Fine Structure of Mammalian Renal Cilia W. A. WEBBER AND J . LEE Department of Anatomy, The University of British Columbia, Vancouver, British Columbia, Canada
ABSTRACT Studies were carried out, using transmission electron microscopy, of the cilia of the nephrons of rat and human kidneys. Cilia were observed in the parietal layer of Bowman's capsule, in the proximal tubule, the distal tubule, including the macula densa, and the collecting duct. They had a number of characteristic features, including the presence of a centriole adjacent to the basal body, long, slender cross-striated rootlets, and a typically organized basal body. The shaft of the cilia differed from the typical 9 2 pattern of organization. Near the base of the cilia the pattern was 9 0. In the middle portion, one or more of the peripheral doublets had been displaced centrally to give an 8 1 or a 7 2 pattern, while towards the tip the pattern became more irregular and the doublets were reduced to single microtubules. We have hypothesized that these cilia may be vestigial. They may, if motile, have some minor stirring function, or they may play a sensory role, as has been postulated for similarly structured cilia in other sites.
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+
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Cilia have long been known to be present in large numbers in the renal tubules of many animal species (Latta et al., '61; Zimmerman, 1898) and have been observed to have a propulsive function where they are numerous (Fawcett and Porter, '54). In the mammalian kidney they have frequently been observed as single, isolated structures to which little significance has been ascribed. Recently, with the extensive use of scanning electron microscopy, a number of authors have reported the general pattern of ciliation in the kidney of both man and rat (Andrews and Porter, '74; Bulger et al., '74; Webber, '74). On the parietal layer of Bowman's capsule, the proximal tubule, the distal tubule, and in the light but not the dark cells of the collecting duct, each cell usually bears a single cilium, although occasionally two are present. This uniform pattern has raised the question as to whether these cilia are vestigial or whether they may have some specific function. Cilia are usually motile and serve either to move the organism or to move material relative to a surface. These cilia typically have an internal structure consisting of nine peripherally placed doublets of microtubules and a pair of single centrally placed microtubules, the 9 2 pattern. A
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ANAT.REC., 182: 339-344.
variety of other patterns has also been described, for example in insect sperm (Baccetti et al., '74). In many sites, however, cilia occur singly in locations where motility would not appear to be an important function (Scherft and Daems, '67). A number of these sites are in sense organs or part of the nervous system, some are in contractile cells, notably myoepithelial cells where they may be always present (Tandler et al., '70), while others are in a variety of cells with neither contractile nor sensory functions, such as chondrocytes (Scherft and Daems, '67). In the cilia associated with the nervous system, it has been reported that the fine structure differs from the typical 9 2 pattern in that the central pair of single microtubules are absent, giving a 9 0 pattern. In addition, these cilia have a centriole adjacent to the basal body which is not usually seen in typical motile cilia except during development (Dahl, '63). In certain respects, the cells of the nephron resemble contractile cells. For example, they frequently contain basally located fibrils which have been shown to be actin-like (Harper et al., '70; Newstead, '71; Pease, '68; Ross and Reith, '70). Contractility has not, however, been
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Received Dec. 10, '74. Accepted Feb. 24, '75.
339
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W . A. WEBBER AND J. LEE
conclusively demonstrated. Furthermore, in the macula densa of the nephron it has been postulated that some form of sensing device exists which allows monitoring and response to the composition of the tubular fluid (Thurau, '66). Because of these associations, and the fact that it seemed unlikely that the renal cilia perform a major propulsive role, we were interested in determining whether or not these cilia had the typical 9 2 pattern or some other arrangement of microtubules, and whether they showed any other special ultrastructural characteristics. We were also interested in knowing whether their structure was uniform throughout the nephron and whether it was similar in both man and rat. We therefore undertook a study of the ultrastructure of the cilia in a number of parts of the nephron in these two species.
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MATERIALS AND METHODS
Male and female rats of the Wistar strain were used. The animals were anaesthetized with a combination of phenobarbital and pentobarbital, and the left carotid artery cannulated. The kidney was fixed by retrograde aortic perfusion with Karnovsky's fixative (Karnovsky, '65) diluted 50% with cacodylate buffer at 100 mm Hg. Following perfusion, the tissue was prepared for either scanning or transmission electron microscopy using standard techniques (Webber and Blackbourn, '71). The sections were examined in either a Philips 200 or 300 electron microscope. Cross sections of cilia were examined in the parietal layer of Bowman's capsule, in the proximal tubule, in the distal tubule generally, and in the macula densa and in the collecting duct. Human tissue obtained at autopsy after being fixed by immersion was processed and examined in similar fashion to the rat kidney. Comparison was then possible of the structural features of the cilia from the various segments of the nephron in both species. Since the cilia are relatively sparsely distributed, ideal cross sections are rarely encountered. The results reported here are the result of observations of at least 50 cross sections of cilia in each site on the tubule in both species studied.
RESULTS
The fine structure of the cilia was found to be similar in all segments of the nephron in both species. The following structural Eeatures were seen: ( a ) There were fine cross striated rootIets (R) extending into the cytoplasm from the basal body (fig. 1). ( b ) In appropriate sections, there was a centriole (C) present, oriented at right angles to the basal body (fig. 1). Th'is association has been observed previously in the tubule by Latta ('61) and in the parietal layer of Bowman's capsule (Tyson and Bulger, '72). ( c ) The basal body (B) was structurally typical, having nine peripherally arranged triads of microtubules (fig. 2). ( d ) The base of the shaft, which ranged from 0.20-0.25 in diameter, was atypical in that it contained nine peripherally located doublets of microtubules but lacked single central microtubules (fig. 3). (e) The pattern rapidly changed to 8 1 or 7 2 pairs of doublets (figs. 4, 5, 6 ) . ( f ) More distally, there was a reduction in the number of doublets, and some doublets changed to single microtubules (fig. 7).
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DISCUSSION
The cilia of the mammalian nephron which are characteristically present as a single structure in each cell are not normal cilia with a 9 2 pattern of microtubules. Rather they resemble the cilia which have been reported singly in a considerable number of sites (Scherft and Daems, '67). Thus our results differ somewhat from those of Latta ('61) who, on the basis of longitudinal sections, reported the cilia of the rat nephron to be typical kinocilia. In at least some of these other sites a relationship with sensory function exists although i t is not known what role, if any, the cilia play in the sensory process. At the present time we know little about the function of the cilia in the nephron. It is unlikely that they play a major propulsive role as they do in lower animals in which they are numerous (Fawcett and Porter, '54). If they are motile, and this has not been unequivocally demonstrated, they could at most have some stirring action on the tubular
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FINE STRUCTURE OF MAMMALIAN RENAL CILIA
34 1
Fig. 1 Longitudinal section of a cilium in the distal tubule of the rat showing the basal body, a n adjacent centriole ( C ) and a striated rootlet (R). X 45,000. ____ Fig. 2 Cross section of a cilium in the distal tubule of the rat showing the appearance of a
the 9
+ 0 pattern of microtubules. x 49,500.
an 8
+ 1 arrangement of microtubules. x 49,500.
342
W. A. WEBBER AND J. LEE
contents. It is of course possible that they are simply vestigial and constitute the remnants of the much more numerous cilia seen in other species. In support of this concept it is worth noting that during development the central microtubules have been reported to develop after the outer ring (Roth, '60) and these cilia may simply be incompletely developed. On the other hand, however, the widespread occurrence of similar cilia in sensory structures, in nervous tissue and in contractile cells, raises the interesting possibility that they may have some sensory role and that they may provide a means by which the tubular contents can be monitored. Such an arrangement would allow for a local response to altered composition of the tubular fluid and provide for one element of a system maintaining glomerulo-tubular balance. In this regard it is fairly well established that individual nephrons tend to maintain glomerulo-tubular balance and i t has been postulated that through the macula densa the juxtaglomerular apparatus is responsive to some parameter of tubular fluid (Thurau, '66). It would appear, therefore, that these cilia which show interesting structural variations warrant detailed functional study. ACKNOWLEDGMENTS
The support of the British Columbia Heart Foundation and the technical assistance of Mrs. Patricia Hollingdale are gratefully acknowledged. LITERATURE CITED Andrews, P. M., and K. R. Porter 1974 A scanning electron microscope study of the nephron. Am. J. Anat., 140: 81-115. Baccetti, R., R. Pallai, F. Giusti and F. Bernini 1974 The spermatozoan of Arthropoda XXIII. The 9+9+3 Spermatozoan of Simulid Diptera. J. Ultrastruct. Res., 46: 427-440. Bulger, R. E., F. L. Siege1 and R. Pendergrass 1974 Scanning and transmission electron microscopy of the rat kidney. Am. J. Anat., 139: 483-501. Dahl, H. A. 1963 Fine structure of cilia in rat Fig. 6 Cross section of a more distal portion of a cilium in the distal tubule of the rat showing a somewhat irregular 7 2 pattern of microtubules. x 49,500. Fig. 7 Cross section of a cilium in the distal tubule of the rat showing reduction of the microtubular doublets to single microtubules in a 7 2 pattern. X 49,500.
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Fig. 5 Cross section of a more distal portion of a cilium from human proximal tubule showing a similar arrangement of microtubules to figure 4. X 49,500.
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FINE STRUCTURE OF MAMMALIAN RENAL CILIA cerebral cortex. 2. Zellforsch. Mikroscop. Anat., 60: 369-386. Fawcett, D. W., and K. R. Porter 1954 A study of the fine structure of ciliated epithelia. J. Morph., 94: 221-281. Harper, J. T., H. Puchtler, S. N. McLoan and M. S. Terry 1970 Light microscopic demonstration of myoid fibrils in renal epithelial, mesangial and interstitial cells. J. Microsc., 91: 7185. Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27: 137A138A. Latta, H., A. V. Maunsbach and S. C. Madden 1961 Cilia i n different segments of the rat nephron. J. Biophys. Biochem. Cytol., 11: 248252. Newstead, J. D. 1971 Filaments in renal parenchymal and interstitial cells. J. Ultrastruct. Res., 34: 316-328. Pease, D. C. 1968 Myoid features of renal corpuscles and tubules. J. Ultrastruct. Res., 23: 304-320. Ross, M. H., and J. R. Reith 1970 Myoid elements in the mammalian nephron and their relationship to other specializations i n the basal part of kidney tubular cells. Am. J. Anat., 129: 399-415.
343
Roth, L. E. 1960 Observations on division stages of the protozoan hypotrich Stylonychia. In: Proc. of the Fourth International Congress of Electron Microscopy, Vol. 11. W. Bargmann, D. Peters and C. Wolpers, eds. Springer, Berlin, 1958. Scherft, J. P., and W. T. Daems 1967 Single cilia in chondrocytes. J. Ultrastruct. Res., 19: 546-555. Tandler, B., C. R. Denning, I. D. Mandel and A. H. Kutscher 1970 Ultrastructure of human labial salivary glands. 111. Myoepithelium and ducts. J. Morph., 130: 227-233. Thurau, K. 1966 The influence of sodium concentration at macula densa cells on tubular sodium load. Ann. N. Y. Acad. Sci., 139: 3 8 8 3 9 9 , Tyson, G . E., and R. E. Bulger 1972 Effect of Vinblastine sulfate on the fine structure of cells of the rat renal corpuscle. Am. J. Anat., 135: 319-344. Webber, W. A., and J. Blackbourn 1971 The Permeability of the parietal layer of Bowman’s capsule. Lab. Invest., 25: 367-373. Webber, W. A., and J. Lee 1974 The ciliary pattern of the parietal layer of Bowman’s capsule. Anat. Rec., 180: 449-456. Zimmerman, K. W. 1898 Beitrage zur kenntnis einiger Driisen und Epithelien. Arch. Mikrosk. Anat., 52: 552-706.
ABSTRACT Studies were carried out, using transmission electron microscopy, of the cilia of the nephrons of rat and human kidneys. Cilia were observed in the parietal layer of Bowman's capsule, in the proximal tubule, the distal tubule, including the macula densa, and the collecting duct. They had a number of characteristic features, including the presence of a centriole adjacent to the basal body, long, slender cross-striated rootlets, and a typically organized basal body. The shaft of the cilia differed from the typical 9 2 pattern of organization. Near the base of the cilia the pattern was 9 0. In the middle portion, one or more of the peripheral doublets had been displaced centrally to give an 8 1 or a 7 2 pattern, while towards the tip the pattern became more irregular and the doublets were reduced to single microtubules. We have hypothesized that these cilia may be vestigial. They may, if motile, have some minor stirring function, or they may play a sensory role, as has been postulated for similarly structured cilia in other sites.
+
+
+
+
Cilia have long been known to be present in large numbers in the renal tubules of many animal species (Latta et al., '61; Zimmerman, 1898) and have been observed to have a propulsive function where they are numerous (Fawcett and Porter, '54). In the mammalian kidney they have frequently been observed as single, isolated structures to which little significance has been ascribed. Recently, with the extensive use of scanning electron microscopy, a number of authors have reported the general pattern of ciliation in the kidney of both man and rat (Andrews and Porter, '74; Bulger et al., '74; Webber, '74). On the parietal layer of Bowman's capsule, the proximal tubule, the distal tubule, and in the light but not the dark cells of the collecting duct, each cell usually bears a single cilium, although occasionally two are present. This uniform pattern has raised the question as to whether these cilia are vestigial or whether they may have some specific function. Cilia are usually motile and serve either to move the organism or to move material relative to a surface. These cilia typically have an internal structure consisting of nine peripherally placed doublets of microtubules and a pair of single centrally placed microtubules, the 9 2 pattern. A
+
ANAT.REC., 182: 339-344.
variety of other patterns has also been described, for example in insect sperm (Baccetti et al., '74). In many sites, however, cilia occur singly in locations where motility would not appear to be an important function (Scherft and Daems, '67). A number of these sites are in sense organs or part of the nervous system, some are in contractile cells, notably myoepithelial cells where they may be always present (Tandler et al., '70), while others are in a variety of cells with neither contractile nor sensory functions, such as chondrocytes (Scherft and Daems, '67). In the cilia associated with the nervous system, it has been reported that the fine structure differs from the typical 9 2 pattern in that the central pair of single microtubules are absent, giving a 9 0 pattern. In addition, these cilia have a centriole adjacent to the basal body which is not usually seen in typical motile cilia except during development (Dahl, '63). In certain respects, the cells of the nephron resemble contractile cells. For example, they frequently contain basally located fibrils which have been shown to be actin-like (Harper et al., '70; Newstead, '71; Pease, '68; Ross and Reith, '70). Contractility has not, however, been
+
+
Received Dec. 10, '74. Accepted Feb. 24, '75.
339
340
W . A. WEBBER AND J. LEE
conclusively demonstrated. Furthermore, in the macula densa of the nephron it has been postulated that some form of sensing device exists which allows monitoring and response to the composition of the tubular fluid (Thurau, '66). Because of these associations, and the fact that it seemed unlikely that the renal cilia perform a major propulsive role, we were interested in determining whether or not these cilia had the typical 9 2 pattern or some other arrangement of microtubules, and whether they showed any other special ultrastructural characteristics. We were also interested in knowing whether their structure was uniform throughout the nephron and whether it was similar in both man and rat. We therefore undertook a study of the ultrastructure of the cilia in a number of parts of the nephron in these two species.
+
MATERIALS AND METHODS
Male and female rats of the Wistar strain were used. The animals were anaesthetized with a combination of phenobarbital and pentobarbital, and the left carotid artery cannulated. The kidney was fixed by retrograde aortic perfusion with Karnovsky's fixative (Karnovsky, '65) diluted 50% with cacodylate buffer at 100 mm Hg. Following perfusion, the tissue was prepared for either scanning or transmission electron microscopy using standard techniques (Webber and Blackbourn, '71). The sections were examined in either a Philips 200 or 300 electron microscope. Cross sections of cilia were examined in the parietal layer of Bowman's capsule, in the proximal tubule, in the distal tubule generally, and in the macula densa and in the collecting duct. Human tissue obtained at autopsy after being fixed by immersion was processed and examined in similar fashion to the rat kidney. Comparison was then possible of the structural features of the cilia from the various segments of the nephron in both species. Since the cilia are relatively sparsely distributed, ideal cross sections are rarely encountered. The results reported here are the result of observations of at least 50 cross sections of cilia in each site on the tubule in both species studied.
RESULTS
The fine structure of the cilia was found to be similar in all segments of the nephron in both species. The following structural Eeatures were seen: ( a ) There were fine cross striated rootIets (R) extending into the cytoplasm from the basal body (fig. 1). ( b ) In appropriate sections, there was a centriole (C) present, oriented at right angles to the basal body (fig. 1). Th'is association has been observed previously in the tubule by Latta ('61) and in the parietal layer of Bowman's capsule (Tyson and Bulger, '72). ( c ) The basal body (B) was structurally typical, having nine peripherally arranged triads of microtubules (fig. 2). ( d ) The base of the shaft, which ranged from 0.20-0.25 in diameter, was atypical in that it contained nine peripherally located doublets of microtubules but lacked single central microtubules (fig. 3). (e) The pattern rapidly changed to 8 1 or 7 2 pairs of doublets (figs. 4, 5, 6 ) . ( f ) More distally, there was a reduction in the number of doublets, and some doublets changed to single microtubules (fig. 7).
+
+
DISCUSSION
The cilia of the mammalian nephron which are characteristically present as a single structure in each cell are not normal cilia with a 9 2 pattern of microtubules. Rather they resemble the cilia which have been reported singly in a considerable number of sites (Scherft and Daems, '67). Thus our results differ somewhat from those of Latta ('61) who, on the basis of longitudinal sections, reported the cilia of the rat nephron to be typical kinocilia. In at least some of these other sites a relationship with sensory function exists although i t is not known what role, if any, the cilia play in the sensory process. At the present time we know little about the function of the cilia in the nephron. It is unlikely that they play a major propulsive role as they do in lower animals in which they are numerous (Fawcett and Porter, '54). If they are motile, and this has not been unequivocally demonstrated, they could at most have some stirring action on the tubular
+
FINE STRUCTURE OF MAMMALIAN RENAL CILIA
34 1
Fig. 1 Longitudinal section of a cilium in the distal tubule of the rat showing the basal body, a n adjacent centriole ( C ) and a striated rootlet (R). X 45,000. ____ Fig. 2 Cross section of a cilium in the distal tubule of the rat showing the appearance of a
the 9
+ 0 pattern of microtubules. x 49,500.
an 8
+ 1 arrangement of microtubules. x 49,500.
342
W. A. WEBBER AND J. LEE
contents. It is of course possible that they are simply vestigial and constitute the remnants of the much more numerous cilia seen in other species. In support of this concept it is worth noting that during development the central microtubules have been reported to develop after the outer ring (Roth, '60) and these cilia may simply be incompletely developed. On the other hand, however, the widespread occurrence of similar cilia in sensory structures, in nervous tissue and in contractile cells, raises the interesting possibility that they may have some sensory role and that they may provide a means by which the tubular contents can be monitored. Such an arrangement would allow for a local response to altered composition of the tubular fluid and provide for one element of a system maintaining glomerulo-tubular balance. In this regard it is fairly well established that individual nephrons tend to maintain glomerulo-tubular balance and i t has been postulated that through the macula densa the juxtaglomerular apparatus is responsive to some parameter of tubular fluid (Thurau, '66). It would appear, therefore, that these cilia which show interesting structural variations warrant detailed functional study. ACKNOWLEDGMENTS
The support of the British Columbia Heart Foundation and the technical assistance of Mrs. Patricia Hollingdale are gratefully acknowledged. LITERATURE CITED Andrews, P. M., and K. R. Porter 1974 A scanning electron microscope study of the nephron. Am. J. Anat., 140: 81-115. Baccetti, R., R. Pallai, F. Giusti and F. Bernini 1974 The spermatozoan of Arthropoda XXIII. The 9+9+3 Spermatozoan of Simulid Diptera. J. Ultrastruct. Res., 46: 427-440. Bulger, R. E., F. L. Siege1 and R. Pendergrass 1974 Scanning and transmission electron microscopy of the rat kidney. Am. J. Anat., 139: 483-501. Dahl, H. A. 1963 Fine structure of cilia in rat Fig. 6 Cross section of a more distal portion of a cilium in the distal tubule of the rat showing a somewhat irregular 7 2 pattern of microtubules. x 49,500. Fig. 7 Cross section of a cilium in the distal tubule of the rat showing reduction of the microtubular doublets to single microtubules in a 7 2 pattern. X 49,500.
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Fig. 5 Cross section of a more distal portion of a cilium from human proximal tubule showing a similar arrangement of microtubules to figure 4. X 49,500.
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FINE STRUCTURE OF MAMMALIAN RENAL CILIA cerebral cortex. 2. Zellforsch. Mikroscop. Anat., 60: 369-386. Fawcett, D. W., and K. R. Porter 1954 A study of the fine structure of ciliated epithelia. J. Morph., 94: 221-281. Harper, J. T., H. Puchtler, S. N. McLoan and M. S. Terry 1970 Light microscopic demonstration of myoid fibrils in renal epithelial, mesangial and interstitial cells. J. Microsc., 91: 7185. Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27: 137A138A. Latta, H., A. V. Maunsbach and S. C. Madden 1961 Cilia i n different segments of the rat nephron. J. Biophys. Biochem. Cytol., 11: 248252. Newstead, J. D. 1971 Filaments in renal parenchymal and interstitial cells. J. Ultrastruct. Res., 34: 316-328. Pease, D. C. 1968 Myoid features of renal corpuscles and tubules. J. Ultrastruct. Res., 23: 304-320. Ross, M. H., and J. R. Reith 1970 Myoid elements in the mammalian nephron and their relationship to other specializations i n the basal part of kidney tubular cells. Am. J. Anat., 129: 399-415.
343
Roth, L. E. 1960 Observations on division stages of the protozoan hypotrich Stylonychia. In: Proc. of the Fourth International Congress of Electron Microscopy, Vol. 11. W. Bargmann, D. Peters and C. Wolpers, eds. Springer, Berlin, 1958. Scherft, J. P., and W. T. Daems 1967 Single cilia in chondrocytes. J. Ultrastruct. Res., 19: 546-555. Tandler, B., C. R. Denning, I. D. Mandel and A. H. Kutscher 1970 Ultrastructure of human labial salivary glands. 111. Myoepithelium and ducts. J. Morph., 130: 227-233. Thurau, K. 1966 The influence of sodium concentration at macula densa cells on tubular sodium load. Ann. N. Y. Acad. Sci., 139: 3 8 8 3 9 9 , Tyson, G . E., and R. E. Bulger 1972 Effect of Vinblastine sulfate on the fine structure of cells of the rat renal corpuscle. Am. J. Anat., 135: 319-344. Webber, W. A., and J. Blackbourn 1971 The Permeability of the parietal layer of Bowman’s capsule. Lab. Invest., 25: 367-373. Webber, W. A., and J. Lee 1974 The ciliary pattern of the parietal layer of Bowman’s capsule. Anat. Rec., 180: 449-456. Zimmerman, K. W. 1898 Beitrage zur kenntnis einiger Driisen und Epithelien. Arch. Mikrosk. Anat., 52: 552-706.
