I. Renal corpuscle (most of which is called glomerulus)

II. Proximal tubule

III. Thin limbs of the loop of Henle (intermediate tubule)

IV. Distal tubule

I. Connecting tubule (CNT)

II. Cortical collecting duct (or tubule) (CCD)

III. Outer medullary-collecting duct (or tubule) (OMCD)

IV. Inner medullary-collecting duct (or tubule) including the papillary collecting ducts (also called the ducts of Bellini; IMCD)

Nephrons lie in characteristic positions (Fig. 1.2), with the renal corpuscles and proximal convoluted segments in the cortex (Figs. 1.3 and 1.4). The straight part of the proximal tubule, the thin limb segments, and the straight part of the distal tubule form the loop of Henle, which enters a medullary ray of the cortex and extends into the medulla, where it bends, returning to the cortex by means of the same medullary ray. The loops of juxtamedullary nephrons directly connect the outer stripe of the medulla without ever being contained in a medullary ray. As the straight part of the distal tubule returns to the cortex, it passes by the renal corpuscle from which the nephron originated, forming the macula densa; then, after a short postmacula densa segment, it continues as the distal convoluted tubule within the cortex.

The morphology of the nephron varies with the position of the renal corpuscle in the cortex. Each nephron is classified as superficial, midcortical, or juxtamedullary, according to the position of its renal corpuscle within the respective regions of the cortex (Fig. 1.2) and the pattern of efferent vessel formation.⁹,¹⁰,¹¹ A given segment tends to occupy a specific region of the kidney, which gives rise to the gross zonation referred to in the preceding text. In the human kidney, superficial nephrons empty singly into a terminal collecting duct, whereas several juxtamedullary nephrons empty into an arched tubular portion (arcade) that courses peripherally in the cortex before it turns to enter a medullary ray. Most midcortical nephrons from humans empty individually as well.⁵,¹² As known from the study of several species (rats and rabbits), an arcade is established by the connecting tubule epithelium (data from studies in humans are not available).

Nephrons also are classified as short or long looped according to the location of the position where their loops of Henle turn within the kidney. Short-looped nephrons arise from renal corpuscles located in superficial and midcortical regions and have loops of Henle that turn within the outer medulla. In humans, some superficial nephrons may have loops within the cortex itself. Short-looped nephrons have
short, thin limb segments that occur only along the descending limb. Long-looped nephrons have loops of Henle that turn within the inner medulla and have thin limb segments in both descending and ascending limbs. Although most species have both long- and short-looped nephrons, some species, such as dogs and cats, have only long ones,¹³ whereas other species, such as beavers, have only short ones.¹⁴,¹⁵ In human kidneys, the ratio of short- to long-looped nephrons is 6:1 to 7:1.

The visceral epithelial cells (nowadays generally called podocytes) are octopus-shaped cells that reside in Bowman’s space and attach to the GBM only by way of their processes (see below). This shape was well described by Zimmermann¹⁹ and is seen to advantage in scanning electron micrographs (SEM) (see later, Fig. 1.10). The exact details of cell shape differ depending
on the species being studied.²⁰,²¹ As a consequence of the high degree of differentiation, podocytes, like neurons, are incapable of regenerative cell replication in the adult. Thus, podocytes that were lost, for any reason, cannot be replaced by new ones.²²,²³,²⁴ On the other hand, in glomerular diseases based on dedifferentiation of podocytes (collapsing glomerulopathy), a vivid proliferation of the dedifferentiated cells accompanies the disease.²⁵

Recent observations in parietal epithelial cells question this view. First, it has been proposed that a niche of glomerular epithelial stem cells resides within the parietal epithelium at the transition to the proximal tubule.²⁶,²⁷ It is an intriguing hypothesis that proliferating stem cells from this locus may transform into podocytes and may reach the tuft via the transitions of the epithelia at the glomerular vascular pole. Migration of parietal cells onto the the vascular pole and subsequent transition into podocytes have been shown to occur in the newborn mouse.²⁸ However, evidence that such a process may be of any relevance in the adult has so far not been presented.²⁸ Moreover in the adult mouse, it has been shown that proliferating parietal epithelial cells may reach the glomerular tuft via tuft-capsule bridges; there they replace podocytes causing the collapse of the concerned tuft area obviously by bringing the local cross talk between podocytes and the endocapillary compartment to an end.²⁹

The cell bodies give rise to large primary processes that may branch another time, finally splitting apart into terminal processes, called foot processes (as seen from SEM pictures, “finger processes” would be a better word) or pedicels (Figs. 1.6, 1.8,1.9,1.10,1.11, and see Fig. 1.13). The foot processes are anchored within the GBM to a depth of about 40 to 60 nm. The foot processes interdigitate in a complicated manner with those from adjacent cells to form an elaborate layer of small processes along the glomerular basement membrane. This interdigitation results in the formation of an extensive series of narrow slits between the foot processes, which provide a long extracellular path for filtration of water and solutes (Fig. 1.10). In transmission electron micrographs, these slits are bridged by a layer of extracellular material (4 to 6 nm thick) called the filtration-slit membrane (see Figs. 1.9 and 1.13). If tannic acid is added to the fixative solution, a highly ordered isoporous substructure is revealed in en face views of the filtration-slit membrane.³⁰ Staggered rodlike units project from the podocyte plasmalemma and connect to a central linear bar. These rodlike units delineate rectangular pores 4 × 14 nm within the slit membrane (i.e., which approximate the size of an albumin molecule).

Generally, the slit diaphragm is considered as an adherenslike intercellular junction.³¹ Intensive research in recent years has uncovered several transmembrane proteins that participate in the formation of the slit membrane (Fig. 1.11)—including P-cadherin,³¹ nephrin,³² Neph1,³³ and FAT.³⁴ Other molecules, such as ZO1,³⁵ Podocin,³⁶ CD2AP,³⁷ and catenins mediate the connection to the actin cytoskeleton (see below). Nephrin is a member of the immunoglobin superfamily (IgCAM); its gene, NPHS1, has been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type.³² In addition to its role as a structural component, nephrin acts as a signaling molecule that can activate MAP kinase cascades.³⁸ Neph1 is considered as a ligand for nephrin. Podocin belongs to the raft-associated stomatin family, whose gene, NPHS2, is mutated in a subgroup of patients with autosomal recessive steroid-resistent nephrotic syndrome.³⁶ These patients show disease onset in early childhood and rapid progression to end-stage renal failure. Podocin interacts with nephrin and CD2AP³⁹ FAT is a novel member of the cadherin superfamily with 34 tandem cadherinlike extracellular repeats and a molecular weight of 516 KDa.⁴⁰ Because FAT has a huge extracellular domain, it is speculated that it dominates the molecular structure of the slit membrane³⁴; the FAT mutant mouse fails to develop a slit membrane.⁴¹ P-cadherin³¹ is thought to mediate with its intracellular domain the linkage to β- and γ-catenin, a complex which then connects to the actin cytoskeleton via α-catenin and α-actinin. Taken together, many components of the slit membrane are known, but an integrative model of its substructure including all components is thus far lacking.

The cell body of podocytes contains a large nucleus that tends to be indented in the region of the large Golgi apparatus. Furthermore, it houses abundant rough-surface endoplasmic reticulum; individual cisternae, generally arranged in one complex per cell, are widened and filled with fine granular material of varying electrondensity—their relevance is unknown. In addition to the synthesis of membrane proteins necessary to supply the huge surface of their processes, podocytes in the adult synthesize and secrete all components of the GBM⁴² (see below). On the other hand, abundant multivesicular bodies (predominantly found in the large cell processes) demonstrate strong catabolic activity in podocytes.

Podocytes contain a well-developed cytoskeleton that accounts for the unique shape of the cells and the maintenance of the processes. In the cell body and the primary processes, microtubules and intermediate filaments, such as vimentin and desmin, dominate, whereas microfilaments are densely accumulated in the foot processes. Here, they are part of a complex contractile apparatus.⁴³ The microfilaments form loop-shaped bundles, with their limbs running along the longitudinal axis of the foot processes. The bends of these loops are located centrally at the transition to the primary processes and are probably connected to the microtubules by the microtubule-associated protein τ.⁴⁴ Peripherally, the actin bundles appear to be anchored in the dense cytoplasm associated with the cell membrane of the soles of foot processes, and are dynamically linked to the slit diaphragm complex (discussed earlier). The importance of the podocyte actin cytoskeleton is emphasized by the discovery of inherited forms of focal segmental glomerulosclerosis (FSGS) caused by
mutations in actin-binding proteins: α-actinin-4 is a widely expressed homodimeric protein that bundles and crosslinks actin, and is highly expressed within podocyte foot processes. It interacts with a variety of other adhesion and signalling molecules. Several mutations in the ACTN4 gene encoding this protein cause a late-onset, autosomal dominant form of kidney failure.⁴⁵,⁴⁶ More recently, nine independent missense mutations in INF2, which encodes a member of the formin family of actin-regulating proteins, were shown to segregate with FSGS in 11 unrelated families.⁴⁷

Specific transmembrane matrix receptors anchor the podocyte foot processes to the GBM. Two systems are so far known. The first is a specific integrin heterodimer consisting of α₃β₁ integrins. Within the GBM, these integrins bind to collagen type IV, fibronectin, and laminin 11.⁴⁸,⁴⁹ Second, a dystroglycan complex connects the intracellular molecule utrophin to laminin 11, agrin, and perlecan in the GBM.⁵⁰,⁵¹ Both integrins and dystroglycans are coupled via adapter molecules (paxillin, vinculin, α-actinin, etc.) to the podocyte cytoskeleton, allowing outside-in and inside-out signaling as well as transmission of mechanical force in both directions.

In addition to the actin cytoskeleton within the foot processes, a subplasmalemmal actin system is found in podocytes.⁴³ This actin network is connected to the transmembrane sialoprotein podocalyxin, which represents the major protein of the negatively charged surface coat of podocytes.⁵²,⁵³ The cell coat has the characteristics of a glycocalyx that contains sialic acid. A decrease in the content of sialic acid is associated with a podocyte foot process effacement and results in protein leakage through the filter, which has been shown under a great variety of circumstances.⁵⁴,⁵⁵,⁵⁶,⁵⁷,⁵⁸

A huge body of data has been accumulated in recent years concerning the inventory of receptors and signaling processes starting from them in podocytes. cGMP signaling (stimulated by atrial natriuretic peptide [ANP], brain natriuretic peptide [BNP], and C-type natriuretic peptide [CNP], as well as by nitric oxide (NO]), cAMP signaling (stimulated by prostaglandin E₂, dopamine, isoproterenol, parathyroid hormone [PTH]/PTH-related peptide (PTHrP]), and Ca²⁺ signaling (stimulated by a huge number of ligands, including angiotensin II, acetylcholine, prostaglandin F₂ (PGF₂), arginine vasopressin [AVP], adenosine triophosphate [ATP], endothelin, histamine, etc.) have been identified.⁵⁹ An example of an ion channel of particular importance in podocytes is transient receptor potential canonical 6 (TRPC6), a nonselective cation channel, which is activated by diacylglycerol in a protein kinase C-dependent manner.⁶⁰ Mutations in TRPC6 were found to cause autosomal dominant, late adult-onset proteinuria, with a similar clinical phenotype as seen in ACTN4-mediated FSGS.³⁷ The major target of this signaling orchestra is the cytoskeleton, the concrete effects of which, however, are poorly understood. Other receptors, such as for C3b,⁶¹ Heymann’s antigen,⁶² transforming growth factor β (TGFβ),⁶³,⁶⁴ fibroblast growth factor 2 (FGF2),⁶⁵ and various other cytokines and chemokines have been shown to be involved in the development of podocyte diseases.⁵⁹

The endothelium consists of a simple squamous layer of fenestrated (porous) cells with the cell nuclei generally located near the axial region of the capillary loop (Fig. 1.6). The fenestrated regions (Fig. 1.12), which compose roughly 55% of the surface area,⁶⁶ have a thin layer of cytoplasm (about 50 nm thick) penetrated by numerous fenestrae of round, oval, or irregular shape and varying sizes. The total area occupied by fenestrae accounts for 13% of the capillary surface. The fenestrated regions outline the pericapillary portions of the glomerular basement membrane, but also may be found adjacent to the mesangium (Fig. 1.8). The fenestrae have a diameter of 50 to 100 nm (thus they are larger than endothelial fenestrae elsewhere in the body) and are not bridged by diaphragms. Thus, this kind of a porous endothelium is unique for glomerular capillaries. Fenestrae with diaphragms are found only in the outflow segment of the efferent arteriole.⁶⁷ Nonfenestrated regions are generally seen over nuclei and mesangial cell regions. Human glomerular endothelial cells also are fenestrated.⁶⁸,⁶⁹ A cell coat that is rich with polyanionic glycoproteins, including podocalyxin, covers the endothelial surface⁷⁰,⁷¹,⁷² and appears to fill the fenstraelike “sieve plugs.”⁷³ In addition, above this “classic” glycocalix a 200-nm thick endothelial surface layer has been revealed consisting of loosely attached plasma compounds.⁷⁴

As elsewhere in the body, glomerular endothelial cells are active participants in processes controlling coagulation, inflammation, and immune processes. Renal endothelial cells express surface antigens of the class 2 histocompatibility complex. Like platelets, glomerular endothelial cells contain components of the coagulation pathway and are capable of binding factors IXa and Xa and synthesize, release, and bind von Willebrand factor (factor VIII).⁶⁹ Glomerular endothelial cells synthesize and release endothelin-1 and endotheliumderived relaxing factor (EDRF).⁷⁵ Glomerular endothelial cells have receptors for vascular endothelial growth factor (VEGF) and angiopoetin I that are produced by podocytes.⁷⁶,⁷⁷ The signaling axis via VEGF appears to have a major relevance for the maintenance of the glomerular tuft. Glomerular endothelial cells, in turn, synthesize and secrete platelet-derived growth factor β (PDGFβ) that acts on adjacent mesangial cells.⁷⁸

The GBM covers the capillary loops except in the axial region, where it is reflected over the mesangium to the next capillary loop, accompanied by the layer of foot processes (Figs. 1.6,1.7,1.8). The endothelial cells do not have a separate basement membrane; thus, the endothelial cells directly abut the mesangium toward axial regions. In adult humans, the basement membrane has a mean diameter of 320 to 340 µm.⁷⁹ It is thinner in young children and most laboratory animals.⁸⁰

The basement membrane is composed of three layers: an outer, less dense subepithelial layer, the lamina rara externa; a central, electron-dense layer, the lamina densa ; and an inner subendothelial layer, the lamina rara interna, which is continuous with the mesangial matrix (see Figs. 1.9 and 1.13).

During glomerulogenesis, the GBM is generated as two separate layers produced by glomerular endothelial and epithelial cells. The two sheets are fused together to form the mature GBM.⁸¹,⁸²,⁸³,⁸⁴ In the adult, the GBM is subject to a continuous turnover,⁸⁵,⁸⁶,⁸⁷,⁸⁸ but few details are known so far about these processes. Podocytes appear to be the dominant cell type to synthesize and probably to degrade the GBM. Podocytes are alone capable to synthesize all the components of the GBM⁴²,⁸⁹,⁹⁰; glomerular endothelial cells and also mesangial cells may contribute to the formation of the GBM.⁹¹ It is less clear how the GBM degrades. In recent years, several extracellular matrix degrading enzymes have been described being produced by podocytes and mesangial cells.⁹²,⁹³,⁹⁴ The relevance of these enzymes for the turnover of the GBM remains to be established.

The GBM is generally considered as a hydrated meshwork consisting of collagen type IV, laminin, entactin/nidogen, and sulfated proteoglycans including agrin and perlecan.⁴²,⁹⁵,⁹⁶,⁹⁷,⁹⁸,⁹⁹ Models of the ultramicroscopic structure of the basement membrane picture the GBM as a mat of collagen type IV. Monomers of type IV collagen consist of a 400-µm triple helix that, at its carboxy-terminal end, has a large noncollagenous globular domain called NC 1. At the aminoterminus the helix possesses a 60-µm triple helical rod, the 7S domain. Interactions between the 7S domain and the NC1 domain allow collagen type IV monomers to form tetramers that, by lateral association of triple helical strands, assemble into a three-dimensional network.¹⁰⁰,¹⁰¹ Laminin forms a second network that is superimposed to the collagenous network. Laminin is a noncollagenous glycoprotein consisting of three polypeptide chains, two of which are glycosylated and cross-linked by disulfide bridges.⁹⁵,¹⁰²,¹⁰³ Laminin binds to specific sites on the polymerized network of type IV collagen as well as the basal endothelial and epithelial integrins (see the preceding). The α-5-, β-2-, γ-1-laminin chains are assembled to form the GBM-specific heterotrimeric laminin 11.⁴⁸ This combined network of collagen type IV and laminin provides mechanical stability to the basement membrane and serves as a basic structure on which other matrix components attach.

The proteoglycans of the basement membrane consist of core proteins and covalently bound glycosaminoglycans, which are concentrated in the laminae rarae internae and externae, where they have been referred to as anionic sites and can be localized with cationic probes.¹⁰⁴ The major proteoglycans of the GBM are of the heparan sulfate type—the most prominent is agrin,⁹⁶ but perlecan also has been shown to occur in the GBM.⁴² Digestion of these molecules with heparinase leads to a dramatic increase in the permeability of the basement membrane to anionic native ferritin used as a probe.⁷⁰

The mesangium consists of mesangial cells that are embedded in a mesangial matrix. The term mesangium was introduced by Zimmermann in 1929¹⁹ to describe the cells that form the stalk of the glomerulus and the axes of its lobules. Glomerular capillaries pursue a tortuous, highly anastomosing course around the mesangial axes. Together with the capillaries, the mesangium occupies the space inside the GBM, frequently termed the “endocapillary region.” Topographically, the mesangium can be subdivided into a juxtacapillary region, where it abuts the capillary endothelium, and more centrally located axial regions, which are bound by the perimesangial GBM (Figs. 1.5,1.6,1.7).¹⁰⁵ The glomerular mesangium is continuous with the extraglomerular mesangium (Polkissen or lacis cells) along the glomerular stalk (Fig. 1.5). Both intraglomerular and extraglomerular mesangial cells have many similarities.

Mesangial cells are quite irregular in shape, with many cytoplasmic processes extending from the cell body toward the GBM. They have structural characteristics similar to those of smooth muscle cells in that they contain many bundles of microfilaments (especially within the cell processes) and peripheral dense bodies. Actin, myosin, and α-actin have been shown by immunocytochemistry to be contained in mesangial cells.¹⁰⁶,¹⁰⁷ The relevance of mesangial cell contractility is discussed in the following text.

The processes of mesangial cells extend toward the GBM, to which they are attached either directly or by the interposition of extracellular bundles of microfibrils (Figs. 1.6 and 1.9). The GBM has to be considered as the effector structure of mesangial contractility.¹⁰⁵,¹⁰⁸ Connections between mesangial cells and the GBM are especially prominent in the juxtacapillary region. At this site, tonguelike mesangial cell processes (packed with microfilament bundles) run underneath the capillary endothelium toward the turning points (mesangial angles) of the GBM, where they are anchored. Generally, two of these processes interconnect the GBM from two opposing mesangial angles (Fig. 1.8). In the axial mesangial region, contractile filament bundles are predominantly found within the numerous fingerlike projections of mesangial cells. These microprojections also run toward the GBM and are anchored to it. As in the juxtacapillary region, these microfilament bundles interconnect opposing parts of the GBM.¹⁰⁵

The mesangial matrix fills the highly irregular spaces between mesangial cells and the perimesangial part of the GBM. In immunocytochemical studies, collagen types IV and V, heparan sulfate proteoglycan, fibronectin, laminin, and entactin have been localized within the mesangial matrix.⁹⁹,¹⁰⁹,¹¹⁰ Among these components, fibronectin is the most abundant and has been shown to be associated with microfibrils.¹⁰⁹,¹¹¹ Fibrillin 1 and other specific elastic fiber proteins have been detected in the glomerular mesangium and have been shown to be produced by mesangial cells.¹¹²,¹¹³

In specimens prepared for transmission electron microscopy according to routine methods, the mesangial matrix appears as basement membranelike material, albeit more fibrillar in character than the basement membrane proper.¹¹⁴ In specimens prepared by a technique that avoids osmium tetroxide and uses tannic acid for staining, the mesangial matrix is seen to contain a dense network of microfibrils.¹⁰⁵,¹¹⁵ Microfibrils are noncollagenous, nonbranching, hollow structures of indeterminate length that are about 15 µm thick.¹¹⁶ Within the mesangium, microfibrils form a three-dimensional network that establishes a solid base of contact between mesangial cells and the GBM, fettering the GBM to mesangial cells. Distinct bundles of microfibrils may be regarded as “microtendons” that allow the transmission of contractile force of mesangial cells to specific sites of the GBM.¹⁰⁵,¹¹⁵ The functional relevance of this system is discussed later.

The relevance of mesangial cells in phagocytosis is well documented. Mesangial cells are able to ingest particular tracers as well as macromolecules, such as thorotrast,¹¹⁷ ferritin,¹¹⁴ and aggregated proteins, as well as immune complexes.¹¹⁸ An increased uptake of such materials by mesangial cells has been noted in proteinuric states.¹¹⁴,¹¹⁹ It appears, however, that mesangial cells proper (i.e., mesangial cells that have contractile properties) are not primarily phagocytotic. A small subpopulation (3% to 7%) of cells in this region has been recognized as bone marrowderived—they represent macrophages that have taken up residence in the mesangium.¹²⁰

The essential components of the glomerular filtration barrier are the endothelium (Fig. 1.13), which is perforated by large open pores, the extracellular matrix feltwork of the GBM membrane, and the slit diaphragms between the podocyte
foot processes. When compared with the barrier established in capillaries elsewhere in the body, a glomerular filtration barrier is quite different in two respects: its permeability to water, small solutes, and ions is extremely high, whereas its permeability to plasma proteins the size of albumin and larger is very low.

The high hydraulic permeability is rooted in the fact that filtration occurs along extracellular routes. All components of this route—the endothelial pores, the highly hydrated GBM, and the slit membrane—can be expected to be quite permeable to water and small solutes. Drummond and Deen¹²⁵ have calculated the hydraulic conductance of the individual layers. According to this calculation, the hydraulic resistance of the endothelium is negligible. The GBM and the filtration slits each make up roughly one-half of the total hydraulic resistance of the filtration barrier.

Any decrease in the length of the filtration slit, and thus in slit area, as in experimental and clinical glomerulopathies along with footprocess effacement, correlates with the decrease in the ultrafiltration coefficient K_f.¹²⁶,¹²⁷ A model simulating those conditions showed, along with a decrease in slit area, its relevance in determining increases in flow resistance. The decrease in filtration slit area caused the average path length for the filtrate, through the basement membrane, to increase, thereby explaining the overall decreased hydraulic permeability.¹²⁸

On the basis of evidence of contractility of mesangial cells exposed to vasoconstrictor stimuli in culture,¹²⁹,¹³⁰ of dimensional changes observed in intact glomeruli ex vivo,¹³¹,¹³² and of changes in ultrafiltration coefficient K_f found in vivo in response to vasoactive substances,¹³³,¹³⁴ some researchers have concluded that mesangial cells contract in situ and that this contraction alters glomerular filtration dynamics by decreasing filtration surface area.

Other considerations speak against this possibility. The geometric arrangement of the mesangial contractile apparatus (Fig. 1.6), however, does not seem to be compatible with the previously mentioned sequence of actions. Shortening of the mesangial cell processes connecting opposing angles would only bring the angles closer together, compressing the mesangial capillary interface, but leaving peripheral capillary wall area (filtration area) unaltered. In addition, with regard to the contractility of mesangial cells ex vivo (in culture as well as preparations of whole glomeruli), it should be remembered that mesangial contraction in these cases is not opposed by intercapillary hydrostatic pressure as it is in situ. These considerations, together with the absence of measurable changes in glomerular tuft dimensions in morphometric studies,¹³⁵,¹³⁶ as well as in response to vasoactive substances in vivo,¹³⁷ have led to the suggestion¹²¹ that the mechanical action of the mesangial cell contraction is essentially static in nature, developing tension that serves to counteract expansile forces on the tuft without inducing significant changes in capillary dimension. If mesangial contractility acutely alters the glomerular ultrafiltration coefficient K_f, it is, therefore, probably not because of an acute change in filtration surface area.

The low permeability of the glomerular filtration barrier to plasma proteins is still poorly understood. Several points seem to be relevant. First, there is no vesicular transport of proteins through this barrier as in most other capillaries elsewhere in the body. The barrier function of the glomerular filter for macromolecules is quite specific and is selective for size, shape, and charge.¹³⁸,¹³⁹,¹⁴⁰

In early extensive studies, Farquhar and associates,¹³⁸,¹⁴¹,¹⁴² as well as Rennke and associates,¹⁴³ used tracers, such as ferritin and dextrans of different sizes and charge, to elucidate the role of the various layers in determining the selectivity of this filtration barrier. When their results are summarized, it appears that the basement membrane may be the major barrier to anionic substances, whereas the most restrictive part for uncharged and cationic substances may be the slit diaphragm. Uncharged macromolecules up to an effective radius of 1.8 µm pass freely through the filter. Larger compounds are more and more restricted (indicated by their fractional clearances, which progressively decrease) and are totally restricted at effective radii of more than 4.0 µm. The effective radius is an empiric value, measured in artificial membranes, that takes into account the shape of micromolecules and also attributes a radius to nonspherical molecules. Plasma albumin has an effective radius of 3.6 µm; without the repulsion because
of the negative charge, plasma albumin would pass through the filter in considerable amounts.¹⁴⁴

Thus, since these early studies, the glomerular barrier has been generally suggested to contain a size- and a chargerestrictive element. Despite more than 40 years of intensive research, the principles of the glomerular barrier function are still controversial and poorly understood—a situation that gives room even to hypotheses claiming that the glomerular barrier itself does not have any restrictive properties to macromolecules, such as albumin.¹⁴⁵,¹⁴⁶,¹⁴⁷ A more realistic and elegant recent barrier hypothesis is based on the observation that filtration—the flow of filtrate through the barrier—creates a potential difference of 0.02 to 0.05 mV negative in Bowman’s space and that this potential difference is sufficient to hinder the negatively charged macromolecules like albumin from passing the filter.¹⁴⁸

Summarizing the present status of knowledge, accumulating evidence suggest that the GBM has little relevance in size restriction, but the slit membrane is the decisive size-restrictive element within the glomerular barrier.¹⁴⁹ The current strong engagement in elucidating the molecular architecture of the slit membrane promises to enlighten the porous pattern of the slit membrane and, hopefully, explain its size-restrictive property. Regarding the charge selectivity, it appears that the proximal portion of the barrier, above all the endothelium, are most important.⁷³,¹⁵⁰,¹⁵¹,¹⁵² Furthermore, prevention of albumin from entering the filter is also dependant on normal hemodynamic conditions in glomerular capillaries, as first shown by Ryan and Karnovsky.¹⁵³

The proximal convoluted tubule is the longest and largest segment of the mammalian nephron. The tubule is lined by cells that have an elaborate cell shape based on extensive basolateral interdigitations (Figs. 1.19,1.20,1.21), a well-developed microvillus border, a prominent intracellular digestive tract (endocytic apparatus and lysosomes (Fig. 1.15), and numerous large peroxisomes (microbodies). The single ovoid nucleus lies in the middle to basal region of the cytoplasm.

The straight part of the proximal tubule (pars recta) begins in the medullary ray and penetrates into the outer stripe of the outer zone of the medulla (Fig. 1.18). The proximal pars recta converts into the thin limb near the junction of the inner and outer stripe. In rats, the pars recta includes the final region of both P₂ and P₃,¹⁶⁹ and the transition
between the two segments occurs at various levels in the medullary ray. This region is marked by a sudden increase in microvillar length, a decrease in endocytic apparatus, and a decrease in interdigitation between adjacent cells. The microvilli of the pars recta cells decrease in height and number in rabbits¹⁵⁸ and humans¹⁶² but remain high in rats (Fig. 1.18).

In general, the P₃ pars recta cells have been described as lower in height with a less elaborate shape. In humans, the pars recta cells have a convex apical surface, and some lipid droplets are found in the basal cytoplasm. The mitochondria are fewer and no longer closely applied to the cell membrane. The lysosomes are smaller and the Golgi and endocytic apparatuses are less well developed. Peroxisomes are more numerous in the pars recta.⁸⁰,¹⁶² The tight junctions of P₃ can be more complex in shape, consisting of several junctional strands in rats, dogs, and cats.¹⁷⁰

The functional relevance of the proximal tubule is manifold and quantitatively enormous. It reabsorbs about 70% of filtered Na⁺, Cl⁺, and water; 95% of bicarbonate; 60% of Ca²⁺; and sodium phosphate. Reabsorption of the filtered glucose is complete, whereas reabsorption of amino acids is almost complete. This occurs either by transcellular transport via specific channels and transporters in both membranes (water, sodium, phosphate, glucose, amino acids, and bicarbonate in a specific enzyme-mediated mode) or predominantly by paracellular transport through the leaky tight junctions (Ca²⁺, Cl⁺). Furthermore, filtered macromolecules undergo reuptake by a prominently developed endocytotic apparatus. In addition, the proximal tubule (predominantly S₃ segments) secretes organic cations and anions, which constitute an extraordinarily diverse array of compounds of physiologic, pharmacologic, and toxicologic importance. The transepithelial transport involves separate entry steps at the basolateral membrane and exit steps at the luminal membrane with specific transporters at both sites.¹⁷¹,¹⁷²,¹⁷³,¹⁷⁴ Other potentially toxic xenobiotic compounds are metabolized within the well-developed smooth endoplasmic reticulum of S₃ segments.¹⁷⁵

Unique to the proximal tubule is the reabsorption and degradation of filtered protein and peptides (Figs. 1.15 and 1.16). As discussed previously, all proteins smaller than albumin leak through the glomerular filter; even albumin, in small amounts, is always contained in the filtrate. All these diverse macromolecules are taken up by proximal tubule cells via receptor-mediated endocytosis and degraded in lysosomes. In proteinuric states, this function is greatly enhanced. The uptake and digestion include the following steps.¹⁷⁶,¹⁷⁷

1. Binding of the filtered protein to two multiligand receptors, megalin and cubulin, which are densely accumulated within clathrin-coated pits in the intermicrovillar areas of the apical membrane.

Megalin is a 600-kDa transmembrane protein belonging to the LDL-receptor family (see reviews).¹⁷⁶,¹⁷⁷ The extracellular domain contains four clusters of cysteine-rich, complement-type repeats, constituting the ligand-binding regions. Cubulin is a 416-kDa peripheral membrane protein identical to the intrinsic factor vitamin B₁₂ receptor, known from the small intestine. It contains 27 CUB domains, which are responsible for the ability to interact with a great variety of ligands.

2. Small apical vesicals pinch off from the tubular invagination at the intermicrovillar areas to ferry the protein to the next component.

3. Large apical vacuoles located in the apical cytoplasm are formed by fusion of small vesicles representing the early endocytotic compartment.

4. Condensing vacuoles form in which the protein is condensed. These vacuoles move basally in the cell and acquire hydrolytic enzymes by fusion either with primary or secondary lysosomes. The proteins sequestered within lysosomes appear to be broken down into amino acids that are reused by the cell.

5. Already sequestered in the early endosomal compartment, the receptors are concentrated in dense, apical tubules by which they are returned to the apical plasma membrane.

This process of megalin-mediated internalization appears to be of even much wider relevance. As mentioned previously, the proximal tubule reabsorbs phosphate, representing a key element in phosphate homeostasis. Depending on the amount of sodium phosphate cotransporters present in the brush border cell membrane, the reabsorption varies. Internalization of the phosphate transporter type II (e.g., PTH induced to downregulate reabsorption) occurs via megalin-mediated endocytosis.¹⁷⁸,¹⁷⁹ The proximal tubule has also endocrine relevance. In response to stimulation by PTH, it produces, by adding a second hydroxyl group, the active vitamin D₃, calcitriol.

Thin limbs can be short, occurring only along the descending limb, or they can be long, reaching varying distances into the inner medulla. In the long-looped variety, thin limb segments compose part of both the descending and ascending limbs. Four types of thin limb segments are routinely identified: (1) descending thin limbs of short-looped nephrons (SDTL) (Figs. 1.23,1.24,1.25), (2) upper portions of descending thin limbs of long-looped nephrons (LDTL up), (3) lower portions of descending thin limbs of long-looped nephrons (LDTL lp), and (4) ascending thin limbs of long-looped nephrons (ATL) (Figs. 1.24 and 1.25). This pattern has been observed in rats,¹⁸⁰,¹⁸¹ mice,¹⁸² syric hamsters,¹⁸³ the desert rodent Psammomys obesus,¹⁸⁴,¹⁸⁵ and the rabbit.¹⁵⁸ Thin limbs have not been studied as carefully in humans as
in several other animal species.¹³,¹⁸⁶,¹⁸⁷ An additional subsegment of thin limbs has been identified in chinchillas.¹⁸⁸

Surprisingly, as seen by light microscopy, these simple looking epithelia are strikingly different from each other— not only the ascending from the descending limbs but, most remarkably, the descending limbs of short from those of long loops. Furthermore, within the descending segments, the proximal portion, although structurally not different from the distal portion, express a different pattern of transporters than the distal portion. Even islets or interposed limb pieces of functionally different cells are encountered within an otherwise homogenous thin limb segment.¹⁸⁹

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