Glomerular Capillary Loops Research Articles

IgA nephropathy in two adolescent sisters heterozygous for Fabry disease

We report a 16-year-old girl and her one-year-younger sister, both heterozygous for the c.34del24 mutation of the GLA (alpha-galactosidase A) gene, which they inherited from their father who is affected by Fabry disease (FD). Both girls presented with macrohematuria and rapidly progressing proteinuria. Urine analysis revealed glomerular hematuria and a nephrotic range of proteinuria suggesting a concomitant glomerulonephritis. Light microscopy of kidney biopsy was characteristic of IgA nephropathy (IgA deposits in mesangial areas and glomerular capillary loops, and mesangial hypercellularity), whereas electron microscopy showed changes typical of Fabry disease (multiple osmiophilic inclusions in the subendothelial and mesangial areas). These two cases and similar reports in the literature suggest that IgA nephropathy in FD is not merely coincidental.

Anomalous Renal Effects of Tin Protoporphyrin in a Murine Model of Sickle Cell Disease

In human and murine models of sickle cell disease (SCD), heme oxygenase-1 (HO-1) is induced in the kidney, an organ commonly involved in SCD. The present study assessed the role of HO-1 by using a competitive inhibitor of HO activity, tin protoporphyrin (SnPP), in protocols affording a composite, clinically relevant analysis of the kidney in SCD under unstressed and stressed conditions. Whereas short-term administration of SnPP exerted comparable renal hemodynamic effects in wild-type and sickle mice, chronic administration of SnPP exerted divergent effects: SnPP provoked tubulointerstitial inflammation and up-regulation of injury-related genes in wild-type mice, whereas in sickle mice SnPP reduced expression of injury-related genes and vascular congestion without provoking tubulointerstitial inflammation. SnPP also protected against the heightened sensitivity to renal ischemia observed in sickle mice, preventing ischemia-induced worsening of renal injury in sickle mice above that observed in wild-type mice. Effective and comparable inhibition of HO activity by SnPP in wild-type and sickle mice was confirmed. These findings suggest that induction of HO-1, at least as assessed by this approach, may contribute to renal injury in this murine model of SCD and uncover an experimental maneuver that protects the kidney in murine SCD.

Immunolocalization of serum proteins in living mouse glomeruli under various hemodynamic conditions by “in vivo cryotechnique”

Distribution of serum proteins in renal glomeruli is important for histopathology in medical and biological fields, but mechanisms of their passage through glomerular capillary loops (GCL) are still difficult to clarify. We have tried to visualize topographical changes of the serum proteins passing through GCL by "in vivo cryotechnique" in combination with immunohistochemistry. Albumin and immunoglobulin G (IgG), Ig kappa light chain and IgG1 heavy chain were mainly immunolocalized in GCL, but not colocalized with zonula occludens-1 (ZO-1) under normotensive condition. Under heart-arrest condition and in quick-frozen fresh tissues, albumin and kappa light chain were immunolocalized in Bowman's space, indicating their passage caused by the stoppage of blood supply. However, under acute hypertensive condition, they were more clearly immunolocalized along basement membranes and in the Bowman's space, indicating their increased passage through GCL. IgG was also more clearly localized in mesangial areas under acute hypertension, compared with that under the normotensive or heart-arrest condition. This study is the first direct visualization for glomerular passage of serum proteins under abnormal hemodynamic conditions by the "in vivo cryotechnique", and the experimental protocol will be useful for morphofunctional examination of living mouse GCL and immunohistochemical analyses of dynamically changing proteins.

Association of glomerular podocytopathy and nephrotic proteinuria in mesangial lupus nephritis

We investigated a series of patients with systemic lupus erythematosus (SLE), who had sparse subepithelial and mesangial immune deposits. Our goal was to determine structure: function correlation. We examined whether proteinuria correlated with either capillary wall immune aggregate formation or abnormal podocyte morphology. Renal biopsies from patients with sparse (two or fewer subepithelial or intramembranous electron dense deposits per glomerular capillary loop) immune deposits and podocyte effacement were studied. Patients fulfilled criteria for the diagnosis of SLE. Cases were excluded if the biopsy showed endocapillary proliferation or necrosis. Eighteen biopsies were studied, five from patients with nephrotic range proteinuria (> or =3 g/day) and 13 from patients with non-nephrotic proteinuria (<3 g/day). The five nephrotic patients had a mean foot process effacement of 48% +/- 39% (range 10-100%). Thirteen non-nephrotic patients had a mean foot process effacement of 11.7% +/- 8% (range 0-20%). The only distinguishing morphologic finding associated with nephrotic range proteinuria was diffuse foot process effacement. No correlation between subepithelial deposits and proteinuria was observed. There were no other histologic differences between the nephrotic and non-nephrotic patients. Among these patients, the nephrotic syndrome appears best correlated with podocytopathy rather than subepithelial electron dense deposits, mesangial deposits, or mesangial hypercellularity.

Expression of canonical transient receptor potential (TRPC) proteins in human glomerular mesangial cells

Mesangial cells are located within glomerular capillary loops and contribute to the physiological regulation of glomerular hemodynamics. The function of mesangial cells is controlled by a variety of ion channels in the plasma membrane, including nonselective cation channels, receptor-operated Ca2+ channels, and recently identified store-operated Ca2+ channels. Although the significance of these channels has been widely acknowledged, their molecular identities are still unknown. Recently, the members of the canonical transient receptor potential (TRPC) protein family have been demonstrated to behave as cation channels. The present study was performed to identify the isoforms of endogenous TRPC proteins in human mesangial cells (HMCs) and their interactions. Western blotting showed that TRPC1, 3, 4, and 6 were expressed in cultured HMCs. Consistently, immunofluorescent confocal microscopy revealed specific stainings for TRPC1, 3, 4, and 6 with predominant intracellular localization. However, TRPC5 and 7 were not detectable at protein level by either Western blotting or immunofluorescent staining. The expression of TRPC1, 3, 4, and 6 was also observed in rat and human glomeruli using fluorescent immunohistochemistry. Furthermore, coimmunoprecipitation experiments and immunofluorescent double staining displayed that TRPC1 had physical interaction with TRPC4 and 6, while no interactions were detected among other isoforms of TRPCs. Ca2+ fluorescent ratiometry measurement showed that store-operated Ca2+ entry in HMCs was significantly reduced by knocking down TRPC1, but enhanced by overexpressing TRPC1. These results suggest that HMCs specifically express isoforms of TRPC1, 3, 4, and 6 proteins. These isoforms of TRPCs might selectively assemble to form functional complexes.

Application of periodic acid-Schiff fluorescence emission for immunohistochemistry of living mouse renal glomeruli by an "in vivo cryotechnique"

To identify the distribution of endogenous serum proteins in living mouse renal glomeruli under various hemodynamic conditions, we used the periodic acid-Schiff (PAS) and its fluorescence emission as a marker for the glomerular basement membrane (GBM). The immunostaining for collagen type IV was hardly observed without microwave treatment in specimens prepared by an "in vivo cryotechnique". However, PAS staining and its fluorescence emission could be clearly visualized at the GBM with the "in vivo cryotechnique". Under normotensive conditions, immunoreaction products of albumin and immunoglobulin G heavy and light chains (IgG(H+L)) were localized within glomerular capillary loops (GCL) but not colocalized with the PAS fluorescence emission of the GBM. Under heart-arrest conditions and with quick-freezing of resected tissues, albumin, IgG (H+L), immunoglobulin kappa light chain, and IgG1 heavy chain (IgG1) were immunolocalized within the GCL and mesangial areas, but only albumin and the kappa light chain were additionally immunolocalized in Bowman's space, indicating their passage through the GBM. Under acute hypertensive conditions, both albumin and the kappa light chain, but not IgG1, were clearly immunolocalized along the GBM and in the Bowman's space, indicating their increased passage through the GBM. The overlapping areas of PAS fluorescence emission and the albumin or kappa light chain appeared to be larger with quick-freezing and under the heart arrest or acute hypertensive conditions than under normal circulation, whereas those of PAS emission and IgG1 did not differ among these conditions. The serum proteins passing through the GBM were clearly visualized with the "in vivo cryotechnique", immunofluorescence staining, and PAS fluorescence emission.

Gender differences in the relation between ambulatory blood pressure and left ventricular mass in hypertensive outpatients

Renal injury in DOCA-salt hypertensive C5-sufficient and C5-deficient mice. We induced hypertension by uninephrectomy and treatment with desoxycorticosterone (DOCA) and 1% NaCl in the drinking water in congenic mice that differ in the single gene locus responsible for the presence or absence of the complement component C5 and compared them to uninephrectomized normotensive (no DOCA-NaCl) mice. In contrast to C5-sufficient (C5S) mice, C5-deficient (C5D) mice can neither generate C5a nor assemble C5b-9. After four weeks of treatment, DOCA-C5S and -C5D mice developed similar degrees of hypertension; mice receiving no DOCA remained normotensive. Only hypertensive mice developed glomerular injury. Hypertensive DOCA-C5D mice developed more glomerular capillary loop dilatation and larger glomerular capillary tuft volumes than DOCA-C5S mice (1.0 ± 0.1 vs. 0.7 ± 0.03 × 106 µm3, respectively, P < 0.05). However, DOCA-C5S mice, compared to DOCA-C5D mice, had significantly more glomerular, cell proliferation (64.5 ± 2 vs. 42 ± 3 nuclei/glomerulus), cell necrosis (injury score 22 ± 1 vs. 17 ± 1), extracapillary proliferation (26 ± 4 vs. 2.5 ± 2% of glomeruli) and proteinuria (5.9 ± 0.8 vs. 3.7 ± 0.5 mg/24 hr; all P < 0.05). By immunofluorescence microscopy both DOCA-C5S and -C5D had mesangial C3 deposits but only DOCA-C5S mice had C9 deposits. After 16 weeks of DOCA-NaCl C5S mice, in comparison to C5D mice, had more severe glomerulosclerosis (injury score 50 ± 6 vs. 12 ± 4), proteinuria (16.6 ± 0.1 vs. 9 ± 0.1 mg/24 hr), and renal insufficiency (serum creatinine 0.25 vs. 0.15 mg/dl), all P < 0.05. These changes occurred despite levels of hypertension that were similar in DOCA-NaCl C5S and C5D throughout the whole study period. We conclude that C5a and/or C5b-9 may play an important role in hypertensive glomerular injury. Moreover, these studies demonstrate that differences in host responses may determine target organ susceptibility to similar injurious mechanisms.

Immunohistochemical analyses on albumin and immunoglobulin in acute hypertensive mouse kidneys by "in vivo cryotechnique".

The purpose of this study is to visualize topographical changes of serum proteins, albumin and immunoglobulin, passing through mouse glomerular capillary loops and their reabsorption in renal proximal tubules by immunohistochemistry in combination with our "in vivo cryotechnique". The "in vivo cryotechnique" was performed on left mouse kidneys under normotensive, experimentally acute hypertensive and heart-arrest conditions. The cryofixed tissues by the technique were routinely processed for freeze-substitution. Serial deparaffinized sections were stained with hematoxylin-eosine and immunostained with anti-mouse albumin, immunoglobulin G (IgG), kappa or lambda light chain and IgG1 heavy chain antibodies. Under the normotensive and heart-arrest conditions, albumin and IgG were clearly immunolocalized in blood vessels and slightly in apical cytoplasmic parts of some proximal tubules. Under the acute hypertensive condition, the albumin and kappa or lambda light chains, but not IgG1 heavy chain, were strongly immunolocalized in the apical cytoplasm of almost all proximal tubules. This study is the first in vivo visualization for glomerular passage of serum proteins and their transtubular absorption. Thus, the "in vivo cryotechnique" with freeze-substitution can be used for clarifying not only the functional morphology of living animal cells, but also in situ immunohistochemical localization of their components.

ANCA-negative pauci-immune renal vasculitis: histology and outcome

Pauci-immune renal vasculitis with focal glomerular necrosis and crescent formation is usually associated with anti-neutrophil cytoplasmic antibodies (ANCAs). However, ANCA's are absent in up to 10% of cases, which constitutes a rarely studied variant of renal vasculitis. This retrospective multicentre cohort study analyzed the presenting features, renal histology and outcome in 20 patients with pauci-immune crescentic necrotizing renal vasculitis in whom indirect immunofluorescence did not detect ANCA. Renal histology revealed a high percentage of active glomerular lesions (50%), mainly cellular crescents, 28% of them with glomerular necrosis. Chronic tissue damage with glomerulosclerosis (21%) and diffuse interstitial fibrosis (40%) was already present at diagnosis, more prominent than in historical PR3-positive patients. Infiltrates of polymorphonuclear neutrophils in glomerular capillary loops were observed in 40% of all biopsies, mainly in necrotic lesions. The subsets of interstitially infiltrating leukocytes similar to ANCA-associated disease. Microscopic polyangiitis was diagnosed in 17 patients, Wegener's granulomatosis in two and renal-limited vasculitis in one. The patients median disease extent index (DEI) of 5 (range 4-11) reflected a systemic vasculitis. ANCA-negative vasculitis was not associated with infection or malignancy. Renal outcome was correlated to DEI (P = 0.032) and serum creatinine at diagnosis (P = 0.04). The mortality rate was high (35%) and closely related to age above 65 years at diagnosis (P = 0.014). Conclusions. The histological findings and prognosis in ANCA-negative renal vasculitis are comparable with those of ANCA-positive disease. Our data underline the importance of the exact diagnosis in an active vasculitic disease process even in the absence of ANCAs.

Real-time observation of glomerular hemodynamic changes in diabetic rats: Effects of insulin ARB

The progression of diabetic nephropathy is closely related to disturbances in glomerular hemodynamics, such as glomerular hypertension and/or hyperperfusion. The aim of this study was to observe and to analyze glomerular hemodynamics in rats with diabetes mellitus (DM) in vivo using confocal laser scan microscopy (CLSM). We also examined the effects of candesartan cilexetil (TCV-116), a selective angiotensin II type 1 receptor blocker (ARB), on glomerular hemodynamics in DM. Munich-Wistar rats were divided into six groups: (1) four-day control; (2) four-day DM; (3) 28-day control; (4) 28-day DM; (5) DM treated with insulin; (6) DM treated with TCV-116. The kidney-to-body weight ratio, glomerular volume, and proteinuria were estimated. Glomerular hemodynamic changes were observed using CLSM and renal expression of endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) was evaluated by immunofluorescence. The kidney-to-body weight ratio, glomerular volume, the diameters of afferent arterioles (AA) and efferent arterioles (EA), erythrocyte velocities within glomeruli, and volume flow in glomerular capillary loops in four-day DM were significantly higher than in control rats, and increases were even more pronounced in the 28-day DM. TCV-116 treatment ameliorated all these findings and significantly decreased proteinuria, but there was no effect on the blood glucose level. On the other hand, insulin treatment was followed by normalization of all these changes induced in DM. Enhanced renal expression of eNOS in DM was suppressed when treated with either TCV-116 or insulin, while expression of nNOS was unaltered among the four groups. This imaging procedure allowed us to evaluate glomerular microcirculation in vivo, including the diameters of AA and EA, erythrocyte velocity, and volume flow. DM significantly induced glomerular hemodynamic alteration and renal hypertrophy. DM treated with either insulin or ARB ameliorated these changes. This study shows that progress in imaging technology promises to make major contributions to revealing the involvement of hemodynamic changes in glomerular diseases, aiding prognosis and the monitoring of therapeutic effects, as well.

Glomerular Capillary Tenascin Staining in Renal Allografts and Its Correlation with Capillary Loop ‘Double Contours’, Proteinuria and Graft Outcome

Background: Glomerular capillary injuries result in proteinuria, previously reported to be a risk factor for renal allograft dysfunction. According to the Banff 97 Working Classification of Renal Allograft Pathology, ‘double contours’ in glomerular capillary loops (GC-DC) were suggested to be most specific for chronic transplant glomerulopathy. Tenascin-C (TN), a component of extracellular matrix, is known to be overexpressed in various conditions. Methods: TN immunostaining was performed by the labeled streptavidin biotin method. The correlations between glomerular capillary TN (GC-TN) and proteinuria, and between GC-DC and GC-TN, were studied in 27 biopsies showing chronic allograft nephropathy (CAN) lesions. In 14 patients (serum creatinine <2.0 mg/dl at biopsy), graft outcome was studied. Graft function 3 years after biopsy was classified as stable or deteriorated; the term ‘deteriorated’ was used if serum creatinine had increased 20% over baseline, or if the patient required dialysis. Results: In CAN, GC-TN correlated with GC-DC. Proteinuria in patients with intense GC-TN tended to increase during 1 year after biopsy. In graft outcome analysis, GC-TN was significantly lower in stable grafts than in deteriorated ones. Furthermore, all 3 patients demonstrating weak GC-TN without GC-DC returned to dialysis. Conclusion: TN immunostaining is a sensitive method to detect glomerular capillary injury with a predictive value for renal allograft dysfunction.

In situ evaluation of podocin in normal and glomerular diseases

Mutations of the NPHS2 gene are responsible for autosomal-recessive steroid-resistant nephrotic syndrome. Its product, podocin, faces the slit diaphragm area with its two ends in the cytoplasm of foot processes. We generated rabbit polyclonal antibodies against conjugated peptides from human podocin N- and C-termini, and studied podocin and synaptopodin using kidney tissues of normal humans and those with glomerular diseases. Antipodocin antibodies detected the original 42 kD fragment and an extra smaller fragment by Western blot analysis using human isolated mature glomeruli. RNA analysis showed two bands, the original and the other of a decreased length. Immunohistochemically, podocin was detected in a linear pattern along the glomerular capillary loop. Antipodocin antibody (C-terminal) stained the smooth muscles of renal arterioles and aorta. Among 42 patients, podocin was normally expressed in glomeruli in purpura nephritis, IgA nephropathy (IgAN), and minimal-change disease (MCD), while it was either decreased or absent in most subjects with focal segmental glomerulosclerosis (FSGS). The expression of synaptopodin was similar to that of podocin, although some discrepancy existed. Although indirect, our data suggest the existence of a vascular isoform of podocin with a different molecular mass. We propose that examination of podocin expression may help differentiate MCD from FSGS.

Morphological insights into the origin of glomerular endothelial and mesangial cells and their precursors.

Glomerular endothelial and mesangial cells may originate from the metanephric mesenchyme. We used the MAb Thy1.1, a mesangial cell marker in the adult rat kidney, and rat endothelial cell markers MAb RECA-1, MAb PECAM-1 (CD31), and MAb Flk-1 as potential markers to characterize the spatial and temporal distribution of mesangial and endothelial cell precursors during nephrogenesis in the rat. At early stages of glomerulogenesis, RECA-1- and Thy1.1-positive cells were detected in the metanephric blastema at 14 days post conception (dpc) embryos and 15 dpc, respectively, with Thy1.1 expression in cells surrounding the ureteric bud. At 17 and 18 dpc, both RECA-1- and Thy1.1-positive cells were found in the cleft of the S-shaped bodies and in the capillary loops of maturing glomeruli. Double staining for BrdU, a marker of proliferation, and for RECA-1 or BrdU and Thy1.1 also localize in the cleft of S-shaped bodies and in glomerular capillary loops at later stages of development. PDGFRbeta co-localizes in cells expressing endothelial or mesangial markers. The data suggest that endothelial and mesangial cell precursors share common markers during the course of glomerulogenesis and that full differentiation of these cells occurs at late stages of glomerular maturation. Thy1.1- and RECA-1-positive cells may be derived from the metanephric blastemal cells at early stages of kidney development. A subpopulation of these Thy1.1- or RECA-1-positive cells may be precursors that can migrate into the cleft of comma and S-shaped bodies and proliferate in situ to form glomerular capillary tufts.

Glomerular C4d deposition indicates in situ classic complement pathway activation, but is not a marker for lupus nephritis activity.

This study was designed to evaluate whether glomerular C4d deposition may be a useful marker of lupus nephritis activity. Twenty-one patients diagnosed as having lupus nephritis (WHO class III: 4 cases; IV: 12 cases; V: 5 cases) were included. Mean patient age was 29.3 +/- 13.5 years (range: 7-55 years). The presence and intensity of glomerular C4d deposition were compared with the corresponding histologic activity index for each case. Immunofluorescence for C4d showed diffusely granular staining along glomerular capillary loops, in all cases examined (1+, in 8 cases; 2+, in 7 cases; 3+, in 6 cases). In eight cases, C4d deposition was found in the absence of capillary or mesangial C4 deposits. Moreover, the intensity of C4d deposits correlated with those of capillary IgG, IgA, C4, C1q, and fibrinogen deposits. However, C4d staining intensity did not correlate with the lupus nephritis activity index. Although glomerular capillary C4d deposition is a sensitive marker of classic complement pathway activation, it is not a sensitive marker for active lupus nephritis.

Glomerular endothelial fenestrae in vivo are not formed from caveolae.

Previous reports indicate that endothelial fenestrae in vitro can form by fusion of caveolae or caveolae-like vesicles. The principal aim of this study was to determine whether formation of glomerular endothelial cell fenestrae in vivo similarly involves caveolae and caveolin-1. Whereas caveolin-1 immunofluorescence was found around the circumference of human and mouse glomerular capillary loops, it co-localized only partially with the endothelium-specific lectin Ulex Europaeus I in human glomeruli, leaving portions of the endothelium devoid of caveolin-1. Immunogold electron microscopy, used to definitively localize caveolin-1 in glomeruli, showed that caveolin-1 was completely excluded from the fenestrated portion of the endothelium. Moreover, in caveolin-1-deficient mice, which cannot form caveolae, the ultrastructure of glomerular endothelial fenestrae appeared entirely normal. Interestingly, strong caveolin-1 immunogold labeling was observed in podocytes, where some caveolin-1 localized to filtration slits. Caveolin-1 co-immunoprecipitated with the podocyte slit diaphragm proteins nephrin and CD2AP, and dual immunofluorescence confirmed co-localization of caveolin-1 and nephrin. Nevertheless, in caveolin-1-deficient mice, podocyte ultrastructure appeared normal, and the podocyte proteins synaptopodin, nephrin, and podocin were expressed normally. In addition, blood urea nitrogen concentrations and urinary protein excretion in these mice were similar to those in wild-type mice. Thus, unlike caveolae formation, glomerular endothelial cell fenestrae formation in vivo does not require caveolin-1, ruling out the previous hypothesis that endothelial fenestrae represent fused caveolae, at least for glomerular endothelial cells. Localization of caveolin-1 to podocytes and their filtration slits is consistent with the view that the filtration slit plasma membrane represents a type of lipid raft microdomain.

PDZ domain-mediated interaction of rabbit podocalyxin and Na(+)/H(+) exchange regulatory factor-2.

The transmembrane sialoglycoprotein podocalyxin is thought to be essential in the fine interdigitating foot process structure of the podocyte. The intracellular COOH-terminal amino acids Asp-Thr-His-Leu (DTHL) of podocalyxin comprise a putative ligand for a type I PSD95-Dlg-zona occludens-1 (PDZ) domain. A 20-amino acid synthetic peptide containing this motif was used to screen a cDNA library, and clones of rabbit Na(+)/H(+) exchange regulatory factor-2 (NHERF-2) were obtained. In vitro analysis demonstrated that each PDZ domain of NHERF-2 could bind podocalyxin independently. NHERF-2 coprecipitated from glomerular extracts with podocalyxin, and podocalyxin and NHERF-2 colocalized in the glomerular capillary loops, indicating that podocalyxin and NHERF-2 may interact in vivo. Podocalyxin peptide missing the terminal leucine (-DTHL) failed to interact with NHERF-2 in vitro. Podocalyxin localized to the apical membrane of transfected Madin-Darby canine kidney (MDCK) cells. However, mutant podocalyxin (missing a functional DTHL COOH-terminal motif) showed cytoplasmic and apical membrane localization in transfected cells and was also less stable at the apical membrane, as assessed by confocal microscopy and biotinylation studies. Mutant podocalyxin did lower the transepithelial resistance of MDCK cell monolayers, albeit to a lesser extent than full-length podocalyxin. We conclude that podocalyxin can interact with both PDZ domains of NHERF-2 and that this interaction requires the intact COOH terminus of podocalyxin, which is also responsible for the efficient apical localization of podocalyxin in transfected MDCK cells. These results suggest that the interaction of podocalyxin with NHERF-2 may function to efficiently retain podocalyxin at the apical surface of the podocyte and provide a mechanism linking podocalyxin to the actin cytoskeleton.

Glomerular endothelial cells and podocytes jointly synthesize laminin-1 and -11 chains

The glomerular basement membrane (GBM) originates in development from fusion of subendothelial and subepithelial matrices. Subsequently, newly synthesized subepithelial matrix is added as glomerular capillary loops expand. During GBM assembly, the laminin-1 heterotrimer (alpha 1, beta 1, and gamma 1 chains), initially expressed in vascular clefts of comma- and S-shaped bodies, is eventually replaced by laminin-11 (alpha 5, beta 2, and gamma 1 chains), which persists into maturation. The cellular source(s) of these laminins is not known and prompted this study. To determine which cells synthesize the various laminin chains, postfixation immunoelectron microscopy of developing mouse kidney was performed using monoclonal and polyclonal antibodies that specifically recognized laminin alpha 1, beta 1, alpha 5, or beta 2 chains. Intracellular labeling for laminin alpha 1, beta 1 (laminin-1), and alpha 5 and beta 2 (laminin-11) chains was observed in developing glomerular endothelial cells and podocytes of comma- and S-shaped nephric figures. Laminin-1 was also seen in unfused GBMs at this stage, whereas laminin-11 was only found intracellularly. In capillary loop stage GBMs, laminin alpha 1 chain was completely absent, whereas labeling for laminin alpha 5 was intense, indicating rapid substitution between alpha chains. In contrast, laminin beta 1 chain labeling remained strong both intracellularly and in GBMs of capillary loop stage glomeruli, and beta 2 was up-regulated as well. In maturing stage glomeruli, beta 1 labeling declined, and alpha 5 and beta 2 remained at high levels intracellularly in both endothelial cells and podocytes and in GBMs. Our results show that both endothelial cells and podocytes synthesize laminin-1 and -11 chains throughout glomerular development. The sustained and comparatively high level of laminin synthesis by endothelial cells was unexpected, suggesting that the endothelium may be an important source of GBM proteins in glomerular disease.

Transplantation of rat metanephroi into mice.

To determine whether transplanted metanephroi grow and differentiate after implantation into the omentum in hosts of a different species, we implanted metanephroi from embryonic day 15 (E15) rat embryos into uninephrectomized mice (hosts). Some host mice received human CTLA4Ig (hCTLA4Ig), anti-CD45RB, and anti-CD154 (tolerance-inducing agents). E15 metanephroi contained only metanephric blastema, segments of ureteric bud, and primitive nephrons with no glomeruli. Rat metanephroi did not grow or differentiate in mice that received no tolerance-inducing agents. However, by 2 wk posttransplantation in mice that received hCTLA4Ig, anti-CD45RB, and anti-CD154, metanephroi from E15 rats had enlarged, become vascularized, and formed mature tubules and glomeruli. Rat metanephroi contained cells that stained specifically for mouse CD31, a marker for sprouting endothelial cells. Some rat glomerular capillary loops stained positively for mouse CD31. Here, we show that chimeric kidneys develop from metanephroi transplanted rat-->mouse and that glomeruli are vascularized, at least in part, by host vessels.

Participation of endothelial cells and transformed mesangial cells in remodeling of glomerular capillary loops in Thy-1 nephritis.

The relationship between mesangial cells (MC) and endothelial cells (EC) in the remodeling of glomerular capillary loops was investigated in a rat model of anti-Thy-1 antibody (Ab)-induced glomerulonephritis. Immunohistochemical analysis showed that cells positive for alpha-smooth muscle actin (alpha-SMA) appeared in the mesangial stalks at day three, and had increased in number at day seven, after injection of Thy-1 Ab. Double staining for alpha-SMA and proliferating cell nuclear antigen (PCNA) showed that some MC expressing PCNA were negative for alpha-SMA at day three, but by day seven almost all PCNA-positive MC expressed alpha-SMA. Western blotting for alpha-SMA from isolated glomeruli was negative at day one after injection of Thy-1 Ab, but positive at day seven. Type III collagen appeared at day seven, followed by an increase of EC in the capillary loops, as determined by double immunofluorescent staining for rat endothelial cell antigen-1 (RECA-1) and type III collagen. RECA-1-positive cells increased rapidly in number after day seven and eventually showed the same distribution pattern as that in control rats. Both type I and type III collagens were expressed in the mesangial and the ballooning area of the glomerulus at day seven. Electron microscopy revealed that immature MC and EC forming small capillary lumina appeared in the enlarged mesangial area at day seven. In accordance with the increase of capillaries and the enlargement of the lumina, the number of MC and the amount of mesangial matrix decreased gradually, and most of the glomeruli returned to a normal structure by week 4. These data show that type I and type III collagen produced by transformed MC may be of benefit to proliferation of EC and remodeling of the capillary in Thy-1-induced nephritis.

Impaired angiogenesis in the aging kidney: Vascular endothelial growth factor and thrombospondin-1 in renal disease

<h2>Abstract</h2> We investigated the relationship of changes in the microvasculature to age-related structural and functional changes in the kidney to determine whether there was evidence of impaired angiogenesis and whether the loss of microvasculature could be accounted for by changes in the local production of angiogenic or antiangiogenic factors. Glomerular and peritubular capillary number, density, and endothelial cell proliferation were determined in aging (24 months; n=9) and young (3 months; n=8) rat kidneys and correlated with renal functional and structural changes and alterations in renal expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). Aging rats showed a focal decrease in both peritubular capillary (peritubular capillary staining, 5.4% ± 1.8% versus 11.3% ± 2.0% per 100 tubules; rarefaction index, 10.6% ± 4.6% versus 0.6% ± 0.1%, aging versus young rats; <i>P</i> < 0.05 and <i>P</i> < 0.001, respectively) and glomerular capillary loops (27.3 ± 6.9 versus 50.7 ± 7.4/glomerulus, aging versus young rats; <i>P</i> < 0.001). The number of proliferating endothelial cells was decreased in aging rats compared with young rats (glomerular, 0.04 ± 0.01 versus 0.15 ± 0.03 positive cells/glomerular cross-section; peritubular, 0.7 ± 0.2 versus 4.3 ± 2.6 positive cells/mm²; <i>P</i> < 0.05). In the aging kidney, VEGF expression was focally increased in the cortex compared with young rats, whereas a profound decrease was observed in the outer and inner medulla (total area of VEGF expression, 19.2% ± 11.4% versus 39.3% ± 7.6%; <i>P</i> < 0.05). Tubular VEGF expression correlated with peritubular capillary density (<i>r²</i> = 0.57; <i>P</i> < 0.01) and inversely correlated with tubular osteopontin (<i>r²</i> = –0.55; <i>P</i> < 0.05) and macrophage infiltration (<i>r²</i> = –0.64; <i>P</i> < 0.01). TSP-1 staining was increased in the glomeruli and tubulointerstitium of the aging rats. Glomerular TSP-1 score correlated inversely with glomerular capillary number (<i>r²</i> = –0.89; <i>P</i> < 0.001). Tubulointerstitial TSP-1 also correlated with percentage of positive staining of peritubular capillary (<i>r²</i> = –0.59; <i>P</i> < 0.001). Glomerular capillary number showed significant correlation with glomerulosclerosis score, as well as with 24-hour urinary protein excretion. Peritubular capillary density also inversely correlated with interstitial fibrosis score and urinary protein excretion. In conclusion, glomerular and peritubular capillary loss in the aging kidney correlate with alterations in VEGF and TSP-1 expression and also with the development of glomerulosclerosis and tubulointerstitial fibrosis. These findings suggest that impaired angiogenesis associated with progressive loss in renal microvasculature may have a pivotal role in age-related nephropathy.