Cell renewal of glomerular cell types in normal rats. An autoradiographic analysis

Normal adult Sprague-Dawley rats received either a single or repetitive injection of the DNA precursor 3H-thymidine (3H-TdR). For autoradiography semi-thin sections were prepared 2 hr to 14 days after labeling. The majority of labeled cells noted in glomerular tufts were endothelial cells. Mesangial cells had a lower production rate. Podocytes revealed no evidence of proliferation. Bowman's capsule cells showed a higher labeling index than tuft cells at all times. Neither the urinary nor the vascular pole was found to be a proliferative zone for Bowman's capsule cells. The flash and repetitive labeling experiments demonstrated a constant rate of cell renewal of about 1% per day, resulting in a long life span for endothelial and mesangial cells as well as Bowman's capsule cells. These data provide a basis for cell kinetic studies in models of glomerular diseases.     

Pathways to nephron loss starting from glomerular diseases-insights from animal models

Studies of glomerular diseases in animal models show that progression toward nephron loss starts with extracapillary lesions, whereby podocytes play the central role. If injuries remain bound within the endocapillary compartment, they will undergo recovery or be repaired by scaring. Degenerative, inflammatory and dysregulative mechanisms leading to nephron loss are distinguished. In addition to several other unique features, the dysregulative mechanisms leading to collapsing glomerulopathy are particular in that glomeruli and tubules are affected in parallel. In contrast, in degenerative and inflammatory diseases, tubular injury is secondary to glomerular lesions. In both of the latter groups of diseases, the progression starts in the glomerulus with the loss of the separation between the tuft and Bowman's capsule by forming cell bridges (parietal cells and/or podocytes) between the glomerular and the parietal basement membranes. Cell bridges develop into tuft adhesions to Bowman's capsule, which initiate the formation of crescents, either by misdirected filtration (proteinaceous crescents) or by epithelial cell proliferation (cellular crescents). Crescents may spread over the entire circumference of the glomerulus and, via the glomerulotubular junction, may extend onto the tubule. Two mechanisms concerning the transfer of a glomerular injury onto the tubulointerstitium are discussed: (1) direct encroachment of extracapillary lesions and (2) protein leakage into tubular urine, resulting in injury to the tubule and the interstitium. There is evidence that direct encroachment is the crucial mechanism. Progression of chronic renal disease is underlain by a vicious cycle which passes on the damage from lost and/or damaged nephrons to so far healthy nephrons. Presently, two mechanisms are discussed: (1) the loss of nephrons leads to compensatory mechanisms in the remaining nephrons (glomerular hypertension, hyperfiltration, hypertrophy) which increase their vulnerability to any further challenge (overload hypothesis); and (2) a proteinuric glomerular disease leads, by some way or another, to tubulointerstitial inflammation and fibrosis, accounting for the further deterioration of renal function (fibrosis hypothesis). So far, no convincing evidence has been published that in primary glomerular diseases fibrosis is harmful to healthy nephrons. The potential of glomerular injuries to regenerate or to be repaired by scaring is limited. The only option for extracapillary injuries with tuft adhesion is repair by formation of a segmental adherent scar (i.e., segmental glomerulosclerosis).     

Three-dimensional morphometric analysis of segmental glomerulosclerosis in the rat

In idiopathic nephrotic syndrome, and in experimental models of nephrosis, changes of visceral epithelial cells involve the entire glomerular population while segmental sclerotic changes are reported to affect only a certain number of glomeruli. Because conventional determination of the percentage of glomeruli affected by sclerotic lesions is usually based on the examination of randomly selected sections, we wondered whether glomeruli appearing normal in a given section could be affected by sclerosis in other regions of the capillary tuft (CT). To assess the real incidence and the spatial extension of sclerotic changes at the level of single glomerulus, we used serial-section morphological analysis to measure the volume of the glomerulus and that of sclerosis lesions. In glomeruli from control rats and in glomeruli from adriamycin (ADR) treated rats surface area of Bowman's capsule (BC), CT and sclerotic regions were measured using stereology techniques in all the consecutive sections containing each individual glomerulus, and corresponding volumes were then calculated. Mean volume of BC and CT were not significantly different between control and ADR rats (0.71 +/- 0.03 and 0.53 +/- 0.03 vs. 0.76 +/- 0.04 and 0.53 +/- 0.02 microns 3 x 10(-6), respectively). The distribution of glomerular volume parameters in the ADR rats were more spread out than in control animals, indicating that some glomeruli became smaller while other became larger. No sclerotic changes were found in control rats, while in the three ADR rats 94, 90 and 92% of glomeruli, respectively, were affected by sclerotic lesions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The parietal epithelial cell: a key player in the pathogenesis of focal segmental glomerulosclerosis in Thy-1.1 transgenic mice

Focal segmental glomerulosclerosis (FSGS) is a hallmark of progressive renal disease. Podocyte injury and loss have been proposed as the critical events that lead to FSGS. In the present study, the authors have examined the development of FSGS in Thy-1.1 transgenic (tg) mice, with emphasis on the podocyte and parietal epithelial cell (PEC). Thy-1.1 tg mice express the Thy-1.1 antigen on podocytes. Injection of anti-Thy-1.1 mAb induces an acute albuminuria and development of FSGS lesions that resemble human collapsing FSGS. The authors studied FSGS lesions at days 1, 3, 6, 7, 10, 14, and 21, in relation to changes in the expression of specific markers for normal podocytes (WT-1, synaptopodin, ASD33, and the Thy-1.1 antigen), for mouse PEC (CD10), for activated podocytes (desmin), for macrophages (CD68), and for proliferation (Ki-67). The composition of the extracellular matrix (ECM) that forms tuft adhesions or scars was studied using mAb against collagen IV alpha2 and alpha4 chains and antibodies directed against different heparan sulfate species. The first change observed was severe PEC injury at day 1, which increased in time, and resulted in denuded segments of Bowman's capsule at days 6 and 7. Podocytes showed foot process effacement and microvillous transformation. There was no evidence of podocyte loss or denudation of the GBM. Podocytes became hypertrophic at day 3, with decreased expression of ASD33 and synaptopodin and normal expression of WT-1 and Thy-1.1. Podocyte bridges were formed by attachment of hypertrophic podocytes to PEC and podocyte apposition against denuded segments of Bowman's capsule. At day 6, there was a marked proliferation of epithelial cells in Bowman's space. These proliferating cells were negative for desmin and all podocyte markers, but stained for CD10, and thus appeared to be PEC. The staining properties of the early adhesions were identical to that of Bowman's capsule, suggesting that the ECM in the adhesions was produced by PEC. In conclusion, the authors propose the following sequence of events leading to FSGS lesions in the Thy1.1 tg mice: (1) PEC damage and denudation of Bowman's capsule segments; (2) podocyte hypertrophy and bridging; and (3) PEC proliferation with ECM production.     

Renal structural abnormalities following recovery from acute puromycin nephrosis

BACKGROUND: Rats that recover from acute puromycin nephrosis later develop widespread glomerular and tubulointerstitial injury. The current study sought to identify structural changes present in the recovery phase that could precipitate progressive renal disease. METHODS: Stereologic studies were performed 10 weeks after administration of puromycin (PAN) or saline (Cont). Serial sections were examined to assess glomerular structure. RESULTS: Rats receiving puromycin developed heavy proteinuria that returned nearly to control levels at 10 weeks. Kidneys in these animals were moderately enlarged and exhibited expansion of the interstitium (PAN, 254 +/- 47 mm3; Cont, 152 +/- 23 mm3; P < 0.05). The average glomerular volume was not different from control (PAN, 1.90 +/- 0.38 x 10(6) microm3; Cont, 2.07 +/- 0.47 x 10(6) microm3), but a subpopulation of glomeruli of about half normal size was found in PAN rats. Serial sections revealed that most of these glomeruli were not connected to normal tubule segments. Serial sections also revealed that more than 90% of glomeruli in rats recovering from nephrosis had synechias joining the tuft to Bowman's capsule. Synechias occupied an average of 8 +/- 11% of the Bowman's capsule surface in PAN animals versus less than 1% of the surface in controls. The appearance of synechias was not associated with a reduction in the mean number of visceral or parietal epithelial cells per glomerulus. CONCLUSIONS: Acute puromycin nephrosis does not cause a notable reduction in visceral epithelial cell number. However, widespread glomerular injury characterized by synechia between the tuft and Bowman's capsule is present following remission of proteinuria. Progression of this residual glomerular injury could contribute to the late development of glomerular segmental sclerosis following recovery from acute nephrosis.     

Effects of human Cu, Zn-superoxide dismutase in aminonucleoside nephrosis--evaluation of the morphology and glomerular basement membrane anionic charge sites

It was examined whether superoxide dismutase (SOD) had protective effects in puromycin aminonucleoside nephrosis (PAN). Nephrotic rats were induced by a single intraperitoneal injection of puromycin aminonucleoside (PA, 60 mg/kg). Subcutaneous administration of SOD (30 mg/kg) was started the day before PA injection and continued every 24 hours. Animals were sacrificed 10 days later to assess the morphology and glomerular basement membrane anionic charge sites (CSs). GBM CSs were stained in vitro with polyethyleneimine (PEI) and studied by electron microscopic examination. The SOD-injected group had a significant reduction of urinary protein excretion compared to the PA-treated group. Light microscopy revealed that vast majority of glomeruli in PA-treated group had segmental mesangial expansion and epithelial cell adhesions of the glomerular tuft to Bowman's capsule. Contrasted to these changes in the PA group, glomeruli from SOD-treated group showed less severe morphologic changes. On electron microscopy, the PA-treated rats exhibited marked glomerular epithelial loss of foot processes, epithelial attenuation, cytoplasmic vacuolization and protein reabsorption droplets. The SOD-treated animals demonstrated a lesser degree of epithelial loss of foot processes with a few protein reabsorption droplets. There was also a significant decrease in GBM lamina rara externa CSc stained with PEI in PAN compared to the control. The SOD-treated rats had a significant increase in GBM CSs compared to those of the PAN rats. SOD has protective effects in urinary protein excretion, the morphology and GBM CSc in PAN, which indicate indirect evidence that superoxide and/or its metabolites are responsible for glomerular injury.     

Proliferating cells in HIV and pamidronate-associated collapsing focal segmental glomerulosclerosis are parietal epithelial cells

Collapsing focal segmental glomerulosclerosis (cFSGS) is characterized by hyperplasia of glomerular epithelial cells. In a mouse model of FSGS and in a patient with recurrent idiopathic FSGS, we identified the proliferating cells as parietal epithelial cells (PECs). In the present study, we have evaluated the origin of the proliferating cells in cFSGS associated with human immunodeficiency virus (HIV) and pamidronate. We performed a detailed study of glomerular lesions in biopsies of two patients with HIV-associated cFSGS and a nephrectomy specimen of a patient with pamidronate-associated cFSGS. Glomeruli were studied by serial sectioning using light and electron microscopy and immunohistochemistry to determine the epithelial cell phenotype. We used Synaptopodin, vascular endothelial growth factor, and CD10 as podocyte markers, CK8 and PAX2 as PEC markers and Ki-67 as marker of cell proliferation. The newly deposited extracellular matrix was characterized using antiheparan sulfate single-chain antibodies. The proliferating cells were negative for the podocyte markers, but stained positive for the PEC markers and the cell proliferation marker Ki-67. The proliferating PAX-2 and CK8 positive cells that covered the capillary tuft were always in continuity with PAX-2/CK8 positive cells lining Bowman's capsule. The matrix deposited by these proliferating cells stained identically to Bowman's capsule. Our study demonstrates that PECs proliferate in HIV and pamidronate-associated cFSGS. Our data do not support the concept of the proliferating, dedifferentiated podocyte.     

Glomerular damage after uninephrectomy in young rats. II. Mechanical stress on podocytes as a pathway to sclerosis.

In a preceding study [1], we showed that within six months after UNX in young rats, glomeruli in the remnant kidney undergo a sequence of serious changes which finally lead to focal segmental glomerulosclerosis (FGS). The formation of abnormally-shaped capillary channels was shown to result from local mesangial failure and is considered to be a nidus for the development of more severe lesions. In the present paper, the development of characteristic lesions in podocyte structure is described and analyzed. Concomitant with overall glomerular growth after UNX, a pronounced hypertrophy of podocytes was observed, while the mean number of podocytes per glomerulus did not change. It appears that podocytes cannot sustain the same degree of growth as the tuft as a whole; podocyte hypertrophy is soon followed by maladaptive changes which eventually lead to cell destruction. The following sequence of pathologic changes can be suggested: cell bodies enlarge in volume and area associated with a dramatic attenuation to cytoplasmic sheets. Primary processes are thinned out and frequently extend to remote capillaries. As a whole, the capillary area served by a single podocyte is dramatically enlarged. Furthermore, the expanding cytoplasmic sheets (derived from podocyte cell bodies) cover an increasingly large proportion of the outer capillary surface, that is, of the filtration area. Consequently, an increasing amount of filtrate is delivered into the subcellbody space. Obstruction of the efflux of this filtrate into the urinary space causes bulging of the overlying cytoplasmic sheets into pseudocysts. Podocytes overlying abnormally-shaped and dilated capillary channels are generally the most seriously affected. Tuft hypertrophy, pseudocyst formation and local capillary expansion cause wide-spread apposition of podocytes to Bowman's capsule. Appositions are a prerequisite for the development of tuft adhesion. Local detachment of a podocyte from the GBM in those areas allows access of parietal cells to the GBM. In early adhesions the connection of the tuft to Bowman's capsule is established by single parietal cells which attach to both the GBM and the basement membrane of Bowman's capsule. An adhesion is considered as a nidus for segmental sclerosis; as the adhesion progresses, the related tuft regions turn into sclerosis. In the present model FGS develops exclusively in areas of tuft adhesion.     

Focal segmental glomerulosclerosis plays a major role in the progression of IgA nephropathy. I. Immunohistochemical studies

IgA nephropathy (IgAN) often shows lesions morphologically identical with those of focal segmental glomerulosclerosis (FSGS). In order to determine the possible role of FSGS in IgAN lesions, we measured glomerular capsular adhesions, often the first step toward FSGS, in biopsies from 127 patients with IgAN, 100 with lupus nephritis, and 26 with primary FSGS. Capsular adhesions with no lesions in the underlying tuft, consistent with podocyte abnormality or loss, were found regularly in FSGS and IgAN, but infrequently in lupus. Fifteen biopsies of patients with IgAN were studied immunohistochemically using markers for podocytes, Bowman's parietal epithelial cells, proliferating cells, and macrophages. Cytokeratins CK-8 and C2562 differentiated normal podocytes (negative) from parietal epithelial cells (variably positive). There was focal loss of the podocyte markers synaptopodin, glomerular epithelial protein 1 (GLEPP-1), nephrin, and vascular endothelial growth factor (VEGF), particularly at sites of capsular adhesions in otherwise histologically normal glomeruli. Cells displaying the parietal epithelial cell markers PAX2 (paired box gene 2) and the cytokeratins were also positive for the proliferating cell marker, proliferating cell nuclear antigen. These cells gathered at sites of adhesion, and in response to active lesions in the tuft, grew inward along the adhesion onto the tuft, forming a monolayer positive for parietal markers and the podocyte marker Wilms tumor protein-1 (WT-1). These cells deposited a layer of collagen over the sclerosing tuft. Thus, all biopsies of patients with IgAN had changes basically identical to those classically described in FSGS. Hence, our study strongly suggests that podocytopathy of a type similar to that in primary FSGS occurs frequently in IgAN.     

Regression of glomerular injury by losartan in experimental diabetic nephropathy

Many features of chronic kidney disease may be reversed, but it is unclear whether advanced lesions, such as adhesions of sclerotic glomerular tufts to Bowman's capsule (synechiae), can resolve during treatment. We previously showed, using a renal ablation model, that the renoprotective effect of the AT-1 receptor blocker, losartan, is dose-dependent. Here we determined if moderate and advanced glomerular lesions, associated with streptozotocin-induced diabetes, regress with conventional or high-dose losartan treatment. Using daily insulin injection for 10 months, we maintained diabetic adult male Munich-Wistar rats in a state of moderate hyperglycemia. Following this period, some rats continued to receive insulin with or without conventional or high-dose losartan for an additional 2 months. Diabetic rats pretreated with insulin for 10 months and age-matched non-diabetic rats served as controls. Mesangial expansion was found in the control diabetic rats and was exacerbated in those rats maintained on only insulin for an additional 2 months. Conventional and high-dose losartan treatments reduced this mesangial expansion and the severity of synechiae lesions below that found prior to treatment; however, the frequency of the latter was unchanged. There was no dose-response effect of losartan. Our results show that regression of mesangial expansion and contraction of sclerotic lesions is feasible in the treatment of diabetes, but complete resolution of advanced glomerulosclerosis may be hard to achieve.     

Parietal epithelial cells participate in the formation of sclerotic lesions in focal segmental glomerulosclerosis

The pathogenesis of the development of sclerotic lesions in focal segmental glomerulosclerosis (FSGS) remains unknown. Here, we selectively tagged podocytes or parietal epithelial cells (PECs) to determine whether PECs contribute to sclerosis. In three distinct models of FSGS (5/6-nephrectomy + DOCA-salt; the murine transgenic chronic Thy1.1 model; or the MWF rat) and in human biopsies, the primary injury to induce FSGS associated with focal activation of PECs and the formation of cellular adhesions to the capillary tuft. From this entry site, activated PECs invaded the affected segment of the glomerular tuft and deposited extracellular matrix. Within the affected segment, podocytes were lost and mesangial sclerosis developed within the endocapillary compartment. In conclusion, these results demonstrate that PECs contribute to the development and progression of the sclerotic lesions that define FSGS, but this pathogenesis may be relevant to all etiologies of glomerulosclerosis.     

Glomerular damage after uninephrectomy in young rats. I. Hypertrophy and distortion of capillary architecture.

Uninephrectomy (UNX) results in a higher incidence of focal glomerular sclerosis (FGS) in young rats than it does in adults. The reason for this higher susceptibility in young animals is not fully understood, but this does suggest that UNX in young rats may represent a particularly promising model in which to study the development of FGS. In the present study 10-day-old rats were subjected to UNX. After 4, 12 and 24 weeks, glomerular hypertrophy, structural lesions and function were analyzed in comparison with sham-operated controls. Up to the twelfth week, remnant kidney growth and glomerular growth proceeded in parallel; thereafter, kidney growth ceased, whereas glomerular growth continued undiminished. Twenty-four weeks after UNX, glomerular tuft volume in experimental animals exceeded that in controls by 80%. Twelve weeks after surgery, total GFR in UNX rats was approximately 80% of that in controls, a value maintained until the end of the observation period. Twenty-four weeks after surgery, heavy proteinuria was present in UNX animals. Structural abnormalities in glomeruli of UNX animals were already encountered 12 weeks after surgery; they were present to a much lesser extent in controls. In UNX animals these proceeded to the FGS stage by the end of the observation period. Three major groups of glomerular lesions were observed: (1) changes in the width and shape of glomerular capillaries. (2) changes in podocyte structure, and (3) tuft adhesions to Bowman's capsule with or without segmental sclerosis. The structural changes are analyzed in this and an accompanying paper [1]. The present paper deals with the widespread formation of irregular, giant capillary loops. They occur predominantly at the tuft periphery with a clear predilection for the vascular pole region. They are not a result of compensatory growth, but rather an expansion of single capillaries due to failure of the mesangium. Local disconnection of the mesangium from its anchoring points at the GBM leads to bulging and "coalescence" of capillary loops, resulting in abnormally-shaped vascular channels. This process is associated with a rearrangement of the corresponding mesangium. In our view, the appearance of dilated capillaries represents a local event pivotal to the development of more severe lesions, such as tuft adhesions and FGS.     

A role for oxygen free radicals in aminonucleoside nephrosis

The cellular processes responsible for the proteinuria induced by the aminonucleoside of puromycin (PA) remain inadequately defined. Hypoxanthine is both a metabolic breakdown product of PA as well as a substrate for xanthine oxidase, which catalyzes its enzymatic conversion to xanthine and uric acid, yielding the superoxide anion in the process. We examined whether oxygen free radical production contributes to the development of proteinuria in this model. Seven groups of male Sprague-Dawley rats were studied. Proteinuria was quantitated and histology examined 7 days after rats were treated with PA intravenously over 5 min. PA-treated animals received either saline, dimethyl sulfoxide, superoxide dismutase, or catalase over 30 min prior to and 30 min following PA administration. Another group received allopurinol over 4 hr prior to PA. The superoxide dismutase and allopurinol treatment groups had a significant suppression of urinary protein excretion compared to the PA control group. There were also less severe glomerular morphologic changes in the superoxide dismutase group vs. the PA controls, which demonstrated a pathologic pattern that included epithelial cell blebbing, segmental mesangial cell proliferation and matrix expansion, loss of glomerular capillary lumina, and occasional adhesions between the glomerular tuft and Bowman's capsule. The allopurinol group exhibited normal glomerular morphology on light microscopy, with the exception of occasional epithelial cell blebs. All groups showed spreading of the epithelial cell cytoplasm along the glomerular basement membrane with loss of foot processes, focal areas of lifting of the epithelial cell from the glomerular basement membrane, cytoplasmic vacuolization, and protein reabsorption droplets; however, allopurinol-treated animals demonstrated these changes to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Posttransplantation relapse of FSGS is characterized by glomerular epithelial cell transdifferentiation

This study examined six cases of idiopathic nephrotic syndrome with primary lesions of focal segmental glomerulosclerosis (FSGS) that relapsed after renal transplantation. The glomerular lesions comprised the cellular, the collapsing, and the scar variants of FSGS and showed shedding of large round cells into Bowman's space and within the tubular lumens. Immunohistochemistry and confocal laser microscopy carried out on kidneys with FSGS relapse disclosed several phenomena. (1) Some podocytes that expressed podocalyxin, synaptopodin, and glomerular epithelial protein-1 were detached from the tuft and were free in the urinary space. (2) In the cellular variant, most podocytes had lost podocyte-specific epitopes (podocalyxin, synaptopodin, glomerular epithelial protein-1, Wilm's tumor protein-1, complement receptor-1, and vimentin). In the scar variant, these podocyte markers were absent from cobblestone-like epithelial cells and from pseudotubules. (3) Podocytes had acquired expression of various cytokeratins (CK; identified by the AE1/AE3, C2562, CK22, and AEL-KS2 monoclonal antibodies) that were not found in the podocytes of control glomeruli. Parietal epithelial cells expressed AE1/AE3 CK that were faintly, if ever, found on the parietal epithelial cells of normal glomeruli. (4) Numerous cells located at the periphery of the tuft or free in Bowman's space and within tubular lumens expressed macrophagic epitopes (identified by PGM1 [CD68], HAM56, and 25F9 monoclonal antibodies). These macrophage-like cells expressed the activation epitopes HLA-DR and CD16. (5) A number of these cells coexpressed podocalyxin + AE1/AE3 CK, podocalyxin + CD68, and CD68 + AE1/AE3. These findings suggest that in primary FSGS relapsing on transplanted kidneys, some "dysregulated" podocytes, occasionally some parietal epithelial cells, and possibly some tubular epithelial cells undergo a process of transdifferentiation. This process of transdifferentiation was especially striking in podocytes that acquired macrophagic and CK epitopes that are absent from normal adult and fetal podocytes.     

Heparin-binding epidermal growth factor-like growth factor is expressed in the adhesive lesions of experimental focal glomerular sclerosis.

BACKGROUND: In this study, we attempted to determine whether heparin-binding epidermal growth factor-like growth factor (HB-EGF) was up-regulated in two chronic models of proteinuria. METHODS: Chronic passive Heymann nephritis (PHN) and puromycin aminonucleoside (PAN) models were induced in Sprague-Dawley rats. HB-EGF expression was studied by Northern blotting, in situ hybridization, and immunohistochemistry. RESULTS: The chronic PAN model was associated with the development of glomerular lesions of focal glomerular sclerosis (FGS), severe interstitial fibrosis, and renal failure. Lesions of FGS were seen in approximately 80% of glomeruli at all time points, with a slight increase in the number of glomeruli showing extensive adhesion between 40 and 90 days. Northern blots of whole kidney tissue showed a 3- to 5.8-fold increased expression of HB-EGF mRNA in the chronic PAN group. Increased mRNA and protein were localized by in situ hybridization and immunohistochemistry to tubules, glomerular epithelial cells (GECs), and cells of Bowman's capsule. HB-EGF mRNA and protein were strongly expressed by epithelial cells involved in the formation of the lesions of FGS. By contrast, in chronic PHN, there was a small increase in HB-EGF, and the extensive lesions of FGS did not develop despite continued, heavy proteinuria. CONCLUSIONS: These data suggest that HB-EGF may contribute to formation of the lesions of FGS, perhaps through stimulation of abortive mitogenesis in GECs or an adhesive interaction between transmembrane HB-EGF and the exposed glomerular basement membrane.     

Tracer studies in the rat demonstrate misdirected filtration and peritubular filtrate spreading in nephrons with segmental glomerulosclerosis

In two genetic models of "classic" focal segmental glomerulosclerosis (FSGS), the Milan normotensive and the Fawn-hooded hypertensive rats, tracer studies were performed to test the hypothesis that misdirected glomerular filtration and peritubular filtrate spreading are relevant mechanisms that contribute to nephron degeneration in this disease. Two exogenous tracers, lissamine green and horse spleen ferritin, were administered by intravenous injection and subsequently traced histologically in serial kidney sections. In contrast to control rats, both tracers in kidneys of Milan normotensive and Fawn-hooded hypertensive rats with established FSGS were found to accumulate extracellularly at the following sites: (1) within tuft adhesions to Bowman's capsule and associated paraglomerular spaces, (2) at the glomerulotubular junction contained within extensions of the paraglomerular spaces onto the tubule, and (3) within subepithelial peritubular spaces eventually encircling the entire proximal convolution of an affected nephron. This distribution strongly suggests the existence of misdirected filtration into tuft adhesions to Bowman's capsule and subsequent spreading of the filtrate around the entire circumference of a glomerulus and, alongside the glomerulotubular junction, onto the outer aspect of the corresponding tubule. This leads to an interstitial response that consists of the formation of a barrier of sheet-like fibroblast processes around the affected nephron, which confines the filtrate spreading and, subsequently, the destructive process to the affected nephron. No evidence was found that either misdirected filtration and peritubular filtrate spreading themselves or the associated tubulo-interstitial process led to the transfer of the injury from an affected nephron to an unaffected nephron. It is concluded that in the context of FSGS development, misdirected filtration and peritubular filtrate spreading are important damaging mechanisms that underlie the extension of glomerular injury to the corresponding tubulointerstitium, thus leading finally to degeneration of both the glomerulus and the tubule of a severely injured nephron.     

Antioxidant ceruloplasmin is expressed by glomerular parietal epithelial cells and secreted into urine in association with glomerular aging and high-calorie diet

Biologic aging is accelerated by high-calorie intake, increased free radical production, and oxidation of key biomolecules. Fischer 344 rats that are maintained on an ad libitum diet develop oxidant injury and age-associated glomerulosclerosis by 24 mo. Calorie restriction prevents both oxidant injury and glomerulosclerosis. Ceruloplasmin (Cp) is a copper-containing ferroxidase that functions as an antioxidant in part by oxidizing toxic ferrous iron to nontoxic ferric iron. Glomerular Cp mRNA and protein expression were measured in ad libitum-fed and calorie-restricted rats at ages 2, 6, 17, and 24 mo. In ad libitum-fed rats, Cp mRNA expression increased six-fold (P < 0.01) and protein expression increased five-fold (P = 0.01) between 2 and 24 mo of age. In calorie-restricted rats, Cp mRNA expression increased three-fold (P < 0.01) and protein expression increased 1.6-fold (NS) between 2 and 24 mo of age. Both the cell-associated alternately spliced variant and secreted variants of Cp were expressed. Immunofluorescent analysis showed that Cp was expressed by the parietal epithelial cells that line the inner aspect of Bowman's capsule in the glomerulus. Cp also was present in urine, particularly of old ad libitum-fed rats with high tissue Cp expression. Cp expression by Bowman's capsule epithelial cells therefore occurred in direct proportion to known levels of oxidant activity (older age and high-calorie diet) and is secreted into the urine. It is suggested that Cp expression at this site may be part of the repertoire of the glomerular parietal epithelial cell to protect the glomerular podocytes and the downstream nephron from toxic effects of filtered molecules, including ferrous iron.     

Correlation between tubulointerstitial nephropathy and glomerular lesions induced by adriamycin.

This paper describes a morphometric study of the evolution of tubulointerstitial nephropathy in adriamycin-induced focal and segmental glomerulosclerosis in Wistar rats over 32 weeks old. The earliest changes were located in the glomeruli. In the 10 week of the study, tubulointerstitial nephropathy appeared and, although the interstitial space increased after the 2nd week, this increase only became statistically significant after the 10 week. Proteinuria showed the highest correlation with the interstitial space, however, the interstitial space showed the highest correlation indices with the total number of glomeruli affected and to a lesser extent with adhesions to and thickening of Bowman's capsule.     

A pitfall of glomerular sieving: profibrotic and matrix proteins derive from the Bowman's capsule and not the glomerular tuft in rats with renovascular hypertension.

BACKGROUND: The glomeruli in the non-clipped kidney of rats with 2-kidney, 1-clip hypertension are a classical model for studying the mechanisms of glomerular injury. METHODS: In the present study, we compared the glomerular expression of PAI-1 and collagen I alpha1 mRNA from glomeruli isolated by the classic technique of sieving with the recently developed technique of tissue laser microdissection. For quantification of mRNA from both methods, real-time PCR was used. RESULTS: Real-time PCR revealed a 9.0 +/- 1.3- and a 7.1 +/- 0.2-fold induction of PAI-1 and collagen I alpha 1, respectively, in the glomeruli from hypertensive rats isolated by sieving. However, in situ hybridization and microdissection revealed that expression of both mRNAs was mainly from the Bowman's capsule and not from the glomerular tuft (10.7 +/- 1.3- and 7.2 +/- 0.6-fold higher induction in whole glomeruli compared with tuft alone). CONCLUSION: This emphasizes that studies focusing on processes in the mesangium, endothelial cells or podocytes should not rely on glomeruli obtained by sieving. Rather, a technique like the laser microdissection or in situ hybridization should be applied which allows the clear separation of different glomerular and periglomerular compartments.     

Glomerular sclerosis in kidneys with congenital nephrotic syndrome (NPHS1)

Congenital nephrotic syndrome of the Finnish type (NPHS1) is a rare genetic disease caused by mutations in the NPHS1 gene encoding a major podocyte slit-diaphragm protein, nephrin. Patients with NPHS1 have severe nephrotic syndrome from birth and develop renal fibrosis in early childhood. In this work, we studied the development of glomerular sclerosis in kidneys removed from 4- to 44-month-old NPHS1 patients. The pathological lesions and expression of glomerular cell markers were studied in nephrectomized NPHS1 and control kidneys using light and electron microscopy and immunohistochemistry. An analysis of 1528 glomeruli from 20 patients revealed progressive mesangial sclerosis and capillary obliteration. Although few inflammatory cells were detected in the mesangial area, paraglomerular inflammation and fibrosis was common. The podocytes showed severe ultrastructural changes and hypertrophy with the upregulation of cyclins A and D1. Podocyte proliferation, however, was rare. Apoptosis was hardly detected and the expression of antiapoptotic B-cell lymphoma-2 and proapoptotic p53 were comparable to controls. Moderate amounts of podocytes were secreted into the urine of NPHS1 patients. Shrinkage of the glomerular tuft was common, whereas occlusion of tubular opening or protrusion of the glomerular tuft into subepithelial space or through the Bowman's capsule were not detected. The results indicate that, in NPHS1 kidneys, the damaged podocytes induce progressive mesangial expansion and capillary obliteration. Podocyte depletion, glomerular tuft adhesion, and misdirected filtration, however, seem to play a minor role in the nephron destruction.