Collective Cell Migration Drives Morphogenesis of the Kidney Nephron

Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase–positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow–dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis.     

Neuropeptide Y inhibits cAMP accumulation in renal proximal convoluted tubules

The effect of neuropeptide Y (NPY) on cAMP accumulation in various segments of the rabbit nephron was examined. NPY inhibited parathyroid hormone-stimulated cAMP accumulation in the proximal convoluted tubule in a concentration-dependent manner. NPY also inhibited forskolin-stimulated cAMP production in this segment of the nephron. In contrast, NPY had no effect on parathyroid hormone or forskolin-stimulated cAMP accumulation in the proximal straight tubule. Similarly, NPY had no effect on forskolin-stimulated cAMP levels along the rest of the nephron. These results are consistent with previous studies which have localized NPY receptors to the proximal convoluted tubule, and suggest that NPY via its effects on cAMP metabolism may play a role in proximal tubule transport.     

Nephron structure and immunohistochemical localization of ion pumps and aquaporins in the kidney of frogs inhabiting different environments

Amphibians inhabit areas ranging from completely aqueous to terrestrial environments and move between water and land. The kidneys of all anurans are similar at the gross morphological level: the structure of their nephrons is related to habitat. According to the observation by light and electron microscopy, the cells that make up the nephron differ among species. Immunohistochemical studies using antibodies to various ATPases showed a significant species difference depending on habitat. The immunoreactivity for Na+,K(+)-ATPase was low in the proximal tubules but high in the basolateral membranes of early distal tubules to collecting ducts in all species. In the proximal tubule, apical membranes of the cells were slightly immunoreactive to H(+)-ATPase antibody in aquatic species. In the connecting tubule and the collecting duct, the apical membrane of intercalated cells was immunoreactive in all species. In aquatic species, H+,K(+)-ATPase immunoreactivity was observed in cell along the proximal, distal tubule to the collecting duct. However, H+,K(+)-ATPase was present along the intercalated cells of the distal segments from early distal to collecting tubules in terrestrial and semi-aquatic species. In the renal corpuscle, the neck segment and the intermediate segment, immunoreactivities to ion pumps were not observed in any of the species examined. Taking together our observations, we conclude that in the aquatic species, a large volume of plasma must be filtered in a large glomerulus and the ultrafiltrate components are reabsorbed along a large and long proximal segment of the nephron. Control of tubular transport may be poorly developed when a small short distal segment of the nephron is observed. On the contrary, terrestrial species have a long and well-developed distal segment and regulation mechanisms of tubular transport may have evolved in these segments. Thus, the development of the late distal segments of the nephron is one of the important factors for the terrestrial adaptation.     

Morphology of the Nephron in the Mesonephros of Bufo bufo (Amphibia,Anura, Bufonidae)

The present study deals with the morphology and ultrastruclure of the nephron in the mesonephros of the toad, Bufo bufo (Linnaeus, 1758). Based on serial sections in paraffin, Araldite and Epon, the position of the different segments of the nephron within the kidney tissue was determined, and a nephron subsequently reconstructed. The nephron consists of the following parts: Malpighian corpuscle, neck segment, proximal tubule, intermediate segment, early distal tubule, late distal tubule and collecting tubule. The late distal tubule was subdivided into three morphologically different sections. The total number of nephrons in the toad mesonephros was estimated at 6000 units. The length of the segments in the reconstructed nephron was calculated. The cytology of the epithelial cells constituting the segments was described using transmission and scanning electron microscopy. Heterocellularity was found in the late distal tubule section I and III and in the collecting tubule. The proportional distribution and number of intercalated (mitochondria-rich) cells in the late distal tubule and collecting tubule was calculated. Only one morphological type of intercalated cell could be distinguished. Late distal tubules were removed from fresh Bufo kidneys for preliminary studies of the intercalated cells with Nomarski optics.     

Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney

Recently, we cloned two Na(+)-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na(+) -coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.     

Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney

Recently, we cloned two Na⁺-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na⁺-coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.     

Evidence for differences in the sensitivity to ouabain of NaK-ATPase along the nephrons of rabbit kidney

To determine the possible intrarenal site of action of an endogenous ouabain-like natriuretic factor, we searched for the presence of NaK-ATPase highly sensitive to ouabain in the kidney, an organ previously reported to display a low sensitivity to ouabain. For this purpose, the sensitivity of NaK-ATPase to ouabain was determined at the level of single, well defined segments of nephron microdissected from rabbit kidney. Results indicated that NaK-ATPase activity is 10- to 30-fold more sensitive to ouabain in the collecting tubule, where final adjustments of sodium excretion take place, than in more proximal segments of the nephron. [3H]Ouabain binding experiments confirmed this finding as the affinity for ouabain increases from the proximal tubule to the collecting tubule. These results suggest that endogenous natriuretic factor may control sodium transport in the collecting tubule preferentially.     

Quantitative immunogold localization of Na, K-ATPase along rat nephron.

Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

Immunocytochemical localization of cathepsin D in lysosomes of cortical collecting tubule cells of the rat kidney

Immunocytochemical localization of cathepsin D in rat renal tubules was investigated by means of indirect immunoenzyme and protein A--gold techniques. By light microscopy, fine granular staining was seen in the mesangial cells of glomeruli. Heavy reaction deposits were present in the cortical tubular segments and some of the medullary collecting tubules. The proximal tubules contained a few positive granules. Other segments were negative for cathepsin D. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were present in cytoplasmic granules and multivesicular bodies of the segment of the cortical collecting tubule. These cytoplasmic granules were presumed to be digestive vacuoles (secondary lysosomes) from their morphological profile. The proximal tubule cells contained the very weakly labeled secondary lysosomes. No specific labeling was noted in other segments of the nephron. Control experiments confirmed the specificity of the immunostaining. Quantitative analysis of the labeling density in each subcellular compartment also confirmed that the main subcellular sites for cathepsin D are the secondary lysosomes and multivesicular bodies. The labeling density in these granules of the lysosomal system varied widely with the individual granules, suggesting that there is a considerable heterogeneity of enzyme content among the granules of the lysosomal system. The prominent presence of cathepsin D in the cortical collecting tubule suggests a certain segment-specific function of this proteinase.     

Purinergic signaling in the lumen of a normal nephron and in remodeled PKD encapsulated cysts

The nephron is the functional unit of the kidney. Blood and plasma are continually filtered within the glomeruli that begin each nephron. Adenosine 5' triphosphate (ATP) and its metabolites are freely filtered by each glomerulus and enter the lumen of each nephron beginning at the proximal convoluted tubule (PCT). Flow rate, osmolality, and other mechanical or chemical stimuli for ATP secretion are present in each nephron segment. These ATP-release stimuli are also different in each nephron segment due to water or salt permeability or impermeability along different luminal membranes of the cells that line each nephron segment. Each of the above stimuli can trigger additional ATP release into the lumen of a nephron segment. Each nephron-lining epithelial cell is a potential source of secreted ATP. Together with filtered ATP and its metabolites derived from the glomerulus, secreted ATP and adenosine derived from cells along the nephron are likely the principal two of several nucleotide and nucleoside candidates for renal autocrine and paracrine ligands within the tubular fluid of the nephron. This minireview discusses the first principles of purinergic signaling as they relate to the nephron and the urinary bladder. The review discusses how the lumen of a renal tubule presents an ideal purinergic signaling microenvironment. The review also illustrates how remodeled and encapsulated cysts in autosomal dominant polycystic kidney disease (ADPKD) and remodeled pseudocysts in autosomal recessive PKD (ARPKD) of the renal collecting duct likely create an even more ideal microenvironment for purinergic signaling. Once trapped in these closed microenvironments, purinergic signaling becomes chronic and likely plays a significant epigenetic and detrimental role in the secondary progression of PKD, once the remodeling of the renal tissue has begun. In PKD cystic microenvironments, we argue that normal purinergic signaling within the lumen of the nephron provides detrimental acceleration of ADPKD once remodeling is complete.     

Renal adrenergic receptors: localization of [125I]prazosin binding sites along the microdissected rat nephron.

Norepinephrine stimulates renal tubular sodium reabsorption, probably through an alpha 1-adrenoceptor-mediated mechanism. Although the distribution of alpha 1-adrenoceptors in the kidney has been studied with autoradiography, the precise location of these receptors in isolated nephron segments is unclear. Using a microassay we determined the specific binding of [125I]iodoarylazidoprazosin ([125I]prazosin), a high specific radioactivity analog of the selective alpha 1-antagonist prazosin, to microdissected glomeruli and tubule segments. Specific binding of [125I]prazosin (3 nM) in the proximal convoluted tubule was time- and concentration-dependent, saturable, and reversible. In this segment the apparent KD by association and dissociation rate constants of [125I]prazosin binding was 0.47 nM, and the maximum receptor density was approximately 0.19 fmol/mm, or 720 fmol/mg protein. Binding specificity was verified in competition studies with excess (3 microM) unlabeled prazosin and probes for alpha 2- (yohimbine), beta- (propranolol), dopamine1- (SCH23390), and dopamine2- (S-sulpiride) receptors. [125I]Prazosin binding was inhibited significantly only by unlabeled prazosin. Mapping of prazosin binding along the nephron revealed that the highest density was in the proximal convoluted tubule, followed by the proximal straight tubule. Lesser binding was found in the thick ascending limb and in the distal convoluted tubule, whereas in the cortical and outer medullary collecting duct and in glomeruli, binding was not significantly different from zero.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relative contribution of OAT1 and OAT3 transport activities in isolated perfused rabbit renal proximal tubules

The expression of both OAT1 and OAT3 along the isolated rabbit renal proximal tubule (RPT) was determined using RT-PCR. They were found to be very strong in S2 segment and weak in S1 and S3 segments. We further examined the relative transport activity of these transporters in isolated perfused rabbit RPT using [(3)H]para-aminohippurate ([(3)H]PAH), and estrone sulfate ([(3)H]ES) as specific substrates for rbOAT1 and rbOAT3, respectively. The transport activity of OAT1 was in the order S2>S1=S3 segments and that of OAT3 was in the order S1=S2>S3 segments. The addition of alpha-ketoglutarate (100 muM) in the bathing medium increased both OAT1 and OAT3 transport activities in all segments of proximal tubule. The kinetics of [(3)H]succinic acid transport, used to measure the activity of sodium dicarboxylate transporter 3 (NaDC3), were examined. The J(max) for succinic acid was in the order S2>S3 and unmeasurable in the S1 segment. Our data indicate that both OAT1 and OAT3 play quantitatively significant roles in the renal transport of organic anions along the proximal tubule but predominately in S2 segment. The relative contribution of both transporters depends on their relative expression levels and may possibly be affected by the activity of NaDC3 in RPT.     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.     

Protein localization of SLC26A2 (DTDST) in rat kidney

The SLC26 family represents a group of integral membrane anion transport proteins. Mutations in one member of this protein family, SLC26A2 (DTDST or diastrophic dysplasia sulfate transporter), result in various chondrodysplasias due to undersulfation of proteoglycans in chondrocytes, a major site of DTDST protein expression. DTDST mRNA has been detected in the kidney, but protein expression has not been characterized. Our objective for this study was to determine the protein localization of this sulfate transporter in the kidney. We used immunofluorescence (IMF) techniques with an anti-DTDST monoclonal antibody to examine kidneys harvested from adult rats. Double labeling was performed with antibodies directed against megalin, which is found in the microvillus membrane and coated pits of the proximal tubule. IMF analysis indicated that DTDST protein expression was limited to the microvillus membrane of proximal tubule cells in the renal cortex but absent in glomeruli and other nephron segments. DTDST was also detected in isolated rat kidney proximal tubule microvillus membranes by Western blot analysis, confirming the immunofluorescent localization of the DTDST transporter to this nephron segment. The functional role of the DTDST protein in the kidney is unknown, but it may play a role in proximal tubule sulfate transport.     

The pelvic kidney of male Ambystoma maculatum (Amphibia,urodela, ambystomatidae) with special reference to the sexual collecting ducts

This study details the gross and microscopic anatomy of the pelvic kidney in male Ambystoma maculatum. The nephron of male Ambystoma maculatum is divided into six distinct regions leading sequentially away from a renal corpuscle: (1) neck segment, which communicates with the coelomic cavity via a ventrally positioned pleuroperitoneal funnel, (2) proximal tubule, (3) intermediate segment, (4) distal tubule, (5) collecting tubule, and (6) collecting duct. The proximal tubule is divided into a vacuolated proximal region and a distal lysosomic region. The basal plasma membrane is modified into intertwining microvillus lamellae. The epithelium of the distal tubule varies little along its length and is demarcated by columns of mitochondria with their long axes oriented perpendicular to the basal lamina. The distal tubule possesses highly interdigitating microvillus lamellae from the lateral membranes and pronounced foot processes of the basal membrane that are not intertwined, but perpendicular to the basal lamina. The collecting tubule is lined by an epithelium with dark and light cells. Light cells are similar to those observed in the distal tuble except with less mitochondria and microvillus lamellae of the lateral and basal plasma membrane. Dark cells possess dark euchromatic nuclei and are filled with numerous small mitochondria. The epithelium of the neck segment, pleuroperitoneal funnel, and intermediate segment is composed entirely of ciliated cells with cilia protruding from only the central portion of the apical plasma membrane. The collecting duct is lined by a highly secretory epithelium that produces numerous membrane bound granules that stain positively for neutral carbohydrates and proteins. Apically positioned ciliated cells are intercalated between secretory cells. The collecting ducts anastomose caudally and unite with the Wolffian duct via a common collecting duct. The Wolffian duct is secretory, but not to the extent of the collecting duct, synthesizes neutral carbohydrates and proteins, and is also lined by apical ciliated cells intercalated between secretory cells. Although functional aspects associated with the morphological variation along the length of the proximal portions of the nephron have been investigated, the role of a highly secretory collecting duct has not. Historical data that implicated secretory activity concordant with mating activity, and similarity of structure and chemistry to sexual segments of the kidneys in other vertebrates, lead us to believe that the collecting duct functions as a secondary sexual organ in Ambystoma maculatum. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Potassium pyroantimonate and osmium-zinc iodide reactivity in the tubular epithelium of the opisthonephric kidney of the sea lamprey,Petromyzon marinus L

Pyroantimonate precipitate indicates that the epithelium of the proximal tubule is the only segment of the tubular nephron of the fresh water lamprey where large accumlations of cations are distributed. Unusually large amounts of reaction product are located within the lateral intercellular spaces and within vesicles closely associated with the plasma membrane at the lateral and basal surfaces. This technique suggests the continuity of these vesicles with the plasma membrane and alludes to the possibility of an endomembranous system of vesicles and the intercellular spaces as vehicles for ion transport. Lateral intercellular spaces of proximal tubules of lower vertebrates may play a different role in kidney function that their counterparts in higher vertebrates. Osmium-zinc iodide has a specificity for certain cells within the proximal, intermediate, and distal segments, but no structural differences are noted when these cells are compared to unstained cells. Smooth endoplasmic reticulum remains unstained in the distal segment but the stain has a strong affinity for elements of the Golgi apparatus, lysosomes, and the nuclear envelope of all cell types. This technique does not suggest a structural or functional similarity between cells of the distal segment and the chloride cells of the gills of teleosts.     

Intrarenal localization of degradation of atrial natriuretic peptide in isolated glomeruli and cortical nephron segments

Using isolated glomeruli and nephron segments obtained from collagenase treated rabbit kidneys, we examined the in vitro degradation of alpha-human atrial natriuretic polypeptide (alpha-hANP). The ANP-degrading activity was measured by the amount of immunoreactive ANP remaining after incubation of about 50 fmoles alpha-hANP with each tissue preparation for 7.5 min. The sequence of degrading activity among isolated nephron segments was as follows: proximal straight tubule greater than proximal convoluted tubule greater than cortical collecting tubule greater than distal convoluted tubule greater than cortical thick ascending limb. A single glomerulus exhibited the degrading activity which was comparable to approximately 50% of the activity of 1 mm proximal convoluted tubule. Phosphoramidon, an inhibitor of endopeptidase, prevented the degradation of ANP in proximal convoluted tubule and glomerulus by 68% and 89%, respectively, but not in cortical thick ascending limb and cortical collecting tubule. From these results, we conclude that the degradation of ANP by endopeptidase occurs mainly in the proximal tubule and glomerulus.