather marked from animal toanimal (Fig. 1), in that rpf did not increase in anappreciable number of animals. Snbf increased from

2 Wehave also conducted parallel estimates of PG by stopflow technique (PSFP = SFP + 7rA) (9) in these same animalsin both hydropenia and mannitol. PSFP was significantlyhigher than the corresponding PG in hydropenia at 51.2±41.2mmHg (n = 30) (P < 0.001), and PSFP was 49.44±1.2 mmHg (n = 33) after mannitol infusion (P > 0.1). Because ofour greater confidence in the validity of direct PG measure-ments, only these values were used in this study. In a pre-vious study, were also noted that PSFP in separate nephronswere higher than PG in hydropenia but noted identical valueswhen measured in the same nephron (9). The specific reasonfor this disparity is not known.

204+7 to 312+19 nl/min/g kidney wt (P < 0.01).Hematocrit during hydropenia was 55+1 and fell to49+1 volume % (P < 0.01) after the infusion ofmannitol.

The arterial pressure in hydropenia was 134+5 mmHg and after mannitol infusion was 125+-6 (P > 0.3).Nephron AR was 0.43+0.03 mmHg/ml/min/g kidneywt in hydropenia and was 0.28+t0.04 (n = 8) after theinfusion of mannitol (P < 0.02).

Influence of mannitol infusion upon C and r. CAwas 5.6+0.1 g/100 ml during hydropenia and fell to4.1+0.2 g/100 ml after the infusion of mannitol. 7rAtherefore fell from 18.3+0.6 to 11.9+0.6 mm Hg(P < 0.001). The individual changes in TrA for eachanimals are demonstrated in Fig. 2. ir fell consistentlyafter mannitol in each study. CE was 8.6+-0.2 inhydropenia and was 6.0+t0.3 g/100 ml after mannitolinfusion. 7rE generated was 35.7+ 1.4 in hydropeniaand 20.7+ 1.7 mmHg after mannitol (P < 0.001).

Mean snff did not change as a result of the infusionof mannitol 0.35+0.01-0.30+0.04, (P > 0.4). However,in those animals in which rpf increased substantially,snff tended to fall, and in those animals in which rpfalterations were minimal or did not change, there wasan increase in snff. In summary, although sngfr rose inmost animals, the increase in plasma flow was quitevariable from animal to animal and was not a consistentdetermining factor in the increase in filtration rate.

Evaluation of the integrated EFP and LpA before andafter mannitol infusion. In hydropenia the EFP at theafferent end of the glomerular capillary (EFPA) was12.1+ 1.5 mmHg. The 7rE for each animal were com-pared with the mean AP and were not statisticallydifferent (P > 0.05) suggesting that filtration pressureequilibrium was attained. However, the mean efferenteffective filtration pressure (EFPE) among animals was-5.3+2.0 mmHg, a value less than SD from zero andtherefore not different from zero. This negative valueis of course biologically unrealistic and represents therange of errors in 7rE and AP when analyzed as the meanof each animal. Since a smaller number of PG observa-tions was obtained in certain studies (Table I), we havealso analyzed EFPA and EFPE as the mean of all ob-servations (n = 23) and they are 12.4+ 1.2 (EFPA)and -3.8+ 1.3 mmHg (EFPE). At filtration pressureequilibrium an infinite number of values for EFP mayexist and only a maximum estimate can be determined.Also LpA cannot be calculated uniquely under theseconditions, but rather a minimum possible value.Maximum EFP in hydropenia was4.2+1.1 mmHg andthe minimum possible LpA 0.099+0.006 nl/s/g kidneywt/mm Hg. When these values were determined fromall values of AP, maximum possible EFP was 5.3+ 1.0mmHg and LpA 0.106+0.011 nl/s/g kidney wt/mmHg (Table III).

11.38 R. C. Blantz

TABLE IIPG and 7r during Hydropenia and after Mannitol Infusion

Hydropenia Mannitol

Rat no. PG AP rrA WE EFPE Rat no. PG AP 'rA rE EFPE

mmHg mmHg mmHg mmHg mmHg mmHg mmHg mmHg mmHg mmHg51.241.742.145.334.636.9

Mean 42.0

41.635.242.7

Mean 39.8

41.641.650.4

Mean 44.5

40.030.530.934.123.425.7

30.8

28.824.732.2

28.6

28.829.639.3

32.6

43.1 25.443.6 26.4

Mean 43.4 25.9

40.0 24.543.2 27.745.6 30.1

Mean 42.9 27.4

44.345.4

Mean 44.8

44.854.440.0

Mean 46.4

33.234.3

33.8

29.643.229.6

34.1

9

Mean

47.144.037.243.2

42.9

29.626.519.725.7

25.4 11.3 15.2 +10.2

16.2 30.6 +0.2

10

18.4 35.7 -7.1Mean

13

20.3 34.7 -2.1 Mean

17Mean

55.2 32.848.8 26.4

52.0 29.6

42.842.440.0

41.7

26.024.420.8

23.7

47.3 29.147.3 29.1

13.1 20.0 +9.6

9.0 25.1 -1.4

11.7 15.6 +13.5

20.2 38.0 -12.1

21

Mean

62.6 41.149.7 35.1

56.2 38.118.6 33.5 -6.1

22 49.1 31.455.8 38.1

16.8 33.1 +0.7 Mean 52.4 34.8

26 48.0 28.850.4 31.2

Mean 49.2 30.016.1 35.6 -1.5

10.4 23.7 +14.4

13.7 20.3 +11.1

11.5 16.6 +13.4

40.0 29.640.0 29.6

2719.8 44.0 -14.4

43.0 : 1.0 30.5 4± .1 18.34±0.6 35.7 ±41.4 -5.3 42.0(a = 23) (n = 23) (n = 8) (n = 8) (a = 8)

Mean

Overallmean

38.4 21.638.4 21.6

47.2 41.6 28.7 41.4(a = 17) (n = 17)P <0.01 P >0.05

14.3 28.8 -7.2

11.9 40.6 20.7 ±1.7(a = 8) (n = 8)

P <0.001 P <0.001

After mannitol infusion, EFPA was 17.24+2.1 mmHgwhich was not significantly different from hydropenia(P > 0.05). When WE and AP in each animal were

compared after mannitol, AP was significantly higherby paired analysis (P < 0.025) and the mean EFPEwas +8.042.8 mmHg, a value significantly differentfrom the value in hydropenia and from zero (P <0.025). Utilizing the mean of all values of AP (n= 17),EFPA was 17.2+1.4 mmHg (P < 0.02 compared tohydropenia) and fell to and EFPE of 8.6+ 1.8 mmHg(P < 0.001).

Since the EFPE was significantly greater than zero

after mannitol, a specific value for both EFP and LpAcan be determined. The EFP by the iterative methodwas 12.8+0.5 and 12.7+:0.5 mmHg by the method ofDeen et al. (10), significantly greater than the maxi-mumpossible value during the hydropenic condition(P < 0.001). The mean LpA after mannitol was 0.065

0.003 nl/s/g kidney wt/mm Hg (0.066 by the methodof Deen et al. [10]), which was significantly lower thanthe minimum possible value during hydropenia. Also,when this value for LpA in mannitol was applied to the

Effect of Mannitol on Glomerular Ultrafiltration

9

10

13

17

21

22

26

27Mean

Overallmean +8.0 42.8

(a = 8)P <0.02

1139

25 -

20 -

EE

15 -

10 -

5-

0-

18.2t0.6

11.9t0.6

P<0.0

HYDROPENIA MANNITOL

FIGURE 2 The rA for each animal, as estimated from serumprotein concentrations, are depicted for hydropenia and aftermannitol infusion. Overall mean values are also given and theP value refers to the paired comparisons.

hydropenic values for 7rA, rpf, and AP, the EFPE thatresulted was greater than zero, and the measuredvalue for sngfr in hydropenia (32.0 nl/min/g kidneywt) could not be attained with this value for LpA(23.2+4.1 nl/min/g kidney wt resulted).

This reduction in LpA is unlikely the result of adecrease in surface area (A) but the exact mechanismfor reduced LpA is not known. Recently Deen et al.have suggested that a concentration polarization phe-nomenon might have a minor but real influence uponthe effective permeability as calculated (26). The lowerconcentration of systemic protein after mannitol wouldtend to magnify the concentration polarization effectby permitting a higher rate of filtration at the capillarymembrane. This effect would produce a calculated LpAbelow the true permeability of the membrane. Also,reduced C might alter the configuration of pores orslits in the membrane if the 7r exerts its effect across thecell membrane of capillary endothelial cells and therebyalters cell volume.

TABLE I I IEFP and Total LpA during Hydropenia and

after Mannitol Infusion

Hydropenia Mannitol

EFP, mmHg 4.2±:1.1* 12.840.5§

L,,A, nl/s/gkidney wt/mm Hg 0.0994t0.0061 0.065±0.003§

Independent evaluation of the factors which increaseEFP. There are two factors which have contributedto the increase in EFP and sngfr; increased rpf and de-creased rA. The AP and LpA did not change in a direc-tion which would act to increase the sngfr. Utilizingthe iterative method for evaluation of the EFP profile,we have separated these two positive contributionsto the increased EFP and quantitated their respectiveinfluences.

Wehave held WAconstant as in hydropenia but per-mitted all other factors to change as a result of mannitolinfusion. The EFP which results was 7.0 mmHg. Whenthe opposite manuever was performed and rpf heldconstant while all other factors have changed to thepostmannitol values, the EFPwas higher at 9.7 mmHg.The measured EFPafter mannitol when all factors werechanged from hydropenia was 12.8 mmHg. Therefore,if we focus upon those two factors which directly in-crease filtration rate, more than one-half (58-66%) ofthis increase was directly a result of a decrease in 7rA.This set of predictions is not dependent upon uniquevalues for EFP and LpA in hydropenia. This predictioncan be conducted both at the maximum possible EFPin hydropenia and as EFP approaches zero as a limit,thus effectively defining the range of percentage in-fluence of the respective positive factors (Table IV).

Decreased 7A mediates its effect by increasing theEFP and by allowing filtration fraction to rise to highervalues even at a constant rate of plasma flow.

The effects of mannitol infusion on total glomerularfiltration rate, plasma flow, and filtration fraction. Totalkidney glomerular filtration rate was 1.00+4-0.05 ml/min/g kidney wt in hydropenia and was 1.02+40.07ml/min/g kidney wt after mannitol infusion (P > 0.8).Total renal plasma flow was 2.90+0.15 in hydropeniaand rose to 3.36+0.29 ml/min/g kidney wt (P < 0.01)after mannitol infusion. Renal blood flow was 6.48+0.34 in hydropenia and was unchanged at 6.57+0.55ml/min/g kidney wt after mannitol infusion (P > 0.3).The total filtration fraction therefore was 0.35+0.01in hydropenia and fell to 0.29+0.02 after mannitolinfusion (P < 0.05). As noted previously, snff wasunchanged after mannitol (P > 0.4).

TABLE IVSeparation and Analysis of the Contribution of Decreased rA

and Increased rpf on the Increased EFP with Mannitol*

EFP after mannitol, mmHg 12.8i0.5

EFP if 7rA held constant, mmHg 7.0

EFP if rpf held constant, mmHg 9.7

* Maximum estimate of EFP in hydropenia is 4.2±1.1mmHg.

1140 R. C. Blantz

* A maximum estimate at filtration pressure equilibrium.t A minimum estimate at filtration pressure equilibrium.§ P < 0.001.

This finding of dissociation of changes in snff andtotal GFR has been observed by others in both ex-panded states and after reduction in renal blood flow(27-29). This finding suggest that filtration rate insuperficial nephrons increases to a greater extent thando nephrons below the surface. This may be due toeither (a) disproportionate reductions in AP due toeither inadequate elevations of PG or disproportionateincreases in tubular pressure, or (b) that filtrationequilibrium may not be attained in nephrons belowthe surface or equilibrium may be attained nearer tothe end of the glomerular capillary. Either explanationmight account for a lesser increase in sngfr.

DISCUSSION

The widespread use of mannitol in clinical medicine inan effort to increase, maintain, or restore glomerularfiltration rate has inspired a large number of studieswhich have examined the mechanism of action of thisuseful agent (1-6, 11-14). Most investigators havefavored an increase in either or both rpf and PG as themajor factors contributing to this beneficial effect. Thepostulated changes were attributed to a vasodilatingeffect of mannitol upon the AR vessels (11-13).

However, a careful examination of previous studiesin both humans and laboratory animals reveals thatglomerular filtration rate fell in some studies, oftenwhen bolus injections were made or urine volume re-placement was potentially inadequate (1, 3). Oneexplanation may be that large losses of urine volumeresulted without adequate replacement and volumedepletion could have produced this decrement inclearance.

Recent studies from this author and Brenner and co-workers have demonstrated that 7rA increases to a valueat the end of the glomerular capillary in hydropeniathat approximates the AP, suggesting filtration equili-brium (9, 10, 15, 16). Under these conditions it isobvious that several EFP curves could exist and aunique value for LpA cannot be calculated with confi-dence. Filtration equilibrium obtained during hydro-penia in the present study, in that paired comparisonof WEand AP in each animal revealed that these valueswere not different.

Although the mean EFPE was -5.3 mmHg, whenanalyzed as the mean of each animal (-3.8 mmHgwhen analyzed as the mean of all observations), thesevalues fall within 1 SD of zero, and this negativevalue represents the errors involved in estimation ofboth 7rE and AP. When three or more values for APwere obtained in a single animal, there was a significantvariance in this determination among glomeruli, whichrepresents both errors of measurement and trueheterogeneity. At filtration equilibrium, this variationin AP should require a similar heterogeneity in WrE. I

Therefore, the accuracy with which filtration pressureequilibrium can be defined within an animal is enhancedwith a greater number of evaluations of AP and limitedwith only single measurements.

Values for the LpA calculated during hydropenia inthis study remain minimum estimates of this value.However, after mannitol, in which a positive forceoccurred at the efferent arteriole, LpA can be calculatedwith confidence. Therefore it seems likely that thisvalue for LpA is an accurate estimate and that mannitolincreases sngfr while it decreases the calculated totalglomerular permeability.

The increase in sngfr was entirely attributable to anincrease in EFP. Since the AP did not increase, hydro-static forces were not a positive influence contributingto this increase in sngfr. Previous studies have demon-strated a high degree of filtration rate dependence uponchanges in rpf, at least when filtration rate is increasedby the infusion of isoncotic rat plasma (30). However,in those studies plus studies with aortic constrictionby Robertson, Deen, Troy, and Brenner, CA and rremain constant (31). The renal plasma flow responseof animals to mannitol was quite variable in the presentstudy and derived from considerable variability inalterations in the AR. This was not attributable tovariations in extracellular volume, since net positivefluid balance did not exceed 1 ml before attaining asteady-state urine excretion in any of the eight studies.In several animals rpf did not increase, yet glomerularfiltration rate rose in these animals.

When the increase in total EFP was analyzed bycomputer, it was noted that if renal plasma flow hadnot increased, then the sngfr would have increased morethan 50% of the observed value after mannitol, duetotally to the decrease in Wr. This consistent effect ofdecreased 7r observed in this study is not simply theresult of an increased EFPA. Because of the nonlinearrelationship of C and Wr, for any given volume of filtrateproduced along the glomerular capillary, the decay inthe slope of the EFP due to the concentration of proteinwill be less at a lower CA. Therefore the isolated effectof a decrease in 7rA, at any EFPA, will be an increase inthe total EFP, and therefore an increase in sngfr.

The second major effect of the decrease in 7rA is topermit a higher snff and thereby a higher sngfr throughan increase in EFP, while plasma flow remains constant.At each value of WrA and AP, the maximum filtrationfraction is defined by filtration equilibrium, or thatvalue of WEwhich equals the AP. Therefore we have twofactors, decreased WAand increased rpf, which both actto increase sngfr by increasing EFP, but the decreasein rA tends to increase filtration fraction, and at leastat higher rates, increases in plasma flow tend to de-crease filtration fraction. Both of these factors havecombined to maintain the snff constant in this study,

Effect of Mannitol on Glomerular Ultrafiltration 1 141

but at a higher EFP and a positive value for EFPEafter mannitol.

The exact quantification of the respective influencesof decreased 7rA and increased rpf to the increasedfiltration rate apply, of course, to the specific conditionsof this study. The relative contributions of these twofactors may vary with markedly different quantitiesof mannitol used and possibly by the rate of administra-tion. The effect of dilution of protein upon filtrationrate is dependent only upon decreased 7rAs, a predictableoccurrence. However, the efficiency of mannitol inproducing afferent arteriolar dilatation and increasedplasma flow is dependent upon the capacity of therenal vasculature to respond, which may vary markedlyin differing clinical and experimental conditions.

The present observations suggest that the majoreffect of dilution of protein and reduced ir with mannitolmay serve to explain, in part, previous findings ofrestoration of filtration rate observed in certain statesof low renal blood flow. In the studies of Flores, DiBona,Beck, and Leaf (12) and Morris, Alexander, Bruns, andLevinsky, the "no reflow" phenomenon was examined indetail. After a period of renal artery occlusion or duringrenal hypoperfusion, the infusion of hypertonic solu-tions containing mannitol produced significant restora-tion of filtration rate, as determined by direct methods(13) and as evidenced by a lesser rise in serum creatininein the study of Flores et al. NaCl or Na2SO4 solutionsof similar hypertonicity also had some beneficial effectupon filtration. The effect of mannitol was attributed tovasodilation at the afferent arteriole, possibly by reduc-ing endothelial cell swelling. Although serum proteinswere not measured in the studies (12, 13), an additiveeffect may have resulted from a large reduction in 7rAwith administration of hypertonic solutions whichproduce a redistribution of fluid from intracellular toextracellular compartments.

Therefore, a major beneficial effect of mannitol canbe explained by mechanisms not totally dependentupon afferent arteriolar dilatation and the resultant in-creased plasma flow. An increase in the AP was not ob-served in this study. Mannitol produced a consistentincrease in sngfr by augmenting plasma volume anddiluting C at the expense of a decreasing intracellularfluid volume. This additional mechanism may explainthe greater effect of mannitol on sngfr than observedwith other agents which also vasodilate the renalvasculature.

APPENDIX

Measured data were analyzed by the method of Deen et al.(10) and by a set of equations which are described here areanalyzed by computer at the University of California atSan Diego Computer Facility. These equations are based upon

the following relationship:

sngfr = (LpA) J EFP dx*,

where A designates the afferent end of the capillary and E isthe efferent end. Since a true glomerular capillary length isnot known a dimensionless normalization of the length canbe applied (x*). A EFP can be determined by the followingequation:

EFP = f (AP - r)dx*,

and that:

sngfr = (LPA)-f (AP - r)dx*.

Utilizing the measurable parameters, the profile of the EFPand the corresponding total membrane hydraulic permeabilitycan be determined through the use of a double iterativetechnique. Calculations require the nephron plasma flow (Q.),CA, CE, and PGand Pt. A block iteration technique is used toobtain an EFP profile for any particular LpA. The initialstep of the curve is determined by:

EFPA = AP - 7rA.

Flux (sngfri) across any small distance Ax*, where Ax* = x*/n(n = 40-100), can be calculated as:

sngfri = (LpA*Ax*)EFPA.This determines the parameters at Ax,*, where Q,&.,*, is equalto the initial nephron plasma flow (Q.) minus sngfri. The newprotein concentration (Ci.1*) is defined by:

C,&,,* = CA(Qo/Q,&x,*)which in turn can be used to determine 7r,,r,*. EFP is thencalculated by:

EFPAx1* = (AP - )axl*,assuming that AP remains constant. The second flux incre-ment is then:

sngfr2 = (LpA -Ax*) * (AP -P)x- *

The process of block iteration continues until the end of thecapillary where x* = 1. The result is a profile of EFP alongcapillary length x* as long as the Ax* is small. This profiledepends on the LpA used. To find a unique solution for LpAan iteration of LpA must be applied. Nesting the block itera-tion technique within the LpA iterations then the followingcriterion must be satisfied with increasing values of L5A:

z sngfr = QO(l - CA/CE).

The final LpA is then the value utilized to satisfy this condi-tion. This particular method will provide a single solution forLA and EFP for any of the given parameters as long as theEFP profile does not reach equilibrium. When equilibriumattains, an infinite number of combinations of EFP and L5Awill meet the specified parameters. A minimum value for LpAand maximum value for EFP is obtained in an equilibratedsituation.

1142 R. C. Blantz

ACKNOWLEDGMENTSThe excellent technical assistance of Ms. Karen Konnen andexcellent secretarial assistance of Ms. Barbara Drysdale isgratefully acknowledged. Wealso thank Mr. Bryan Tuckerwho was instrumental in the development of the computermodel of glomerular ultrafiltration.

These studies were supported by grants from the U. S.Public Health Service (HL 14914) and from the VeteransAdministration.

REFERENCES1. Braun, W. E., and L. Lilienfield. 1963. Renal hemo-

dynamic effects of hypertonic mannitol infusions. Proc.Soc. Exp. Biol. Med. 114: 1-6.

2. Lilien, 0. 1973. The paradoxical reaction of renal vascula-ture to mannitol. Invest. Urol. 10: 346-353.

3. Peters, G., and H. Brunner. 1963. Mannitol diuresis inhemorrhagic hypotension. Am. J. Physiol. 204: 555-562.

4. Gazituia, S., J. B. Scott, B. Swindall, and F. J. Haddy.1971. Resistance responses to local changes in plasmaosmolality in three vascular beds. Am. J. Physiol. 220:384-391.

5. Stahl, W. M. 1965. Effect of mannitol on the kidney.N. Engl. J. Med. 272: 381-386.

6. Parry, W. L., J. Schaefer, and C. Mueller. 1963. Experi-mental studies of acute renal failure. I. The protectiveeffect of mannitol. J. Urol. 89: 1-6.

7. Seely, J. F., and J. H. Dirks. 1969. Micropuncture studyof hypertonic mannitol diuresis in the proximal and distaltubule of the dog kidney. J. Clin. Invest. 48: 2330-2340.

8. Goldberg, M., and M. A. Ramirez. 1967. Effects of salineand mannitol diuresis on the renal concentrating mecha-nism in dogs: alterations in renal tissue solutes and water.Clin. Sci. (Oxf.). 32: 475-493.

9. Blantz, R. C., A. H. Israelit, F. C. Rector, Jr., and D. W.Seldin. 1972. Relation of distal tubular NaCl delivery andglomerular hydrostatic pressure. Kidney Int. 2: 22-32.

10. Deen, W. M., C. R. Robertson, and B. M. Brenner. 1972.A model of glomerular ultrafiltration in the rat. Am. J.Physiol. 223: 1178-1183.

11. Goldberg, A. H., and L. S. Lilienfield. 1965. Effects ofhypertonic mannitol on renal vascular resistance. Proc.Soc. Exp. Biol. Med. 119: 635-642.

12. Flores, J., D. R. DiBona, C. H. Beck, and A. Leaf. 1972.The role of all swelling in ischemic renal damage and theprotective effect of hypertonic solute. J. Clin. Invest. 51:118-126.

13. Morris, C. R., E. A. Alexander, F. J. Bruns, and M. G.Levinsky. 1972. Restoration and maintenance of glomer-ular filtration by mannitol during hypoperfusion of thekidney. J. CGin. Invest. 51: 1555-1564.

14. Coelho, J. B., and S. E. Bradley. 1964. Function of thenephron population during hemorrhagic hypotension inthe dog, with special reference to the effect of osmoticdiuresis. J. Clin. Invest. 43: 386-400.

15. Brenner, B. M., J. L. Troy, and T. M. Daugharty. 1971.The dynamics of glomerular ultrafiltration in the rat.J. Clin. Invest. 50: 1776-1780.

16. Blantz, R. C., F. C. Rector, Jr., and D. WV. Seldin. 1974.Effect of hyperoncotic albumin expansion upon glomerularultrafiltration in the rat. Kidney Int. In press.

17. Blantz, R. C. 1973. Effect of mannitol (M) on glomerularultrafiltration in the hydropenic (H) rat. Abstracts of theAmerican Society of Nephrology, Washington, D. C. 6: 12.

18. Weinman, E. J., M. Kashgarian, and J. P. Hayslett. 1971.Role of peritubular protein concentration in sodium re-absorption. Am. J. Physiol. 221: 1521-1528.

19. Daugharty, T. M., I. F. Ueki, J. I. Troy, and B. M.Brenner. 1971. Direct assessment of renal versus corticalhemodynamics and sodium Na in hydropenic (H) andvolume expanded (VE) rats. Clin. Res. 19: 150. (Abstr.)

20. Barratt, L. J., J. D. Wallin, F. C. Rector, Jr., and D. W.Seldin. 1973. Influence of volume expansion on single-nephron filtration rate and plasma flow in the rat. Am.J. Physiol. 224: 643-650.

21. Wallin, J. D., R. C. Blantz, M. A. Katz, V. E. Andreucci,F. C. Rector, Jr., and D. W. Seldin. 1971. Effect ofsaline diuresis on intrarenal blood flow in the rat. Am. J.Physio. 221: 1297-1304.

22. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the folin phenolreagent. J. Biol. Chem. 193: 265-275.

23. Brenner, B. M., K. H. Falchuk, R. I. Keimowitz, and R.W. Berliner. 1969. The relationship between peritubularcapillary protein concentration and fluid reabsorption bythe renal proximal tubule. J. Clin. Invest. 48: 1519-1531.

24. Landis, E. M., and J. R. Pappenheimer. 1963. Exchangeof substances through the capillary walls. Handb. Physiol.Circulation. 2: (Sect. 2) 961-1034.

25. Snedecor, G. W. 1956. Statistical Methods. Iowa StateUniversity Press, Ames, Iowa.

26. Deen, W. M., C. R. Robertson, and B. M. Brenner. 1973.Concentration polarization phenomena in the glomerularcapillary. Abstracts of the American Society of Nephrol-ogy, Washington, D. C. 6: 30.

27. Herrera-Acosta J., V. E. Andreucci V., F. C. Rector, Jr.,and D. W. Seldin. 1972. Effect of expansion of extracellularvolume on single-nephron filtration rates in the rat. Am.J. Physiol. 222: 938-944.

28. Baines, A. D. 1971. Effect of extracellular fluid volumeexpansion on maximum glucose reabsorption rate andglomerular tubular balance in single rat nephrons. J.Clin. Invest. 50: 2414-2425.

29. Gertz, K. H., M. Brandis, G. Braun-Schubert, and J. W.Boylan. 1969. The effect of saline infusion and hemorrhageon glomerular filtration pressure and single nephronfiltration rate. Pfluegers A rchiv. Gesamte Physiol.Menschen Tiere. 310: 193-200.

30. Brenner, B. M., J. L. Troy, T. M. Daugharty, W. M.Deen, and C. R. Robertson. 1972. Dynamics of glomerularultrafiltration in the rat. II plasma-flow dependence ofG.F.R. Am. J. Physiol. 223: 1184-1190.

31. Robertson, C. R., W. M. Deen, J. L. Troy, and B. M.Brenner. 1972. Dynamics of glomerular ultrafiltration inthe rat. III. hemodynamics and autoregulation. Am. J.Physiol. 223: 1191-1200.

Effect of Mannitol on Glomerular Ultrafiltration 1143