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Composition of Renal Medullary Tissue

Composition of Renal Medullary Tissue

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Kidney International, Vol. 31(1987), pp. 556—561

Composition of renal medullary tissue

RUTH ELLEN BULGER

Renal Research Laboratory, University of Texas Health Science Center at Houston, Graduate School of Biomedical Sciences, Houston, Texas, USA

Concentration of the urine is achieved by osmotic equilibriumtissue chloride concentration in rabbit medullary tissue when of the collecting duct fluid with the hypertonic, medullarycompared to that of the cortex. Investigators soon realized that interstitial fluid. The medullary tissue concentration of variousthere were problems in expressing their determinations using osmotically—active substances, especially and sodiumthe most appropriate reference standard, since both overall chloride, undergoes drastic changes depending on the physio-solute and content were changing. Results are still logical state of the animal. It has been assumed that intracellularexpressed in a variety of units, which only rarely use DNA or and extracellular osmotic pressures and urea concentrations areprotein as a reference standard. In addition, the physiological equal. However, many questions remain unanswered. What arestate of the varies with enormous changes in urine the concentrations of osmotically active molecules in each ofosmolality (and hence of the various solutes and amount of the various compartments? What types and amounts of smallwater present). One might therefore expect to find large differ- organic compounds are present? How does the high osmolalityences in salts and water in the medulla which correlate with the effect the metabolic state of these cells? Are inorganic mole-physiological state of the animal studied. cules bound, that is, what are their activity coefficients? Another problem inherent in the chemical analyses is the Many techniques have been used to approach these questionslarge quantity of fluid in the lumens of the renal medulla, which including chemical analysis of whole slices of tissue, freez-may contain fluid with greatly differing concentrations of dif- ing—point depression studies of tissue slices, microcentrifuga-fusible ions from that in the surrounding tissue (for example low tion to obtain interstitial fluid, micropuncture of the tubules andsodium in the collecting tubule). Some preservation techniques capillaries at various levels within the renal medulla, (rapid freezing) retain this fluid within the lumens while other isolation and culture techniques, and electron microprobe anal-techniques do not. ysis. In this paper, values for the concentration and content of In spite of these problems, general trends were established. solute and water in various physiological states will be reviewedUllrich and coworkers [17, 18, 20] showed that in antidiuresis, with emphasis on the results obtained from electron microprobethe content of sodium, chloride, urea and of the dog analysis. papilla increased, while that of , magnesium, calcium, In 1908 Hirokawa [1] observed that the osmotic pressure ofand did not. The tissue concentration of solutes the renal medulla was extraordinarily variable but the value wasalso increased and reflected the increased concentration of always higher than in the renal cortex. He also noted that theurine. The osmotic pressure at the papillary tip was, in fact, osmotic pressure of the urine increased during passage throughfound to be the same as that of the final urine. In addition, these the loops of Henle and the collecting tubules. Kuhn and Ryffelauthors showed that approximately 75 to 80% of the increased [2] proposed that the loops of Henle functioned as a counter-osmolality in the medullary tissue was accounted for by sodium current multiplier. Using frozen sections, 30 m thick, of ratchloride and urea. During , a decrease in papillary , mounted between cover glasses in a bath whose tem-osmolality was noted. Gardner and Vierling [9] showed that perature was gradually raised, Wirz, Hargitay, and Kuhn [3]papillary content of all solids, and specifically sodium and urea, measured the melting point to determine the osmotic pressureincreased with increasing urinary concentration, but water, of the tubular contents. They demonstrated that the osmoticDNA, and content did not change. They noted that large pressure of the luminal contents rose from the cortico-medul-increases in tissue urea as occurs in antidiuresis (up to 20% of lary boundary to the papillary tip. medullary dry weight), complicate the terminology for expres- Analysis of medullary tissue by several investigators pro-sion of content, and suggested the use of urea—free dry weight. vided the first chemical determinations of the contents ofBray 121], Dicker [22], and Ruiz—Guinazu, Arrizurieta and medulla in and antidiuretic states [4—19] (Table 1). InYelinek [23] all demonstrated that the osmolality of the medulla most of these studies, the sodium and urea concentrations rosedecreased during water diuresis but remained hypertonic to the progressively from the cortico-medullary junction toward the plasma. papilla. Ljungberg [13], demonstrated a sevenfold increase in Careful studies of the content and concentration of the renal medulla at various stages after antidiuretic hormone (ADH) administration were useful in attempts to understand the tem- Received for publication July 9, 1986 poral effects of ADH [4—8, 10—12, 14, 15, 17]. In a chemical ©1987 by the International Society of Nephrology analysis of kidney pieces by Schmidt—Nielsen, Graves and Roth

556 Inorganic content of medulla 557

Table 1. Composition of urine, renal papilla, and cortex Urine Papilla Cortex mMoles/KgH2O mMoles/KgH2O mMoles/KgH2O Reference Dog Water diuresis Osmolality 70.6 16.6 [12] Na 2.8 2.5 118 23.1 68.1 5.7 5,8 3.7 48.6 4.4 77.2 4.0 Urea 42.1 16.4 33.4 9.1 10.0 2.5 2 hr post-ADH administration Osmolality 1012 232 [12] Na 148 34.4 206 27.5 80.5 5.4 120 75.6 45.6 6.1 13.4 3.9 Osmolality 1725 361 [12] Na 153 60 320 48.4 80.8 8.9 162 70 52.7 5.5 80.5 7.9 Urea 991 228 855 160 21.0 4.6 Rat Hydropenia Osmolality 2275 253 [15] Na 76 30 470 148 898 K 178 33 175 56 98 8 Urea 1500 175 871 264 22 4 Water diuresis Osmolality 172 95 [15] Na 3.4 2.1 312 71 89 14 11.8 5.4 190 62 92 14 Urea 137 70 177 89 22 6 Normal Osmolality 2056 [19] Na 181.1 8.8 358.4 33.2 79.2 2.8 K 356.7 22.7 43.2 4.3 93.9 1.7 Urea 839.3 56.9 583.5 47.9 18.0 5.4 insipidus Osmolality 124.0 6.7 [19] Na 9.9 1.1 201.5 31.0 89.5 3.0 18.2 1.4 39.6 8.2 93.9 4.3 Urea 59.6 5.2 40.7 18.4 6.3 3.8 Diabetes insipidus treated with ADH Osmolality 969.0 220.2 [19] Na 84.5 11.4 236.8 36.1 81.6 4.0 K 150.3 33.0 41.5 3.1 90.4 3.6 Urea 479.6 126 282.8 57.3 16.9 6.5 Dehydration Osmolality 3817.0 89.3 [19] Na 247.8 26.4 401.8 26.9 80.9 3.9 K 438.2 24.9 53.9 4.9 90.1 3.8 Urea 2172.0 65.5 811.8 18.3 18.5 2.8

[24], the two most important factors contributing to the rise invarying their sodium content. This finding agreed with the inner medullary osmolality were removal of water and additionearlier results of Ullrich and Pehling [26] who found that the of urea. The addition of urea to the papillary tissue exceededsodium space of the inner medulla approximated the total the contribution of the osmotic equivalent of sodium chloridevolume of the tissue. by a factor of 2.8 in both rats and hamsters. Because a relatively large piece of renal medulla is required Morgan [25] bisected the renal papilla and incubated it infor chemical analysis, small areas of renal medulla cannot be media in which sodium chloride was varied from 150 to 600 mrvistudied. In addition, no direct way to analyze and compare the and potassium chloride was varied from 2 to 20 m. Thecomposition of cellular versus extracellular compartments was increase in sodium chloride to 600 m reduced cell volume byavailable. Therefore, techniques with higher spatial resolution 25%, increased cell sodium content by 250%, but had nowere necessary. significant effect on cell potassium concentration or content. The concentrations of various inorganic substances in spe- Increasing the potassium in the media did not change cell watercific locations within the renal medulla have been determined or sodiuni, but did increase cell potassium. Morgan [25] con-using micropuncture and microanalytical techniques by many cluded that papillary cells maintain osmotic equilibrium byinvestigators. For example, Wirz [27] demonstrated that 558 Bulger

Table 2. Analysis of urine in papillary interstitial fluid from diabetes constant intracellular potassium is of considerable importance insipidus rats [34] considering the sensitivity of intracellular enzymes to potas- Papillaryinterstitial sium concentration [24]. Recent x-ray microprobe studies (Ta- fluid Urine bles 3 and 4) also disclose constant intracellular potassium mmolelliter mmole/liter value. It therefore appears that cellular potassium levels are (means SD) (means SD) reasonably constant throughout the cortex and medulla, and do Na Urea Na Urea not play an important role in the response of the cells to the hyperosmolality of the medulla. Untreated, heterozygous288 61380 123201 28533 48 hr. dehydration heterozygous 486 74810 127298 731469 301 Electron microprobe analysis Untreated, homozygous169 4672 14 25 5 75 103 Electron microprobe analysis has the potential to allow 14 hr dehydration measurements of total solids (mass), and mass fractions of homozygous 179 23284 10322 7414 77 various elements (from which concentrations can be calculated Post-ADH homozygous 231 34347 159170 50495 193 if water content is known) within each cellular and extracellular compartment of the renal medulla. Early studies used freeze dried sections of 15 to 20 regions of antidiuretic rat kidneys. The renal pedicles were clamped and in has a high osmotic pressure at the tip of thethen rapidly frozen in liquid pentane [381. Dry mass was found papilla. He also showed that fluid obtained in the early distalto be constant in the cortex and outer zone of the renal medulla, tubule was always hypotonic. Gottschalk and Mylle [28, 29]decreased abruptly at the inner stripe—inner zone boundary of analyzed the contents of vasa recta, loops of Henle, andthe renal medulla, and continued to rise to a maximum value collecting ducts and showed them to be equally hypertonic attoward the papillary tip. The values of sodium and comparable levels. Small but significant differences have been(elemental content not ionic state) were constant in the cortex, noted between fluid in the ascending thin limb and adjacentincreased in the outer medulla to two to three times that of descending limbs, and between the descending thin limb andcortex, underwent a discontinuity at the border of outer and plasma and adjacent descending vasa recta [30—33].Ininner medulla and then showed an exponential increase to the antidiuretic hamsters, Marsh and Azen [32] found a concentra-papillary tip especially in the last 1000 tm. At the papillary tip, tion of sodium in the descending thin limb to be 736.2 35.1 the concentrations of sodium and chlorine were seven to 14 mEq/liter and in vasa recta plasma to be 696.838 mEq/liter.times and 10 to 15 times higher then the values, respectively, in They assumed that the plasma sodium concentration was thethe cortex. The potassium content expressed by volume, how- same as that of the interstitial fluid. Numerous similar importantever, had the same distribution as the dry mass. studies utilizing these techniques have been instrumental in From their own measurements and those of others [14, 15, 19, understanding many aspects of and are dealt23], Koepsell et al [38] estimated water content profiles for within appropriate articles in this symposium. antidiuretic animals as 77% in the cortex, 90% at the border Another approach to determining concentrations of inorganicbetween inner and outer medullary zones, and 60% at the tip of ions and urea in compartments of the renal medulla is to employthe papilla. This decrease in papillary water content was not of severed renal papilla to obtain a representativenoted in animals undergoing water diuresis. sample of papillary interstitial fluid [34—37]. Table 2 summarizes Recently, two other groups [39—47] have utilized electron values for urine and papillary interstitial sodium and ureamicroprobe analysis at higher resolution than the earlier studies concentration from Lee, Morgan and Williams [34]. An accu-to obtain values of elemental and water content within various mulation of sodium and urea was found after 48 hours ofcellular and extracellular compartments in renal cortex and dehydration which was not present in rats homozygous forpapilla. The group used different techniques for analysis which diabetes insipidus. Schmidt—Nielsen [36] calculated intracellu-involve different assumptions. In comparing results obtained lar concentrations using the centrifugation technique and theusing the two different techniques, it is apparent that some concentration of extracellular marker substance. She deter-variations exist in results reported for the proximal tubule cells mined that intracellular potassium concentration remained atof the cortex (Table 3) [39—43] while widely divergent results are the same level in all zones but intracellular sodium paralleledobtained for certain solutes (sodium and chloride) in the renal the osmolality of the tissues. Therefore, she concluded thatpapilla (see Table 4) [40, 44—47]. These differing results have led sodium was entering the cells as they regulated cell volume. to substantially different interpretations as to the mechanisms Potassium exhibits a pattern of distribution different than thatby which cells adapt to the hypertonic environment of the renal of sodium and chloride. Saikia [14] could not find a definitemedulla. In contrast, the values reported for cellular potassium potassium concentration or content gradient within the me-by the two groups show good agreement [40, 41, 45—48]. dulla. Atherton et a! [6, 7] and Ruiz—Guinazu, Arrizurieta and Saubermann and his associates have used a method in which Yelinek [23], in agreement, found that any difference in potas-analysis of frozen hydrated sections is done first on a cold stage sium concentration could be explained by differences in waterat —185°C to determine total mass (tissue components and content. Potassium levels expressed as mass per volume hadwater) and then the sections are dried within the electron the same pattern as the corresponding dry mass distributionmicroscope and reanalyzed to determine the fraction of total [38]. Schmidt—Nielsen [36] found that kidney potassium levelsmass for each element. Concentrations of elements per wet were generally the same in all zones, but there was a small butweight are then calculated from the loss in mass after drying. significant increase in potassium in the papilla. This ratherThe results indicate that papillary epithelial cells of all types Inorganic content of medulla 559

Table3. Renal proximal tubular cells Dry Na P Cl K weight mMoleslkg mMoles/kg mMoles/kg mMoles/kg wet wt wet wt wet wt wet wt Reference Technique/location g/100 g — Cytoplasm— 33 1.3 22 1.4 179 6.7 33 2.3 136 4.5 nucleus 23 0.8 20 1.2 145 5.4 21 1.7 140 4.5 [42] Nucleus 23 0.3 20 0.4 150 1.6 23 0.7 144 2.0 [49] Nucleus 21 0.5 19 1.3 151 5.2 19 0.7 149 4.3 cytoplasm 30 0.9 18 1.2 193 6.7 34 1.7 142 4.7 [44] Frozen hydrated, —53°C 29 52 1.6 191 3.8 52 1.6 132 2.6 [41] Frozenhydrated —80°C 29 44 2.7 193 5.7 46 2.3 137 5.0 [41] Tissue dipped in albumin 33 0.8 46 1.5 — 44 1.4 116 11.0 [411 standard, —53°C Slice technique (insulin space 22 35 156 [50] 26 0.6%) Microprobe 70 36 90 Chemical 80 494 869 [51] [41, 52] Calculated isolated proximal 20% 41 mEq/liter 156 mEq/liter [53] straight tubules tissue volume tissue volume Calculated from Na pump data 35 [54]

Table 4. Values of dry weights and elements from microprobe analysis Urine Na P Cl K S concen- Location in mOsm/kg mOsm/kg mOsm/kg mOsm/kg mOsm/kg tration papilla Dry wt % wet wt wet wt wet wt wet wt wet wt mOsm Reference PCD 43.3 344 127 252 20 331 108 156 54 29 6 — [45] PCD 19.7 29 1 316 7 76 3 135 3 — 1509 116 [44] PCD (undipped) 31.7 227 16 433 17 269 17 154 7 34 1 —1650 [40, 47] PCD (dipped) 25 124 12 363 13 154 13 125 4 29 1 —-1650 [40, 47] PCD (antidiuretic) 47 186 49 260 82 121 12 2240 308 [46] PCD(diuretic) 19 64 28 77 20 90 23 300 3 [46] PEC 40.5 287 106 242 54 318 79 141 36 39 16 — [45] PEC 20.9 232 333 12 80 4 136 5 — 1509 116 [44] PEC (undipped) 32.2 254 31 433 41 278 32 132 12 40 4 —1650 140, 471 PEC (dipped) 30.4 151 11 403 16 186 11 141 6 392 —1650 [40, 471 PEC (antidiuretic) [46] PEC (diuretic) [46] IC 40.1 898 194 145 26 725 140 79 16 266 — [45] IC 21.0 50 3 310 12 107 4 139 42 — 1509 116 [44] IC (undipped) 31.5 362 28 412 27 401 31 137 8 23 2 —1650 [40, 47] IC (dipped) 33.7 391 23 403 18 429 24 131 6 22 2 —1650 [40, 47] IC (antidiuretic) 48 429 41 504 73 94 31 2242 308 [46] IC (diuretic) 18 154 26 116 34 50 19 322 3 [46] I 22.6 590 119 40 15 445 115 42 8 279 — [45] I 11.8 437 19 15 1 438 20 35 2 — 1509 116 [44] I (undipped) 16.9 654 31 30 3 652 32 38 2 2 4 —1650 [40, 47] I(dipped) 7.6 518 15 39 5 517 15 41 3 9 1 —1650 [40, 47] I (antidiuretic) 20 426 152 461 143 28 10 [46] I (diuretic) 10 99 15 73 21 36 15 [46] Abbreviations are: PCD, papillary collecting duct; PEC, papillary epithelial cells; IC, interstitial cells; I, interstitium. accumulate sodium and chloride, but not potassium duringanalyzed frozen dried sections of tissue which was first dipped adaptation to a hypertonic medullary environment. The dryin 20% albumin solution containing known concentrations of weight fraction of the cells from sections close to the papillaryvarious elements. Under these conditions, the papilla cell tip [45] was about 40%, which agrees closely with the earliersodium and chloride are not greatly elevated despite the studies of Koepsell Ct al [38]. Sections slightly removed fromhypertonic environment (Table 4). The different results of these the papilla, however, had cell dry weights of 31 to 35% [41, 47].groups reflect differences in techniques, experimental condi- It was noted that papillae obtained from animals in watertions and other factors which have been discussed in detail diuresis had much lower values of cell sodium and chloride andelsewhere. higher values for cell water than papillae from antidiuretic To attempt to understand the reasons for these divergent animals [46] (Table 4). Thurau and his associates [42—44, 49]results, Saubermann et al [40, 41, 47] clamped the renal pedicle, 560 Bulger dipped whole kidneys in an albumin solution, rapidly froze the References kidney, and analyzed the proximal tubule cytoplasm using both methods simultaneously by calculating elemental concentra- 1. HIROKAWA W: Uber den osmotischen Druck des Nierenpar- enchyms. Beitr Chem Physiol Path 11:458—478, 1908 tions using the different algorithms. Similar values were ob- 2. KUHN W, RYFFEL K: Herstellung konzentrierter Losungen aus tained in the experiments when using either algorithms for verdunnten durch blosse Memranwirkung. Em Modeilversuch zur elemental analysis of proximal tubule cytoplasm. The content Funktion der Niere. Hoppe-Seyler's Zeitschrift F Physiol Chemie of water and elements in the adherent albumin layer did not 276:145—178, 1942 change significantly from the solution which had been used for 3. Wiaz H, HARGITAY B, KUHN W: Lokalisation des Konzentrierung Sprozesses in dec Niere durch direkte Kryoskopie. Helv Physiol dipping. Hence, the differences in sodium and chloride concen- Acta 9:196—207, 1951 trations of proximal tubular cells between the two laboratories 4. ATHERTON JC, HAl MA, THOMAS S: Effects of water diuresis and were not explained by the algorithm used or by changes in the osmotic (mannitol) diuresis on urinary solute by the albumin layer [40, 41]. conscious rat. J Physiol 197:395—410, 1968 5. ATHERTON JC, HA! MA, THOMAS S: The time course of changes in Having established that both techniques give similar results renal tissue composition during water diuresis in the rat. J Physiol when done simultaneously on the cortex using the same x-ray 197:429—443, 1968 data, Saubermann et al [40, 411 repeated the experiments of 6. ATHERT0N JC, EVANS JA, GREEN R, THOMAS S: Influence of Beck et al [44] on the papilla. They also analyzed the contra- variations in hydration and in solute excretion on the effects of lateral renal papilla without dipping it in the albumin solution. lysine— infusion on urinary and renal tissue composition in the conscious rat. J Physiol 213:311—327, 1971 There were significant differences in the values obtained for 7. ATHERTON JC, GREEN R, THOMAS S: Influence of lysine—vasopres- collecting duct cells, papillary epithelial cells and interstitium sin dosage on the time course of changes in renal tissue and urinary between the dipped and undipped papilla (Table 4). Moreover, composition in the conscious rat. J Physiol 213:291—309, 1971 changes in elemental concentration and water content of the 8. ATHERTON JC, GREEN R, THOMAS 5, WOOD JA: Time course of changes in renal tissue and urinary composition after cessation of adherent albumin were noted. If the values measured in the constant infusion of lysine vasopressin in the conscious, hydrated albumin solution by the freeze hydrated/freeze dried methodol- rat. J Physiol 222:583—595, 1972 ogy were used to calculate the cell elemental distribution, the 9. GARDNER KD JR, VIERLING JM: Solids, water, and solutes in values were closer to those reported by the Saubermann group papillary region of the rat kidney. Am J Physiol 217:58—64, 1969 [45]. Furthermore, the value of sodium content in the intersti-10. HA! MA, THOMAS S: The time—course of changes in renal tissue composition during lysine vasopressin infusion in the rat. Pflügers tium in the study by Saubermann et al [47] was 654 31 Arch 310:297—319, 1969 mEq/liter and not greatly different from the value of 736.2 11. HAT MA, THOMAS S: Influence of prehydration on the changes in 35.1 mEq/liter measured in plasma from ascending vasa recta renal tissue composition induced by water diuresis in the rat. J by Marsh and Azen [32] or the value in the interstitial fluid Physiol 205:599—618, 1969 12. LEvITIN H, GOODMAN A, PIGEON G, EPSTEIN FH: Composition of measured by Lee, Morgan and Williams [34]. the renal medulla during water diuresis. J Clin Invest 41:1145—1151, Finally, the fact that the concentration of cell potassium did 1962 not vary significantly in the two studies reinforces the interpre-13. LJUNGBERG E: On the of chlorides in the kidney of tation that differences in values for sodium and chlorine reflect rabbit. Acta Med Scand 186:169—189, 1947 14. SAIKIA TC: Composition of the renal cortex and medulla of rats methodological problems. The sum of two times the sodium and during water diuresis and antidiuresis. Q J Exp Physiol50:146—157, potassium concentration of the collecting duct cells is 762 1965 mOsM/kg wet weight; of the papillary epithelial cells, 772 15. SAIKIA TC: The acute effect of vasopressin upon the composition of the rat renal cortex and medulla. Q J Exp Physiol 50:158-168, 1965 mOsM/kg; of the interstitial cells 998 mOsM/kg; and of the16. SCHMIDT—NIELSEN B, O'DELL R: Effect of diet on distribution of interstitium 692 mOsM/kg. Assuming a of 1650 urea and electrolytes in kidneys of sheep. Am J Physiol 197: mOsM/liter (actually measured, right kidney 1573 184, left 856—860, 1959 194), and assuming that all the elements are 17. ULLRICH KJ, DRENCKHAHN FO, JARAUSCH KH: Untersuchungen kidney 1690 zum Problem der Harnkonzentrierung und -verdunnung. Pflugers osmotically active, there is still a large difference between the Arch 261:62—77, 1955 urine osmolality and the osmotic contribution of the major18. ULLRICH KJ, JARAUSCH KH: Untersuchungen zum Problem der inorganic elements in urine of 888 mOsm in collecting duct Harnkonzentrierung und Harnverdunnung. Pflugers Arch 262: 537—550, 1956 cells, 878 mOsM/kg wet weight in papillary epithelial cells; 65219. 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